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Tanaka S, Oide H, Ikeda S, Tagaya M, Nagai H, Kubori T, Arasaki K. Subversion of the host endocytic pathway by Legionella pneumophila-mediated ubiquitination of Rab5. J Cell Biol 2025; 224:e202406159. [PMID: 40035702 PMCID: PMC11893168 DOI: 10.1083/jcb.202406159] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2024] [Revised: 11/17/2024] [Accepted: 01/10/2025] [Indexed: 03/06/2025] Open
Abstract
Legionella pneumophila is an intracellular bacterial pathogen that modulates membrane trafficking to survive and proliferate within host cells. After phagocytosis, the L. pneumophila-containing vacuole evades the endocytic pathway by excluding the host GTPase Rab5, a crucial regulator of phagosomal maturation. In this study, we show that the evolutionarily conserved lysine residue K134 of Rab5 undergoes ubiquitination during infection. This modification depends on Lpg2525, an F-box protein from L. pneumophila that acts as a component of the SKP-Cullin-F-box complex. We further demonstrate that Rab5 ubiquitination facilitates the recruitment of RabGAP-5, a Rab5-specific GAP, leading to Rab5 inactivation and subsequent release from the bacterial vacuole. Importantly, the K134 Rab5 mutant limits L. pneumophila replication within host cells. These findings reveal that Lpg2525-mediated Rab5 ubiquitination is a key survival strategy employed by L. pneumophila in infected host cells.
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Affiliation(s)
- Shino Tanaka
- School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan
| | - Hiromu Oide
- School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan
| | - Shumma Ikeda
- School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan
| | - Mitsuo Tagaya
- School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan
| | - Hiroki Nagai
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Japan
- Center for One Medicine Innovative Translational Research (COMIT), Institute for Advanced Study, Gifu University, Gifu, Japan
| | - Tomoko Kubori
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Japan
| | - Kohei Arasaki
- School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Japan
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2
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Solger F, Rauch J, Vormittag S, Fan M, Raykov L, Charki P, Katic A, Letourneur F, Soldati T, Seibel J, Hilbi H. Inter-kingdom signaling by the Legionella autoinducer LAI-1 involves the antimicrobial guanylate binding protein GBP. PLoS Pathog 2025; 21:e1013026. [PMID: 40300029 PMCID: PMC12040241 DOI: 10.1371/journal.ppat.1013026] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2024] [Accepted: 03/07/2025] [Indexed: 05/01/2025] Open
Abstract
The causative agent of Legionnaires' disease, Legionella pneumophila, is an amoebae-resistant environmental bacterium, which replicates intracellularly in a distinct compartment, the "Legionella-containing vacuole" (LCV). L. pneumophila employs the α-hydroxyketone compound LAI-1 (Legionella autoinducer-1) for intra-species and inter-kingdom signaling. LAI-1 promotes intracellular replication and inhibits the migration of mammalian cells and Dictyostelium discoideum. In this study, we revealed that LAI-1 and "clickable" azido-LAI-1 derivatives inhibit the migration of D. discoideum and localize to LCVs. Azido-LAI-1 colocalizes with the LCV markers calnexin, P4C, and AmtA, but not with mitochondrial or lipid droplet markers. Intriguingly, LAI-1-dependent inhibition of D. discoideum migration involves the single guanylate-binding protein (GBP), a member of the GBP family of large GTPases, which in metazoan organisms promote cell autonomous immunity. D. discoideum lacking GBP (Δgnbp) allows more efficient intracellular replication of L. pneumophila, without apparently compromising LCV formation or integrity, and GBP-GFP localizes to the ER at LCV-ER membrane contact sites (MCS). However, the peri-LCV localization of LAI-1 and GBP is not mutually dependent. Synthetic LAI-1 inhibits the expansion/remodeling of LCVs (but not vacuoles harboring avirulent L. pneumophila) in a GBP-dependent manner. Taken together, the work shows that LAI-1 localizes to LCVs, and LAI-1-dependent inter-kingdom signaling involves D. discoideum GBP, which localizes to LCV-ER MCS and acts as an antimicrobial factor by restricting the intracellular growth of L. pneumophila.
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Affiliation(s)
- Franziska Solger
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Jonas Rauch
- Institute of Organic Chemistry, University of Würzburg, Würzburg, Germany
| | - Simone Vormittag
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Mingzhen Fan
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Lyudmil Raykov
- Department of Biochemistry, Faculty of Science, University of Geneva, Geneva, Switzerland
| | - Paul Charki
- Institute of Organic Chemistry, University of Würzburg, Würzburg, Germany
| | - Ana Katic
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | | | - Thierry Soldati
- Department of Biochemistry, Faculty of Science, University of Geneva, Geneva, Switzerland
| | - Jürgen Seibel
- Institute of Organic Chemistry, University of Würzburg, Würzburg, Germany
| | - Hubert Hilbi
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
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3
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Mount HO, Urbanus ML, Zangari F, Gingras AC, Ensminger AW. The Legionella pneumophila effector PieF modulates mRNA stability through association with eukaryotic CCR4-NOT. mSphere 2025; 10:e0089124. [PMID: 39699231 PMCID: PMC11774319 DOI: 10.1128/msphere.00891-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2024] [Accepted: 11/26/2024] [Indexed: 12/20/2024] Open
Abstract
The eukaryotic CCR4-NOT deadenylase complex is a highly conserved regulator of mRNA metabolism that influences the expression of the complete transcriptome, representing a prime target for a generalist bacterial pathogen. We show that a translocated bacterial effector protein, PieF (Lpg1972) of Legionella pneumophila, directly interacts with the CNOT7/8 nuclease module of CCR4-NOT, with a dissociation constant in the low nanomolar range. PieF is a robust in vitro inhibitor of the DEDD-type nuclease, CNOT7, acting in a stoichiometric, dose-dependent manner. Heterologous expression of PieF phenocopies knockout of the CNOT7 ortholog (POP2) in Saccharomyces cerevisiae, resulting in 6-azauracil sensitivity. In mammalian cells, expression of PieF leads to a variety of quantifiable phenotypes: PieF silences gene expression and reduces mRNA steady-state levels when artificially tethered to a reporter transcript, and its overexpression results in the nuclear exclusion of CNOT7. PieF expression also disrupts the association between CNOT6/6L EEP-type nucleases and CNOT7. Adding to the complexities of PieF activity in vivo, we identified a separate domain of PieF responsible for binding to eukaryotic kinases. Unlike what we observe for CNOT6/6L, we show that these interactions can occur concomitantly with PieF's binding to CNOT7. Collectively, this work reveals a new, highly conserved target of L. pneumophila effectors and suggests a mechanism by which the pathogen may be modulating host mRNA stability and expression during infection. IMPORTANCE The intracellular bacterial pathogen Legionella pneumophila targets conserved eukaryotic pathways to establish a replicative niche inside host cells. With a host range that spans billions of years of evolution (from protists to humans), the interaction between L. pneumophila and its hosts frequently involves conserved eukaryotic pathways (protein translation, ubiquitination, membrane trafficking, autophagy, and the cytoskeleton). Here, we present the identification of a new, highly conserved host target of L. pneumophila effectors: the CCR4-NOT complex. CCR4-NOT modulates mRNA stability in eukaryotes from yeast to humans, making it an attractive target for a generalist pathogen, such as L. pneumophila. We show that the uncharacterized L. pneumophila effector PieF specifically targets one component of this complex, the deadenylase subunit CNOT7/8. We show that the interaction between PieF and CNOT7 is direct, occurs with high affinity, and reshapes the catalytic activity, localization, and composition of the complex across evolutionarily diverse eukaryotic cells.
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Affiliation(s)
| | - Malene L. Urbanus
- Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
| | - Francesco Zangari
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
- Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada
| | - Anne-Claude Gingras
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
- Lunenfeld-Tanenbaum Research Institute, Sinai Health, Toronto, Ontario, Canada
| | - Alexander W. Ensminger
- Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
- Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada
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4
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Romanov KA, O'Connor TJ. Legionella pneumophila, a Rosetta stone to understanding bacterial pathogenesis. J Bacteriol 2024; 206:e0032424. [PMID: 39636264 PMCID: PMC11656745 DOI: 10.1128/jb.00324-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/07/2024] Open
Abstract
Legionella pneumophila is an environmentally acquired pathogen that causes respiratory disease in humans. While the discovery of L. pneumophila is relatively recent compared to other bacterial pathogens, over the past 50 years, L. pneumophila has emerged as a powerhouse for studying host-pathogen interactions. In its natural habitat of fresh water, L. pneumophila interacts with a diverse array of protozoan hosts and readily evolve to expand their host range. This has led to the accumulation of the most extensive arsenal of secreted virulence factors described for a bacterial pathogen and their ability to infect humans. Within amoebae and human alveolar macrophages, the bacteria replicate within specialized membrane-bound compartments, establishing L. pneumophila as a model for studying intracellular vacuolar pathogens. In contrast, the virulence factors required for intracellular replication are specifically tailored to individual host cells types, allowing the pathogen to adapt to variation between disparate niches. The broad host range of this pathogen, combined with the extensive diversity and genome plasticity across the Legionella genus, has thus established this bacterium as an archetype to interrogate pathogen evolution, functional genomics, and ecology. In this review, we highlight the features of Legionella that establish them as a versatile model organism, new paradigms in bacteriology and bacterial pathogenesis resulting from the study of Legionella, as well as current and future questions that will undoubtedly expand our understanding of the complex and intricate biology of the microbial world.
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Affiliation(s)
- Katerina A. Romanov
- Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
| | - Tamara J. O'Connor
- Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
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Ma K, Shu R, Liu H, Ge J, Liu J, Lu Q, Fu J, Liu X, Qiu J. Legionella effectors SidC/SdcA ubiquitinate multiple small GTPases and SNARE proteins to promote phagosomal maturation. Cell Mol Life Sci 2024; 81:249. [PMID: 38836877 PMCID: PMC11335287 DOI: 10.1007/s00018-024-05271-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2023] [Revised: 01/30/2024] [Accepted: 05/07/2024] [Indexed: 06/06/2024]
Abstract
Protein ubiquitination is one of the most important posttranslational modifications (PTMs) in eukaryotes and is involved in the regulation of almost all cellular signaling pathways. The intracellular bacterial pathogen Legionella pneumophila translocates at least 26 effectors to hijack host ubiquitination signaling via distinct mechanisms. Among these effectors, SidC/SdcA are novel E3 ubiquitin ligases with the adoption of a Cys-His-Asp catalytic triad. SidC/SdcA are critical for the recruitment of endoplasmic reticulum (ER)-derived vesicles to the Legionella-containing vacuole (LCV). However, the ubiquitination targets of SidC/SdcA are largely unknown, which restricts our understanding of the mechanisms used by these effectors to hijack the vesicle trafficking pathway. Here, we demonstrated that multiple Rab small GTPases and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are bona fide ubiquitination substrates of SidC/SdcA. SidC/SdcA-mediated ubiquitination of syntaxin 3 and syntaxin 4 promotes their unconventional pairing with the vesicle-SNARE protein Sec22b, thereby contributing to the membrane fusion of ER-derived vesicles with the phagosome. In addition, our data reveal that ubiquitination of Rab7 by SidC/SdcA is critical for its association with the LCV membrane. Rab7 ubiquitination could impair its binding with the downstream effector Rab-interacting lysosomal protein (RILP), which partially explains why LCVs avoid fusion with lysosomes despite the acquisition of Rab7. Taken together, our study reveals the biological mechanisms employed by SidC/SdcA to promote the maturation of the LCVs.
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Affiliation(s)
- Kelong Ma
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China
| | - Rundong Shu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China
| | - Hongtao Liu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China
| | - Jinli Ge
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China
| | - Jiayang Liu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China
| | - Qian Lu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China
| | - Jiaqi Fu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China
| | - Xiaoyun Liu
- Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China
- NHC Key Laboratory of Medical Immunology, Peking University, Beijing, China
| | - Jiazhang Qiu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China.
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6
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Lehman SS, Verhoeve VI, Driscoll TP, Beckmann JF, Gillespie JJ. Metagenome diversity illuminates the origins of pathogen effectors. mBio 2024; 15:e0075923. [PMID: 38564675 PMCID: PMC11077975 DOI: 10.1128/mbio.00759-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2023] [Accepted: 02/12/2024] [Indexed: 04/04/2024] Open
Abstract
Recent metagenome-assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. The discovery of basal lineages (novel families Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles exposed an evolutionary timepoint for the transition to host dependency, which seemingly occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales vir homolog (rvh) type IV secretion system and purportedly use rvh to kill congener microbes rather than parasitize host cells as described for later-evolving rickettsial pathogens. MAG analysis also substantially increased diversity for the genus Rickettsia and delineated a sister lineage (the novel genus Tisiphia) that stands to inform on the emergence of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of Rickettsia rvh effectors to ascertain their origins. A sparse distribution of most Rickettsia rvh effectors outside of Rickettsiaceae lineages illuminates unique rvh evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, indicating profound roles for gene duplication and recombination in shaping effector repertoires in Rickettsia pathogens. Lateral gene transfer plays a prominent role in shaping the rvh effector landscape, as evinced by the discovery of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between Rickettsia and Legionella species. Our study exemplifies how MAGs can yield insight into pathogen effector origins, particularly how effector architectures might become tailored to the discrete host cell functions of different eukaryotic hosts.IMPORTANCEWhile rickettsioses are deadly vector-borne human diseases, factors distinguishing Rickettsia pathogens from the innumerable bevy of environmental rickettsial endosymbionts remain lacking. Recent metagenome-assembled genome (MAG) studies revealed evolutionary timepoints for rickettsial transitions to host dependency. The rvh type IV secretion system was likely repurposed from congener killing in basal extracellular species to parasitizing host cells in later-evolving pathogens. Our analysis of MAG diversity for over two dozen rvh effectors unearthed their presence in some non-pathogens. However, most effectors were found in multiple divergent forms with variable architectures, indicating gene duplication and recombination-fashioned effector repertoires of Rickettsia pathogens. Lateral gene transfer substantially shaped pathogen effector arsenals, evinced by the discovery of effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchanges between Rickettsia and Legionella species. Our study exemplifies how MAGs yield insight into pathogen effector origins and evolutionary processes tailoring effectors to eukaryotic host cell biology.
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Affiliation(s)
- Stephanie S. Lehman
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA
| | - Victoria I. Verhoeve
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Timothy P. Driscoll
- Department of Biology, West Virginia University, Morgantown, West Virginia, USA
| | - John F. Beckmann
- Department of Microbiology and Immunology, University of South Alabama, Mobile, Alabama, USA
| | - Joseph J. Gillespie
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
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7
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Lehman SS, Williamson CD, Tucholski T, Ellis NA, Bouchard S, Jarnik M, Allen M, Nita-Lazar A, Machner MP. The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling. Cell Rep 2024; 43:114033. [PMID: 38568811 PMCID: PMC11141579 DOI: 10.1016/j.celrep.2024.114033] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Revised: 01/17/2024] [Accepted: 03/18/2024] [Indexed: 04/05/2024] Open
Abstract
Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.
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Affiliation(s)
- Stephanie S Lehman
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
| | - Chad D Williamson
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
| | - Trisha Tucholski
- Functional Cellular Networks Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Nicole A Ellis
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
| | - Sabrina Bouchard
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
| | - Michal Jarnik
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
| | - Morgan Allen
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
| | - Aleksandra Nita-Lazar
- Functional Cellular Networks Section, Laboratory of Immune System Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Matthias P Machner
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
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8
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Schroeder EA, Toro-Moreno M, Raphemot R, Sylvester K, Colón IC, Derbyshire ER. Toxoplasma and Plasmodium associate with host Arfs during infection. mSphere 2024; 9:e0077023. [PMID: 38349168 PMCID: PMC10964417 DOI: 10.1128/msphere.00770-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2023] [Accepted: 01/17/2024] [Indexed: 03/27/2024] Open
Abstract
The apicomplexans Toxoplasma gondii and Plasmodium are intracellular parasites that reside within a host-derived compartment termed the parasitophorous vacuole (PV). During infection, the parasites must acquire critical host resources and transport them across their PV for development. However, the mechanism by which host resources are trafficked to and across the PV remains uncertain. Here, we investigated host ADP ribosylation factors (Arfs), a class of proteins involved in vesicular trafficking that may be exploited by T. gondii and Plasmodium berghei for nutrient acquisition. Using overexpressed Arf proteins coupled with immunofluorescence microscopy, we found that all Arfs were internalized into the T. gondii PV, with most vacuoles containing at least one punctum of Arf protein by the end of the lytic cycle. We further characterized Arf1, the most abundant Arf inside the T. gondii PV, and observed that active recycling between its GDP/GTP-bound state influenced Arf1 internalization independent of host guanine nucleotide exchange factors (GEFs). In addition, Arf1 colocalized with vesicle coat complexes and exogenous sphingolipids, suggesting a role in nutrient acquisition. While Arf1 and Arf4 were not observed inside the PV during P. berghei infection, our gene depletion studies showed that liver stage development and survival depended on the expression of Arf4 and the host GEF, GBF1. Collectively, these observations indicate that apicomplexans use distinct mechanisms to subvert the host vesicular trafficking network and efficiently replicate. The findings also pave the way for future studies to identify parasite proteins critical to host vesicle recruitment and the components of vesicle cargo. IMPORTANCE The parasites Toxoplasma gondii and Plasmodium live complex intracellular lifestyles where they must acquire essential host nutrients while avoiding recognition. Although previous work has sought to identify the specific nutrients scavenged by apicomplexans, the mechanisms by which host materials are transported to and across the parasite vacuole membrane are largely unknown. Here, we examined members of the host vesicular trafficking network to identify specific pathways subverted by T. gondii and Plasmodium berghei. Our results indicate that T. gondii selectively internalizes host Arfs, a class of proteins involved in intracellular trafficking. For P. berghei, host Arfs were restricted by the parasite's vacuole membrane, but proteins involved in vesicular trafficking were identified as essential for liver stage development. A greater exploration into how and why apicomplexans subvert host vesicular trafficking could help identify targets for host-directed therapeutics.
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Affiliation(s)
- Erin A. Schroeder
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA
| | - Maria Toro-Moreno
- Department of Chemistry, Duke University, Durham, North Carolina, USA
| | - Rene Raphemot
- Department of Chemistry, Duke University, Durham, North Carolina, USA
| | - Kayla Sylvester
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA
| | - Isabel C. Colón
- Department of Chemistry, Duke University, Durham, North Carolina, USA
| | - Emily R. Derbyshire
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA
- Department of Chemistry, Duke University, Durham, North Carolina, USA
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9
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Syriste L, Patel DT, Stogios PJ, Skarina T, Patel D, Savchenko A. An acetyltransferase effector conserved across Legionella species targets the eukaryotic eIF3 complex to modulate protein translation. mBio 2024; 15:e0322123. [PMID: 38335095 PMCID: PMC10936415 DOI: 10.1128/mbio.03221-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Accepted: 01/16/2024] [Indexed: 02/12/2024] Open
Abstract
The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires' disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of "core" effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria's pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro, revealing the potential function of VipF "core" effectors in Legionella's pathogenic strategy.IMPORTANCEBy translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro. These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae.
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Affiliation(s)
- Lukas Syriste
- Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada
| | - Deepak T. Patel
- Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada
| | - Peter J. Stogios
- Department of Chemical Engineering and Applied Chemistry, Toronto University, Toronto, Ontario, Canada
| | - Tatiana Skarina
- Department of Chemical Engineering and Applied Chemistry, Toronto University, Toronto, Ontario, Canada
| | - Dhruvin Patel
- Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada
| | - Alexei Savchenko
- Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada
- Department of Chemical Engineering and Applied Chemistry, Toronto University, Toronto, Ontario, Canada
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10
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Steinbach A, Bhadkamkar V, Jimenez-Morales D, Stevenson E, Jang GM, Krogan NJ, Swaney DL, Mukherjee S. Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila. Mol Biol Cell 2024; 35:ar27. [PMID: 38117589 PMCID: PMC10916871 DOI: 10.1091/mbc.e23-06-0260] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 12/04/2023] [Accepted: 12/13/2023] [Indexed: 12/22/2023] Open
Abstract
The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.
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Affiliation(s)
- Adriana Steinbach
- Department of Microbiology and Immunology, University of California, San Francisco, CA 94143
- George Williams Hooper Foundation, University of California, San Francisco, CA 94143
| | - Varun Bhadkamkar
- Department of Microbiology and Immunology, University of California, San Francisco, CA 94143
- George Williams Hooper Foundation, University of California, San Francisco, CA 94143
| | - David Jimenez-Morales
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA 94158
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
- Department of Medicine, Division of Cardiovascular Medicine, Stanford University, CA 94309
| | - Erica Stevenson
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA 94158
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
- Quantitative Biosciences Institute, University of California, San Francisco, CA 94158
| | - Gwendolyn M. Jang
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA 94158
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
- Quantitative Biosciences Institute, University of California, San Francisco, CA 94158
| | - Nevan J. Krogan
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA 94158
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
- Quantitative Biosciences Institute, University of California, San Francisco, CA 94158
| | - Danielle L. Swaney
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, CA 94158
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158
- Quantitative Biosciences Institute, University of California, San Francisco, CA 94158
| | - Shaeri Mukherjee
- Department of Microbiology and Immunology, University of California, San Francisco, CA 94143
- George Williams Hooper Foundation, University of California, San Francisco, CA 94143
- Chan Zuckerberg Biohub, San Francisco, CA 94158
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11
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Sheet S, Sathishkumar Y, Acharya S, Lee YS. Exposure of Legionella pneumophila to low-shear modeled microgravity: impact on stress response, membrane lipid composition, pathogenicity to macrophages and interrelated genes expression. Arch Microbiol 2024; 206:87. [PMID: 38305908 DOI: 10.1007/s00203-023-03753-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2023] [Revised: 10/31/2023] [Accepted: 11/16/2023] [Indexed: 02/03/2024]
Abstract
Here, we studied the effect of low-shear modeled microgravity (LSMMG) on cross stress resistance (heat, acid, and oxidative), fatty acid content, and pathogenicity along with alteration in expression of stress-/virulence-associated genes in Legionella pneumophila. The stress resistance analysis result indicated that bacteria cultivated under LSMMG environments showed higher resistance with elevated D-values at 55 °C and in 1 mM of hydrogen peroxide (H2O2) conditions compared to normal gravity (NG)-grown bacteria. On the other hand, there was no significant difference in tolerance (p < 0.05) toward simulated gastric fluid (pH-2.5) acid conditions. In fatty acid analysis, our result showed that a total amount of saturated and cyclic fatty acids was increased in LSMMG-grown cells; as a consequence, they might possess low membrane fluidity. An upregulated expression level was noticed for stress-related genes (hslV, htrA, grpE, groL, htpG, clpB, clpX, dnaJ, dnaK, rpoH, rpoE, rpoS, kaiB, kaiC, lpp1114, ahpC1, ahpC2, ahpD, grlA, and gst) under LSMMG conditions. The reduced virulence (less intracellular bacteria and less % of induce apoptosis in RAW 264.7 macrophages) of L. pneumophila under LSMMG conditions may be because of downregulation related genes (dotA, dotB, dotC, dotD, dotG, dotH, dotL, dotM, dotN, icmK, icmB, icmS, icmT, icmW, ladC, rtxA, letA, rpoN, fleQ, fleR, and fliA). In the LSMMG group, the expression of inflammation-related factors, such as IL-1α, TNF-α, IL-6, and IL-8, was observed to be reduced in infected macrophages. Also, scanning electron microscopy (SEM) analysis showed less number of LSMMG-cultivated bacteria attached to the host macrophages compared to NG. Thus, our study provides understandings about the changes in lipid composition and different genes expression due to LSMMG conditions, which apparently influence the alterations of L. pneumophila' stress/virulence response.
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Affiliation(s)
- Sunirmal Sheet
- Department of Wood Science and Technology, College of Agriculture and Life Sciences, Jeonbuk National University, 567, Jeonju-si, Jeollabuk-do, Republic of Korea
| | | | - Satabdi Acharya
- Department of Biomedical Sciences and Institute for Medical Science, Jeonbuk National University Medical School, 567, Jeonju-si, Jeollabuk-do, Republic of Korea
| | - Yang Soo Lee
- Department of Wood Science and Technology, College of Agriculture and Life Sciences, Jeonbuk National University, 567, Jeonju-si, Jeollabuk-do, Republic of Korea.
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12
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Graham CI, MacMartin TL, de Kievit TR, Brassinga AKC. Molecular regulation of virulence in Legionella pneumophila. Mol Microbiol 2024; 121:167-195. [PMID: 37908155 DOI: 10.1111/mmi.15172] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2023] [Revised: 09/15/2023] [Accepted: 09/17/2023] [Indexed: 11/02/2023]
Abstract
Legionella pneumophila is a gram-negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase-like metabolic changes via alternative sigma factors, and two-component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity.
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Affiliation(s)
- Christopher I Graham
- Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada
| | - Teassa L MacMartin
- Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada
| | - Teresa R de Kievit
- Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada
| | - Ann Karen C Brassinga
- Department of Microbiology, Faculty of Science, University of Manitoba, Winnipeg, Manitoba, Canada
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13
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Ma K, Shu R, Liu H, Fu J, Luo ZQ, Qiu J. Ubiquitination of Sec22b by a novel Legionella pneumophila ubiquitin E3 ligase. mBio 2023; 14:e0238223. [PMID: 37882795 PMCID: PMC10746214 DOI: 10.1128/mbio.02382-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2023] [Accepted: 09/14/2023] [Indexed: 10/27/2023] Open
Abstract
IMPORTANCE Protein ubiquitination is one of the most important post-translational modifications that plays critical roles in the regulation of a wide range of eukaryotic signaling pathways. Many successful intracellular bacterial pathogens can hijack host ubiquitination machinery through the action of effector proteins that are injected into host cells by secretion systems. Legionella pneumophila is the etiological agent of legionellosis that is able to survive and replicate in various host cells. The defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IV secretion system of L. pneumophila injects over 330 effectors into infected cells to create an optimal environment permissive for its intracellular proliferation. To date, at least 26 Dot/Icm substrates have been shown to manipulate ubiquitin signaling via diverse mechanisms. Among these, 14 are E3 ligases that either cooperate with host E1 and E2 enzymes or adopt E1/E2-independent catalytic mechanisms. In the present study, we demonstrate that the L. pneumophila effector Legionella ubiquitin ligase gene 15 (Lug15) is a novel ubiquitin E3 ligase. Lug15 is involved in the remodeling of LCV with polyubiquitinated species. Moreover, Lug15 catalyzes the ubiquitination of host SNARE protein Sec22b and mediates its recruitment to the LCV. Ubiquitination of Sec22b by Lug15 promotes its noncanonical pairing with plasma membrane-derived syntaxins (e.g., Stx3). Our study further reveals the complexity of strategies utilized by L. pneumophila to interfere with host functions by hijacking host ubiquitin signaling.
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Affiliation(s)
- Kelong Ma
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Rundong Shu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Hongtao Liu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Jiaqi Fu
- Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, Changchun, China
| | - Zhao-Qing Luo
- Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, Indiana, USA
| | - Jiazhang Qiu
- State Key Laboratory for Diagnosis and Treatment of Severe Zoonotic Infectious Diseases, Key Laboratory for Zoonosis Research of the Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
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14
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Paul P, Tiwari B. Organelles are miscommunicating: Membrane contact sites getting hijacked by pathogens. Virulence 2023; 14:2265095. [PMID: 37862470 PMCID: PMC10591786 DOI: 10.1080/21505594.2023.2265095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2023] [Accepted: 09/25/2023] [Indexed: 10/22/2023] Open
Abstract
Membrane Contact Sites (MCS) are areas of close apposition of organelles that serve as hotspots for crosstalk and direct transport of lipids, proteins and metabolites. Contact sites play an important role in Ca2+ signalling, phospholipid synthesis, and micro autophagy. Initially, altered regulation of vesicular trafficking was regarded as the key mechanism for intracellular pathogen survival. However, emerging studies indicate that pathogens hijack MCS elements - a novel strategy for survival and replication in an intracellular environment. Several pathogens exploit MCS to establish direct contact between organelles and replication inclusion bodies, which are essential for their survival within the cell. By establishing this direct control, pathogens gain access to cytosolic compounds necessary for replication, maintenance, escaping endocytic maturation and circumventing lysosome fusion. MCS components such as VAP A/B, OSBP, and STIM1 are targeted by pathogens through their effectors and secretion systems. In this review, we delve into the mechanisms which operate in the evasion of the host immune system when intracellular pathogens hostage MCS. We explore targeting MCS components as a novel therapeutic approach, modifying molecular pathways and signalling to address the disease's mechanisms and offer more effective, tailored treatments for affected individuals.
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Affiliation(s)
- Pratyashaa Paul
- Department of Biological Sciences, Indian Institute of Science Education and Research, India
| | - Bhavana Tiwari
- Department of Biological Sciences, Indian Institute of Science Education and Research, India
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15
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Steinbach AM, Bhadkamkar VL, Jimenez-Morales D, Stevenson E, Jang GM, Krogan NJ, Swaney DL, Mukherjee S. Cross-family small GTPase ubiquitination by the intracellular pathogen Legionella pneumophila. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.08.03.551750. [PMID: 37577546 PMCID: PMC10418220 DOI: 10.1101/2023.08.03.551750] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/15/2023]
Abstract
The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s arsenal of ~330 secreted effector proteins have been biochemically characterized as ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p hijacks ubiquitin signaling within the host cell, we undertook a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection results in increased ubiquitination of host proteins regulating subcellular trafficking and membrane dynamics, most notably 63 of ~160 mammalian Ras superfamily small GTPases. We determine that these small GTPases predominantly undergo non-degradative monoubiquitination, and link ubiquitination to recruitment to the Legionella-containing vacuole membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central, but likely indirect, role in cross-family small GTPase ubiquitination. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. This work positions L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.
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Affiliation(s)
- Adriana M. Steinbach
- Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America
- George Williams Hooper Foundation, University of California, San Francisco, San Francisco, California, United States of America
| | - Varun L. Bhadkamkar
- Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America
- George Williams Hooper Foundation, University of California, San Francisco, San Francisco, California, United States of America
| | - David Jimenez-Morales
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, California, United States of America
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, United States of America
- Department of Medicine, Division of Cardiovascular Medicine, Stanford University, California, United States of America
| | - Erica Stevenson
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, California, United States of America
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, United States of America
- Quantitative Biosciences Institute, University of California, San Francisco, California, United States of America
| | - Gwendolyn M. Jang
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, California, United States of America
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, United States of America
- Quantitative Biosciences Institute, University of California, San Francisco, California, United States of America
| | - Nevan J. Krogan
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, California, United States of America
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, United States of America
- Quantitative Biosciences Institute, University of California, San Francisco, California, United States of America
| | - Danielle L. Swaney
- Gladstone Institute of Data Science and Biotechnology, J. David Gladstone Institutes, San Francisco, California, United States of America
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California, United States of America
- Quantitative Biosciences Institute, University of California, San Francisco, California, United States of America
| | - Shaeri Mukherjee
- Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, California, United States of America
- George Williams Hooper Foundation, University of California, San Francisco, San Francisco, California, United States of America
- Chan Zuckerberg Biohub, San Francisco, CA, USA
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16
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Nomura K, Imboden LA, Tanaka H, He SY. Multiple host targets of Pseudomonas effector protein HopM1 form a protein complex regulating apoplastic immunity and water homeostasis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.07.31.551310. [PMID: 37577537 PMCID: PMC10418078 DOI: 10.1101/2023.07.31.551310] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/15/2023]
Abstract
Bacterial type III effector proteins injected into the host cell play a critical role in mediating bacterial interactions with plant and animal hosts. Notably, some bacterial effectors are reported to target sequence-unrelated host proteins with unknown functional relationships. The Pseudomonas syringae effector HopM1 is such an example; it interacts with and/or degrades several HopM1-interacting (MIN) Arabidopsis proteins, including HopM1-interacting protein 2 (MIN2/RAD23), HopM1-interacting protein 7 (MIN7/BIG5), HopM1-interacting protein 10 (MIN10/14-3-3ĸ), and HopM1-interacting protein 13 (MIN13/BIG2). In this study, we purified the MIN7 complex formed in planta and found that it contains MIN7, MIN10, MIN13, as well as a tetratricopeptide repeat protein named HLB1. Mutational analysis showed that, like MIN7, HLB1 is required for pathogen-associated molecular pattern (PAMP)-, effector-, and benzothiadiazole (BTH)-triggered immunity. HLB1 is recruited to the trans-Golgi network (TGN)/early endosome (EE) in a MIN7-dependent manner. Both min7 and hlb1 mutant leaves contained elevated water content in the leaf apoplast and artificial water infiltration into the leaf apoplast was sufficient to phenocopy immune-suppressing phenotype of HopM1. These results suggest that multiple HopM1-targeted MIN proteins form a protein complex with a dual role in modulating water level and immunity in the apoplast, which provides an explanation for the dual phenotypes of HopM1 during bacterial pathogenesis.
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Affiliation(s)
- Kinya Nomura
- Department of Biology, Duke University, Durham, NC 27708, USA
- Howard Hughes Medical Institute, Duke University, Durham, NC 27708, USA
| | - Lori Alice Imboden
- Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824, USA
| | - Hirokazu Tanaka
- Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-0033, Japan
| | - Sheng Yang He
- Department of Biology, Duke University, Durham, NC 27708, USA
- Howard Hughes Medical Institute, Duke University, Durham, NC 27708, USA
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17
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Boamah D, Gilmore M, Bourget S, Ghosh A, Hossain M, Vogel J, Cava F, O’Connor T. Peptidoglycan deacetylation controls type IV secretion and the intracellular survival of the bacterial pathogen Legionella pneumophila. Proc Natl Acad Sci U S A 2023; 120:e2119658120. [PMID: 37252954 PMCID: PMC10266036 DOI: 10.1073/pnas.2119658120] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2021] [Accepted: 04/18/2023] [Indexed: 06/01/2023] Open
Abstract
Peptidoglycan is a critical component of the bacteria cell envelope. Remodeling of the peptidoglycan is required for numerous essential cellular processes and has been linked to bacterial pathogenesis. Peptidoglycan deacetylases that remove the acetyl group of the N-acetylglucosamine (NAG) subunit protect bacterial pathogens from immune recognition and digestive enzymes secreted at the site of infection. However, the full extent of this modification on bacterial physiology and pathogenesis is not known. Here, we identify a polysaccharide deacetylase of the intracellular bacterial pathogen Legionella pneumophila and define a two-tiered role for this enzyme in Legionella pathogenesis. First, NAG deacetylation is important for the proper localization and function of the Type IVb secretion system, linking peptidoglycan editing to the modulation of host cellular processes through the action of secreted virulence factors. As a consequence, the Legionella vacuole mis-traffics along the endocytic pathway to the lysosome, preventing the formation of a replication permissive compartment. Second, within the lysosome, the inability to deacetylate the peptidoglycan renders the bacteria more sensitive to lysozyme-mediated degradation, resulting in increased bacterial death. Thus, the ability to deacetylate NAG is important for bacteria to persist within host cells and in turn, Legionella virulence. Collectively, these results expand the function of peptidoglycan deacetylases in bacteria, linking peptidoglycan editing, Type IV secretion, and the intracellular fate of a bacterial pathogen.
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Affiliation(s)
- David Boamah
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD21205
| | - Michael C. Gilmore
- Department of Molecular Biology, Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, Umeå90187, Sweden
| | - Sarah Bourget
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD21205
| | - Anushka Ghosh
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD21205
| | - Mohammad J. Hossain
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD21205
| | - Joseph P. Vogel
- Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO63110
| | - Felipe Cava
- Department of Molecular Biology, Laboratory for Molecular Infection Medicine Sweden, Umeå Centre for Microbial Research, Umeå University, Umeå90187, Sweden
| | - Tamara J. O’Connor
- Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD21205
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18
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Vormittag S, Hüsler D, Haneburger I, Kroniger T, Anand A, Prantl M, Barisch C, Maaß S, Becher D, Letourneur F, Hilbi H. Legionella- and host-driven lipid flux at LCV-ER membrane contact sites promotes vacuole remodeling. EMBO Rep 2023; 24:e56007. [PMID: 36588479 PMCID: PMC9986823 DOI: 10.15252/embr.202256007] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2022] [Revised: 12/12/2022] [Accepted: 12/14/2022] [Indexed: 01/03/2023] Open
Abstract
Legionella pneumophila replicates in macrophages and amoeba within a unique compartment, the Legionella-containing vacuole (LCV). Hallmarks of LCV formation are the phosphoinositide lipid conversion from PtdIns(3)P to PtdIns(4)P, fusion with ER-derived vesicles and a tight association with the ER. Proteomics of purified LCVs indicate the presence of membrane contact sites (MCS) proteins possibly implicated in lipid exchange. Using dually fluorescence-labeled Dictyostelium discoideum amoeba, we reveal that VAMP-associated protein (Vap) and the PtdIns(4)P 4-phosphatase Sac1 localize to the ER, and Vap also localizes to the LCV membrane. Furthermore, Vap as well as Sac1 promote intracellular replication of L. pneumophila and LCV remodeling. Oxysterol binding proteins (OSBPs) preferentially localize to the ER (OSBP8) or the LCV membrane (OSBP11), respectively, and restrict (OSBP8) or promote (OSBP11) bacterial replication and LCV expansion. The sterol probes GFP-D4H* and filipin indicate that sterols are rapidly depleted from LCVs, while PtdIns(4)P accumulates. In addition to Sac1, the PtdIns(4)P-subverting L. pneumophila effector proteins LepB and SidC also support LCV remodeling. Taken together, the Legionella- and host cell-driven PtdIns(4)P gradient at LCV-ER MCSs promotes Vap-, OSBP- and Sac1-dependent pathogen vacuole maturation.
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Affiliation(s)
- Simone Vormittag
- Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland
| | - Dario Hüsler
- Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland
| | - Ina Haneburger
- Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland
| | - Tobias Kroniger
- Institute of MicrobiologyUniversity of GreifswaldGreifswaldGermany
| | - Aby Anand
- Division of Molecular Infection Biology and Center for Cellular NanoanalyticsUniversity of OsnabrückOsnabrückGermany
| | - Manuel Prantl
- Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland
| | - Caroline Barisch
- Division of Molecular Infection Biology and Center for Cellular NanoanalyticsUniversity of OsnabrückOsnabrückGermany
| | - Sandra Maaß
- Institute of MicrobiologyUniversity of GreifswaldGreifswaldGermany
| | - Dörte Becher
- Institute of MicrobiologyUniversity of GreifswaldGreifswaldGermany
| | - François Letourneur
- Laboratory of Pathogen Host InteractionsUniversité de Montpellier, CNRS, INSERMMontpellierFrance
| | - Hubert Hilbi
- Institute of Medical MicrobiologyUniversity of ZürichZürichSwitzerland
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19
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Verhoeve VI, Lehman SS, Driscoll TP, Beckmann JF, Gillespie JJ. Metagenome diversity illuminates origins of pathogen effectors. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.02.26.530123. [PMID: 36909625 PMCID: PMC10002696 DOI: 10.1101/2023.02.26.530123] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/02/2023]
Abstract
Recent metagenome assembled genome (MAG) analyses have profoundly impacted Rickettsiology systematics. Discovery of basal lineages (Mitibacteraceae and Athabascaceae) with predicted extracellular lifestyles reveals an evolutionary timepoint for the transition to host dependency, which occurred independent of mitochondrial evolution. Notably, these basal rickettsiae carry the Rickettsiales vir homolog (rvh) type IV secretion system (T4SS) and purportedly use rvh to kill congener microbes rather than parasitize host cells as described for derived rickettsial pathogens. MAG analysis also substantially increased diversity for genus Rickettsia and delineated a basal lineage (Tisiphia) that stands to inform on the rise of human pathogens from protist and invertebrate endosymbionts. Herein, we probed Rickettsiales MAG and genomic diversity for the distribution of Rickettsia rvh effectors to ascertain their origins. A sparse distribution of most Rickettsia rvh effectors outside of Rickettsiaceae lineages indicates unique rvh evolution from basal extracellular species and other rickettsial families. Remarkably, nearly every effector was found in multiple divergent forms with variable architectures, illuminating profound roles for gene duplication and recombination in shaping effector repertoires in Rickettsia pathogens. Lateral gene transfer plays a prominent role shaping the rvh effector landscape, as evinced by the discover of many effectors on plasmids and conjugative transposons, as well as pervasive effector gene exchange between Rickettsia and Legionella species. Our study exemplifies how MAGs can provide incredible insight on the origins of pathogen effectors and how their architectural modifications become tailored to eukaryotic host cell biology.
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Affiliation(s)
- Victoria I Verhoeve
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
| | - Stephanie S Lehman
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA
| | - Timothy P Driscoll
- Department of Biology, West Virginia University, Morgantown, West Virginia, USA
| | - John F Beckmann
- Microbiology and Immunology, University of South Alabama, Mobile, AL, USA
| | - Joseph J Gillespie
- Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, USA
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20
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Helminiak L, Mishra S, Keun Kim H. Pathogenicity and virulence of Rickettsia. Virulence 2022; 13:1752-1771. [PMID: 36208040 PMCID: PMC9553169 DOI: 10.1080/21505594.2022.2132047] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Revised: 09/16/2022] [Accepted: 09/29/2022] [Indexed: 12/24/2022] Open
Abstract
Rickettsiae include diverse Gram-negative microbial species that exhibit obligatory intracellular lifecycles between mammalian hosts and arthropod vectors. Human infections with arthropod-borne Rickettsia continue to cause significant morbidity and mortality as recent environmental changes foster the proliferation of arthropod vectors and increased exposure to humans. However, the technical difficulties in working with Rickettsia have delayed our progress in understanding the molecular mechanisms involved in rickettsial pathogenesis and disease transmission. Recent advances in developing genetic tools for Rickettsia have enabled investigators to identify virulence genes, uncover molecular functions, and characterize host responses to rickettsial determinants. Therefore, continued efforts to determine virulence genes and their biological functions will help us understand the underlying mechanisms associated with arthropod-borne rickettsioses.
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Affiliation(s)
| | | | - Hwan Keun Kim
- Center for Infectious Diseases, Department of Microbiology and Immunology, Stony Brook University, Stony Brook, NY, USA
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21
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Tomaskovic I, Gonzalez A, Dikic I. Ubiquitin and Legionella: From bench to bedside. Semin Cell Dev Biol 2022; 132:230-241. [PMID: 35177348 DOI: 10.1016/j.semcdb.2022.02.008] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2021] [Revised: 02/02/2022] [Accepted: 02/07/2022] [Indexed: 12/15/2022]
Abstract
Legionella pneumophila, a Gram-negative intracellular bacterium, is one of the major causes of Legionnaires' disease, a specific type of atypical pneumonia. Despite intensive research efforts that elucidated many relevant structural, molecular and medical insights into Legionella's pathogenicity, Legionnaires' disease continues to present an ongoing public health concern. Legionella's virulence is based on its ability to simultaneously hijack multiple molecular pathways of the host cell to ensure its fast replication and dissemination. Legionella usurps the host ubiquitin system through multiple effector proteins, using the advantage of both conventional and unconventional (phosphoribosyl-linked) ubiquitination, thus providing optimal conditions for its replication. In this review, we summarize the current understanding of L. pneumophila from medical, biochemical and molecular perspectives. We describe the clinical disease presentation, its diagnostics and treatment, as well as host-pathogen interactions, with the emphasis on the ability of Legionella to target the host ubiquitin system upon infection. Furthermore, the interdisciplinary use of innovative technologies enables better insights into the pathogenesis of Legionnaires' disease and provides new opportunities for its treatment and prevention.
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Affiliation(s)
- Ines Tomaskovic
- Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
| | - Alexis Gonzalez
- Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany
| | - Ivan Dikic
- Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Riedberg Campus, Max-von-Laue Straße 15, 60438 Frankfurt am Main, Germany.
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22
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Loose M, Auer A, Brognara G, Budiman HR, Kowalski L, Matijević I. In vitro
reconstitution of small
GTPase
regulation. FEBS Lett 2022; 597:762-777. [PMID: 36448231 DOI: 10.1002/1873-3468.14540] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2022] [Revised: 10/27/2022] [Accepted: 11/07/2022] [Indexed: 12/05/2022]
Abstract
Small GTPases play essential roles in the organization of eukaryotic cells. In recent years, it has become clear that their intracellular functions result from intricate biochemical networks of the GTPase and their regulators that dynamically bind to a membrane surface. Due to the inherent complexities of their interactions, however, revealing the underlying mechanisms of action is often difficult to achieve from in vivo studies. This review summarizes in vitro reconstitution approaches developed to obtain a better mechanistic understanding of how small GTPase activities are regulated in space and time.
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Affiliation(s)
- Martin Loose
- Institute of Science and Technology Austria (ISTA) Klosterneuburg Austria
| | - Albert Auer
- Institute of Science and Technology Austria (ISTA) Klosterneuburg Austria
| | - Gabriel Brognara
- Institute of Science and Technology Austria (ISTA) Klosterneuburg Austria
| | | | - Lukasz Kowalski
- Institute of Science and Technology Austria (ISTA) Klosterneuburg Austria
| | - Ivana Matijević
- Institute of Science and Technology Austria (ISTA) Klosterneuburg Austria
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23
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Huang D, Luo J, OuYang X, Song L. Subversion of host cell signaling: The arsenal of Rickettsial species. Front Cell Infect Microbiol 2022; 12:995933. [PMID: 36389139 PMCID: PMC9659576 DOI: 10.3389/fcimb.2022.995933] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2022] [Accepted: 10/04/2022] [Indexed: 10/10/2023] Open
Abstract
Rickettsia is a genus of nonmotile, Gram-negative, non-spore-forming, highly pleomorphic bacteria that cause severe epidemic rickettsioses. The spotted fever group and typhi group are major members of the genus Rickettsia. Rickettsial species from the two groups subvert diverse host cellular processes, including membrane dynamics, actin cytoskeleton dynamics, phosphoinositide metabolism, intracellular trafficking, and immune defense, to promote their host colonization and intercellular transmission through secreted effectors (virulence factors). However, lineage-specific rickettsiae have exploited divergent strategies to accomplish such challenging tasks and these elaborated strategies focus on distinct host cell processes. In the present review, we summarized current understandings of how different rickettsial species employ their effectors' arsenal to affect host cellular processes in order to promote their own replication or to avoid destruction.
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Affiliation(s)
- Dan Huang
- Department of Respiratory Medicine, Center of Pathogen Biology and Infectious Disease, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, China
| | - Jingjing Luo
- Department of Respiratory Medicine, Center of Pathogen Biology and Infectious Disease, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, China
| | - Xuan OuYang
- State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
| | - Lei Song
- Department of Respiratory Medicine, Center of Pathogen Biology and Infectious Disease, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital of Jilin University, Changchun, China
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24
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Legionella pneumophila Infection of Human Macrophages Retains Golgi Structure but Reduces O-Glycans. Pathogens 2022; 11:pathogens11080908. [PMID: 36015029 PMCID: PMC9415278 DOI: 10.3390/pathogens11080908] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2022] [Revised: 08/02/2022] [Accepted: 08/08/2022] [Indexed: 11/30/2022] Open
Abstract
Legionella pneumophila is an accidental pathogen that replicates intracellularly within the Legionella-containing vacuole (LCV) in macrophages. Within an hour of infection, L. pneumophila secretes effectors to manipulate Rab1 and intercept ER-derived vesicles to the LCV. The downstream consequences of interrupted ER trafficking on the Golgi of macrophages are not clear. We examined the Golgi structure and function in L. pneumophila-infected human U937 macrophages. Intriguingly, the size of the Golgi in infected macrophages remained similar to uninfected macrophages. Furthermore, TEM analysis also did not reveal any significant changes in the ultrastructure of the Golgi in L. pneumophila-infected cells. Drug-induced Golgi disruption impacted bacterial replication in human macrophages, suggesting that an intact organelle is important for bacteria growth. To probe for Golgi functionality after L. pneumophila infection, we assayed glycosylation levels using fluorescent lectins. Golgi O-glycosylation levels, visualized by the fluorescent cis-Golgi lectin, Helix pomatia agglutinin (HPA), significantly decreased over time as infection progressed, compared to control cells. N-glycosylation levels in the Golgi, as measured by L-PHA lectin staining, were not impacted by L. pneumophila infection. To understand the mechanism of reduced O-glycans in the Golgi we monitored UDP-GalNAc transporter levels in infected macrophages. The solute carrier family 35 membrane A2 (SLC35A2) protein levels were significantly reduced in L. pneumophila-infected U937 and HeLa cells and L. pneumophila growth in human macrophages benefitted from GalNAc supplementation. The pronounced reduction in Golgi HPA levels was dependent on the translocation apparatus DotA expression in bacteria and occurred in a ubiquitin-independent manner. Thus, L. pneumophila infection of human macrophages maintains and requires an intact host Golgi ultrastructure despite known interference of ER–Golgi trafficking. Finally, L. pneumophila infection blocks the formation of O-linked glycans and reduces SLC35A2 protein levels in infected human macrophages.
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25
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Lockwood DC, Amin H, Costa TRD, Schroeder GN. The Legionella pneumophila Dot/Icm type IV secretion system and its effectors. MICROBIOLOGY (READING, ENGLAND) 2022; 168. [PMID: 35639581 DOI: 10.1099/mic.0.001187] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
To prevail in the interaction with eukaryotic hosts, many bacterial pathogens use protein secretion systems to release virulence factors at the host–pathogen interface and/or deliver them directly into host cells. An outstanding example of the complexity and sophistication of secretion systems and the diversity of their protein substrates, effectors, is the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) Type IVB secretion system (T4BSS) of
Legionella pneumophila
and related species.
Legionella
species are facultative intracellular pathogens of environmental protozoa and opportunistic human respiratory pathogens. The Dot/Icm T4BSS translocates an exceptionally large number of effectors, more than 300 per
L. pneumophila
strain, and is essential for evasion of phagolysosomal degradation and exploitation of protozoa and human macrophages as replicative niches. Recent technological advancements in the imaging of large protein complexes have provided new insight into the architecture of the T4BSS and allowed us to propose models for the transport mechanism. At the same time, significant progress has been made in assigning functions to about a third of
L. pneumophila
effectors, discovering unprecedented new enzymatic activities and concepts of host subversion. In this review, we describe the current knowledge of the workings of the Dot/Icm T4BSS machinery and provide an overview of the activities and functions of the to-date characterized effectors in the interaction of
L. pneumophila
with host cells.
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Affiliation(s)
- Daniel C Lockwood
- Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, BT9 7BL, Northern Ireland, UK
| | - Himani Amin
- MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, SW7 2AZ, UK
| | - Tiago R D Costa
- MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, SW7 2AZ, UK
| | - Gunnar N Schroeder
- Wellcome-Wolfson Institute for Experimental Medicine, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, BT9 7BL, Northern Ireland, UK
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26
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Ge Z, Yuan P, Chen L, Chen J, Shen D, She Z, Lu Y. New Global Insights on the Regulation of the Biphasic Life Cycle and Virulence Via ClpP-Dependent Proteolysis in Legionella pneumophila. Mol Cell Proteomics 2022; 21:100233. [PMID: 35427813 PMCID: PMC9112007 DOI: 10.1016/j.mcpro.2022.100233] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2021] [Revised: 02/17/2022] [Accepted: 04/07/2022] [Indexed: 01/11/2023] Open
Abstract
Legionella pneumophila, an environmental bacterium that parasitizes protozoa, causes Legionnaires’ disease in humans that is characterized by severe pneumonia. This bacterium adopts a distinct biphasic life cycle consisting of a nonvirulent replicative phase and a virulent transmissive phase in response to different environmental conditions. Hence, the timely and fine-tuned expression of growth and virulence factors in a life cycle–dependent manner is crucial for survival and replication. Here, we report that the completion of the biphasic life cycle and bacterial pathogenesis is greatly dependent on the protein homeostasis regulated by caseinolytic protease P (ClpP)-dependent proteolysis. We characterized the ClpP-dependent dynamic profiles of the regulatory and substrate proteins during the biphasic life cycle of L. pneumophila using proteomic approaches and discovered that ClpP-dependent proteolysis specifically and conditionally degraded the substrate proteins, thereby directly playing a regulatory role or indirectly controlling cellular events via the regulatory proteins. We further observed that ClpP-dependent proteolysis is required to monitor the abundance of fatty acid biosynthesis–related protein Lpg0102/Lpg0361/Lpg0362 and SpoT for the normal regulation of L. pneumophila differentiation. We also found that the control of the biphasic life cycle and bacterial virulence is independent. Furthermore, the ClpP-dependent proteolysis of Dot/Icm (defect in organelle trafficking/intracellular multiplication) type IVB secretion system and effector proteins at a specific phase of the life cycle is essential for bacterial pathogenesis. Therefore, our findings provide novel insights on ClpP-dependent proteolysis, which spans a broad physiological spectrum involving key metabolic pathways that regulate the transition of the biphasic life cycle and bacterial virulence of L. pneumophila, facilitating adaptation to aquatic and intracellular niches.
ClpP is the major determinant of biphasic life cycle–dependent protein turnover. ClpP-dependent proteolysis monitors SpoT abundance for cellular differentiation. ClpP-dependent regulation of life cycle and bacterial virulence is independent. ClpP-dependent proteolysis of T4BSS and effector proteins is vital for virulence.
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Affiliation(s)
- Zhenhuang Ge
- School of Chemistry, Sun Yat-sen University, Guangzhou, China; School of Life Sciences, Sun Yat-sen University, Guangzhou, China; Run Ze Laboratory for Gastrointestinal Microbiome Study, Sun Yat-sen University, Guangzhou, China
| | - Peibo Yuan
- Microbiome Medicine Center, Division of Laboratory Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Lingming Chen
- Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China
| | - Junyi Chen
- School of Life Sciences, Sun Yat-sen University, Guangzhou, China; Run Ze Laboratory for Gastrointestinal Microbiome Study, Sun Yat-sen University, Guangzhou, China
| | - Dong Shen
- School of Life Sciences, Sun Yat-sen University, Guangzhou, China
| | - Zhigang She
- School of Chemistry, Sun Yat-sen University, Guangzhou, China
| | - Yongjun Lu
- School of Life Sciences, Sun Yat-sen University, Guangzhou, China; Run Ze Laboratory for Gastrointestinal Microbiome Study, Sun Yat-sen University, Guangzhou, China.
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27
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Hochstrasser R, Michaelis S, Brülisauer S, Sura T, Fan M, Maaß S, Becher D, Hilbi H. Migration of Acanthamoeba through Legionella biofilms is regulated by the bacterial Lqs-LvbR network, effector proteins and the flagellum. Environ Microbiol 2022; 24:3672-3692. [PMID: 35415862 PMCID: PMC9544456 DOI: 10.1111/1462-2920.16008] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2022] [Revised: 04/05/2022] [Accepted: 04/08/2022] [Indexed: 11/30/2022]
Abstract
The environmental bacterium Legionella pneumophila causes the pneumonia Legionnaires' disease. The opportunistic pathogen forms biofilms and employs the Icm/Dot type IV secretion system (T4SS) to replicate in amoebae and macrophages. A regulatory network comprising the Legionella quorum sensing (Lqs) system and the transcription factor LvbR controls bacterial motility, virulence and biofilm architecture. Here we show by comparative proteomics that in biofilms formed by the L. pneumophila ΔlqsR or ΔlvbR regulatory mutants the abundance of proteins encoded by a genomic ‘fitness island’, metabolic enzymes, effector proteins and flagellar components (e.g. FlaA) varies. ∆lqsR or ∆flaA mutants form ‘patchy’ biofilms like the parental strain JR32, while ∆lvbR forms a ‘mat‐like’ biofilm. Acanthamoeba castellanii amoebae migrated more slowly through biofilms of L. pneumophila lacking lqsR, lvbR, flaA, a functional Icm/Dot T4SS (∆icmT), or secreted effector proteins. Clusters of bacteria decorated amoebae in JR32, ∆lvbR or ∆icmT biofilms but not in ∆lqsR or ∆flaA biofilms. The amoeba‐adherent bacteria induced promoters implicated in motility (PflaA) or virulence (PsidC, PralF). Taken together, the Lqs‐LvbR network (quorum sensing), FlaA (motility) and the Icm/Dot T4SS (virulence) regulate migration of A. castellanii through L. pneumophila biofilms, and – apart from the T4SS – govern bacterial cluster formation on the amoebae.
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Affiliation(s)
- Ramon Hochstrasser
- Institute of Medical Microbiology, University of Zürich, Gloriastrasse 30, 8006, Zürich, Switzerland
| | - Sarah Michaelis
- Institute of Medical Microbiology, University of Zürich, Gloriastrasse 30, 8006, Zürich, Switzerland
| | - Sabrina Brülisauer
- Institute of Medical Microbiology, University of Zürich, Gloriastrasse 30, 8006, Zürich, Switzerland
| | - Thomas Sura
- Institute of Microbiology, University of Greifswald, Felix-Hausdorff-Strasse 8, 17489, Greifswald, Germany
| | - Mingzhen Fan
- Institute of Medical Microbiology, University of Zürich, Gloriastrasse 30, 8006, Zürich, Switzerland
| | - Sandra Maaß
- Institute of Microbiology, University of Greifswald, Felix-Hausdorff-Strasse 8, 17489, Greifswald, Germany
| | - Dörte Becher
- Institute of Microbiology, University of Greifswald, Felix-Hausdorff-Strasse 8, 17489, Greifswald, Germany
| | - Hubert Hilbi
- Institute of Medical Microbiology, University of Zürich, Gloriastrasse 30, 8006, Zürich, Switzerland
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28
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López-Gómez M, García de Santiago B, Delgado-López PD, Malmierca E, González-Olmedo J, Gómez-Raposo C, Sandoval C, Ruiz-Seco P, Escribano N, Gómez-Cerezo JF, Casado E. Gastrointestinal tumors and infectious agents: A wide field to explore. World J Meta-Anal 2021; 9:505-521. [DOI: 10.13105/wjma.v9.i6.505] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/01/2021] [Revised: 08/26/2021] [Accepted: 12/23/2021] [Indexed: 02/06/2023] Open
Abstract
Infection is currently one of the main contributors to carcinogenesis. In fact, the International Agency for Research on Cancer has categorized eleven biological agents as group I carcinogens. It is estimated that around 16% of the 12.7 million new cancers diagnosed in 2008 were attributable to infectious agents. Although underdeveloped regions carry the highest incidence rates, about 7.4% of infection-related cancer cases occur in developed areas. Physicians are increasingly aware of the potential carcinogenic role of common virus like the Human Papilloma virus in cervical cancer, or the hepatitis B and C viruses in hepatocarcinoma. However, the carcinogenic role of several other infectious agents is less recognized. Given that gastrointestinal malignancies carry an overall poor prognosis, a better understanding of the carcinogenic mechanisms triggered by infectious agents is key to decrease the rate of cancer related deaths. Preventive measures directed to such infections would ideally impact survival. In this paper we review the main pathogenic mechanisms related to the development of gastrointestinal malignancies induced by infectious microorganisms and other pathogens which are currently under investigation.
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Affiliation(s)
- Miriam López-Gómez
- Medical Oncology Department. Precision Oncology Laboratory, Infanta Sofía University Hospital, San Sebastián de los Reyes 28231, Madrid, Spain
| | - Belén García de Santiago
- Pharmacy Department, Infanta Sofia University Hospital, San Sebastián de los Reyes 28703, Madrid, Spain
| | | | - Eduardo Malmierca
- Internal Medicine Department, Infanta Sofía University Hospital, San Sebastián de los Reyes 28703, Madrid, Spain
| | - Jesús González-Olmedo
- Medical Oncology Department, Infanta Sofia University Hospital, San Sebastián de los Reyes 28703, Madrid, Spain
| | - César Gómez-Raposo
- Medical Oncology Department, Infanta Sofia University Hospital, San Sebastián de los Reyes 28703, Madrid, Spain
| | - Carmen Sandoval
- Medical Oncology Department, Infanta Sofia University Hospital, San Sebastián de los Reyes 28703, Madrid, Spain
| | - Pilar Ruiz-Seco
- Internal Medicine Department, Infanta Sofía University Hospital, San Sebastián de los Reyes 28703, Madrid, Spain
| | - Nora Escribano
- Intensive Care Unit, Jiménez Díaz Foundation, Madrid 28040, Madrid, Spain
| | - Jorge Francisco Gómez-Cerezo
- Internal Medicine Department, Infanta Sofía University Hospital, San Sebastián de los Reyes 28703, Madrid, Spain
| | - Enrique Casado
- Medical Oncology Department, Infanta Sofia University Hospital, San Sebastián de los Reyes 28703, Madrid, Spain
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29
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Vaughn B, Abu Kwaik Y. Idiosyncratic Biogenesis of Intracellular Pathogens-Containing Vacuoles. Front Cell Infect Microbiol 2021; 11:722433. [PMID: 34858868 PMCID: PMC8632064 DOI: 10.3389/fcimb.2021.722433] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2021] [Accepted: 10/25/2021] [Indexed: 12/12/2022] Open
Abstract
While most bacterial species taken up by macrophages are degraded through processing of the bacteria-containing vacuole through the endosomal-lysosomal degradation pathway, intravacuolar pathogens have evolved to evade degradation through the endosomal-lysosomal pathway. All intra-vacuolar pathogens possess specialized secretion systems (T3SS-T7SS) that inject effector proteins into the host cell cytosol to modulate myriad of host cell processes and remodel their vacuoles into proliferative niches. Although intravacuolar pathogens utilize similar secretion systems to interfere with their vacuole biogenesis, each pathogen has evolved a unique toolbox of protein effectors injected into the host cell to interact with, and modulate, distinct host cell targets. Thus, intravacuolar pathogens have evolved clear idiosyncrasies in their interference with their vacuole biogenesis to generate a unique intravacuolar niche suitable for their own proliferation. While there has been a quantum leap in our knowledge of modulation of phagosome biogenesis by intravacuolar pathogens, the detailed biochemical and cellular processes affected remain to be deciphered. Here we discuss how the intravacuolar bacterial pathogens Salmonella, Chlamydia, Mycobacteria, Legionella, Brucella, Coxiella, and Anaplasma utilize their unique set of effectors injected into the host cell to interfere with endocytic, exocytic, and ER-to-Golgi vesicle traffic. However, Coxiella is the main exception for a bacterial pathogen that proliferates within the hydrolytic lysosomal compartment, but its T4SS is essential for adaptation and proliferation within the lysosomal-like vacuole.
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Affiliation(s)
- Bethany Vaughn
- Department of Microbiology and Immunology, University of Louisville, Louisville, KY, United States
| | - Yousef Abu Kwaik
- Department of Microbiology and Immunology, University of Louisville, Louisville, KY, United States.,Center for Predictive Medicine, College of Medicine, University of Louisville, Louisville, KY, United States
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30
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The Legionella pneumophila Effector RavY Contributes to a Replication-Permissive Vacuolar Environment during Infection. Infect Immun 2021; 89:e0026121. [PMID: 34543123 DOI: 10.1128/iai.00261-21] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Legionella pneumophila is the causative agent of Legionnaires' disease and is capable of replicating inside phagocytic cells, such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication-permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, and RavY does not contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild-type L. pneumophila. Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.
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31
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The Legionella Effector SdjA Is a Bifunctional Enzyme That Distinctly Regulates Phosphoribosyl Ubiquitination. mBio 2021; 12:e0231621. [PMID: 34488448 PMCID: PMC8546864 DOI: 10.1128/mbio.02316-21] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Legionella pneumophila promotes its survival and replication in phagocytes by actively modulating cellular processes using effectors injected into host cells by its Dot/Icm type IV secretion system. Many of these effectors function to manipulate the ubiquitin network of infected cells, thus contributing to the biogenesis of the Legionella-containing vacuole (LCV), which is permissive for bacterial replication. Among these, members of the SidE effector family (SidEs) catalyze ubiquitination of functionally diverse host proteins by a mechanism that is chemically distinct from the canonical three-enzyme cascade. The activity of SidEs is regulated by two mechanisms: reversal of the phosphoribosyl ubiquitination by DupA and DupB and direct inactivation by SidJ, which is a calmodulin-dependent glutamylase. In many L. pneumophila strains, SidJ belongs to a two-member protein family. Its homolog SdjA appears to function differently from SidJ despite the high-level similarity in their primary sequences. Here, we found that SdjA is a bifunctional enzyme that exhibits distinct activities toward members of the SidE family. It inhibits the activity of SdeB and SdeC by glutamylation. Unexpectedly, it also functions as a deglutamylase that reverses SidJ-induced glutamylation on SdeA. Our results reveal that an enzyme can catalyze two completely opposite biochemical reactions, which highlights the distinct regulation of phosphoribosyl ubiquitination by the SidJ effector family. IMPORTANCE One unique feature of L. pneumophila Dot/Icm effectors is the existence of protein families with members of high-level similarity. Whereas members of some families are functionally redundant, as suggested by their primary sequences, the relationship between SidJ and SdjA, the two members of the SidJ family, has remained mysterious. Despite their sharing 57% identity, sdjA cannot complement the defects in virulence displayed by a mutant lacking sidJ. SidJ inhibits the activity of the SidE family by a calmodulin (CaM)-dependent glutamylase activity. Here, we found that SdjA is a dual function protein: it is a CaM-dependent glutamylase against SdeB and SdeC but exhibits deglutamylase activity toward SdeA that has been modified by SidJ, indicating that SdjA functions to fine-tune the activity of SidEs. These findings have paved the way for future structural and functional analysis of SdjA, which may reveal novel mechanism for isopeptide bond cleavage and provide insights into the study of protein evolution.
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Budowa IV systemu sekrecji Legionella pneumophilai jego znaczenie w patogenezie. POSTEP HIG MED DOSW 2021. [DOI: 10.2478/ahem-2021-0023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Abstrakt
Bakterie Legionella pneumophila w środowisku naturalnym pasożytują wewnątrz komórek wybranych gatunków pierwotniaków, a po przedostaniu się do sztucznych systemów dystrybucji wody stają się ważnym czynnikiem etiologicznym zapalenia płuc u ludzi. Główną cechą determinującą patogenność tych bakterii jest zdolność do życia i replikacji w makrofagach płucnych, czyli w komórkach wyspecjalizowanych do fagocytozy, zabijania i trawienia mikroorganizmów. Warunkiem wstępnym rozwoju infekcji jest przełamanie mechanizmów bójczych makrofagów i utworzenie wakuoli replikacyjnej LCV (Legionella containing vacuole). Biogeneza wakuoli LCV jest możliwa dzięki sprawnemu funkcjonowaniu IV systemu sekrecji Dot/Icm, który jest wielobiałkowym, złożonym kompleksem umiejscowionym w wewnętrznej i zewnętrznej membranie osłony komórkowej bakterii. System Dot/Icm liczy 27 elementów, na które składają się m.in. kompleks rdzeniowo-transmembranowy, tworzący strukturalny szkielet całego systemu oraz kompleks białek sprzęgających. Geny kodujące komponenty systemu Dot/Icm są zorganizowane na dwóch regionach chromosomu bak-teryjnego. System sekrecji Dot/Icm umożliwia L. pneumophila wprowadzenie do cytozolu komórki gospodarza ponad 300 białek efektorowych, których skoordynowane działanie powoduje utrzymanie integralności błony wakuoli replikacyjnej oraz pozwala na manipulowanie różnymi procesami komórki. Ważnym elementem strategii wewnątrzkomórkowego namnażania się L. pneumophila jest modulowanie transportu pęcherzykowego, interakcja z retikulum endoplazmatycznym oraz zakłócenie biosyntezy białek, procesów autofagii i apoptozy komórki gospodarza. Poznanie złożonych mechanizmów regulacji i funkcji białek efektorowych systemu Dot/Icm ma decydujące znaczenie w zapobieganiu i leczeniu choroby legionistów.
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Striednig B, Lanner U, Niggli S, Katic A, Vormittag S, Brülisauer S, Hochstrasser R, Kaech A, Welin A, Flieger A, Ziegler U, Schmidt A, Hilbi H, Personnic N. Quorum sensing governs a transmissive Legionella subpopulation at the pathogen vacuole periphery. EMBO Rep 2021; 22:e52972. [PMID: 34314090 PMCID: PMC8419707 DOI: 10.15252/embr.202152972] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2021] [Revised: 07/01/2021] [Accepted: 07/08/2021] [Indexed: 01/24/2023] Open
Abstract
The Gram‐negative bacterium Legionella pneumophila is the causative agent of Legionnaires' disease and replicates in amoebae and macrophages within a distinct compartment, the Legionella‐containing vacuole (LCV). The facultative intracellular pathogen switches between a replicative, non‐virulent and a non‐replicating, virulent/transmissive phase. Here, we show on a single‐cell level that at late stages of infection, individual motile (PflaA‐GFP‐positive) and virulent (PralF‐ and PsidC‐GFP‐positive) L. pneumophila emerge in the cluster of non‐growing bacteria within an LCV. Comparative proteomics of PflaA‐GFP‐positive and PflaA‐GFP‐negative L. pneumophila subpopulations reveals distinct proteomes with flagellar proteins or cell division proteins being preferentially produced by the former or the latter, respectively. Toward the end of an infection cycle (˜ 48 h), the PflaA‐GFP‐positive L. pneumophila subpopulation emerges at the cluster periphery, predominantly escapes the LCV, and spreads from the bursting host cell. These processes are mediated by the Legionella quorum sensing (Lqs) system. Thus, quorum sensing regulates the emergence of a subpopulation of transmissive L. pneumophila at the LCV periphery, and phenotypic heterogeneity underlies the intravacuolar bi‐phasic life cycle of L. pneumophila.
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Affiliation(s)
- Bianca Striednig
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Ulrike Lanner
- Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland
| | - Selina Niggli
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Ana Katic
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Simone Vormittag
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Sabrina Brülisauer
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Ramon Hochstrasser
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Andres Kaech
- Center for Microscopy and Image Analysis, University of Zürich, Zürich, Switzerland
| | - Amanda Welin
- Division of Inflammation and Infection, Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden
| | - Antje Flieger
- Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, Wernigerode, Germany
| | - Urs Ziegler
- Center for Microscopy and Image Analysis, University of Zürich, Zürich, Switzerland
| | - Alexander Schmidt
- Proteomics Core Facility, Biozentrum, University of Basel, Basel, Switzerland
| | - Hubert Hilbi
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
| | - Nicolas Personnic
- Institute of Medical Microbiology, University of Zürich, Zürich, Switzerland
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Serine-ubiquitination regulates Golgi morphology and the secretory pathway upon Legionella infection. Cell Death Differ 2021; 28:2957-2969. [PMID: 34285384 PMCID: PMC8481228 DOI: 10.1038/s41418-021-00830-y] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2021] [Revised: 07/02/2021] [Accepted: 07/05/2021] [Indexed: 12/31/2022] Open
Abstract
SidE family of Legionella effectors catalyze non-canonical phosphoribosyl-linked ubiquitination (PR-ubiquitination) of host proteins during bacterial infection. SdeA localizes predominantly to ER and partially to the Golgi apparatus, and mediates serine ubiquitination of multiple ER and Golgi proteins. Here we show that SdeA causes disruption of Golgi integrity due to its ubiquitin ligase activity. The Golgi linking proteins GRASP55 and GRASP65 are PR-ubiquitinated on multiple serine residues, thus preventing their ability to cluster and form oligomeric structures. In addition, we found that the functional consequence of Golgi disruption is not linked to the recruitment of Golgi membranes to the growing Legionella-containing vacuoles. Instead, it affects the host secretory pathway. Taken together, our study sheds light on the Golgi manipulation strategy by which Legionella hijacks the secretory pathway and promotes bacterial infection.
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Kitao T, Taguchi K, Seto S, Arasaki K, Ando H, Nagai H, Kubori T. Legionella Manipulates Non-canonical SNARE Pairing Using a Bacterial Deubiquitinase. Cell Rep 2021; 32:108107. [PMID: 32905772 DOI: 10.1016/j.celrep.2020.108107] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2019] [Revised: 05/30/2020] [Accepted: 08/12/2020] [Indexed: 10/23/2022] Open
Abstract
The intracellular bacterial pathogen Legionella pneumophila uses many effector proteins delivered by the bacterial type IV secretion system (T4SS) to hijack the early secretory pathway to establish its replicative niche, known as the Legionella-containing vacuole (LCV). On LCV biogenesis, the endoplasmic reticulum (ER) vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNARE) Sec22b is recruited to the bacterial phagosome and forms non-canonical pairings with target membrane SNAREs (t-SNAREs) from the plasma membrane. Here, we identify a Legionella deubiquitinase (DUB), LotB, that can modulate the early secretory pathway by interacting with coatomer protein complex I (COPI) vesicles when ectopically expressed. We show that Sec22b is ubiquitinated upon L. pneumophila infection in a T4SS-dependent manner and that, subsequently, LotB deconjugates K63-linked ubiquitins from Sec22b. The DUB activity of LotB stimulates dissociation of the t-SNARE syntaxin 3 (Stx3) from Sec22b, which resides on the LCV. Our study highlights a bacterial strategy manipulating the dynamics of infection-induced SNARE pairing using a bacterial DUB.
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Affiliation(s)
- Tomoe Kitao
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Gifu 501-1194, Japan
| | - Kyoichiro Taguchi
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Gifu 501-1194, Japan; Laboratory of Veterinary Microbiology, Faculty of Applied Biological Science, Gifu University, Gifu, Gifu 501-1193, Japan
| | - Shintaro Seto
- Department of Pathophysiology and Host Defense, The Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, Kiyose, Tokyo 204-8533, Japan
| | - Kohei Arasaki
- School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
| | - Hiroki Ando
- G-CHAIN, Gifu University, Gifu, Gifu 501-1194, Japan; Laboratory of Phage Biologics, Graduate School of Medicine, Gifu University, Gifu, Gifu 501-1194, Japan
| | - Hiroki Nagai
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Gifu 501-1194, Japan; G-CHAIN, Gifu University, Gifu, Gifu 501-1194, Japan.
| | - Tomoko Kubori
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Gifu 501-1194, Japan; G-CHAIN, Gifu University, Gifu, Gifu 501-1194, Japan.
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36
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De Niz M, Caldelari R, Kaiser G, Zuber B, Heo WD, Heussler VT, Agop-Nersesian C. Hijacking of the host cell Golgi by Plasmodium berghei liver stage parasites. J Cell Sci 2021; 134:jcs252213. [PMID: 34013963 PMCID: PMC8186485 DOI: 10.1242/jcs.252213] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/27/2020] [Accepted: 04/12/2021] [Indexed: 12/28/2022] Open
Abstract
The intracellular lifestyle represents a challenge for the rapidly proliferating liver stage Plasmodium parasite. In order to scavenge host resources, Plasmodium has evolved the ability to target and manipulate host cell organelles. Using dynamic fluorescence-based imaging, we here show an interplay between the pre-erythrocytic stages of Plasmodium berghei and the host cell Golgi during liver stage development. Liver stage schizonts fragment the host cell Golgi into miniaturized stacks, which increases surface interactions with the parasitophorous vacuolar membrane of the parasite. Expression of specific dominant-negative Arf1 and Rab GTPases, which interfere with the host cell Golgi-linked vesicular machinery, results in developmental delay and diminished survival of liver stage parasites. Moreover, functional Rab11a is critical for the ability of the parasites to induce Golgi fragmentation. Altogether, we demonstrate that the structural integrity of the host cell Golgi and Golgi-associated vesicular traffic is important for optimal pre-erythrocytic development of P. berghei. The parasite hijacks the Golgi structure of the hepatocyte to optimize its own intracellular development. This article has an associated First Person interview with the first author of the paper.
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Affiliation(s)
- Mariana De Niz
- Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland
| | - Reto Caldelari
- Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland
| | - Gesine Kaiser
- Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland
| | - Benoit Zuber
- Institute for Anatomy, University of Bern, CH-3012 Bern, Switzerland
| | - Won Do Heo
- Dept. of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
| | - Volker T. Heussler
- Institute of Cell Biology, University of Bern, CH-3012 Bern, Switzerland
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Chauhan D, Shames SR. Pathogenicity and Virulence of Legionella: Intracellular replication and host response. Virulence 2021; 12:1122-1144. [PMID: 33843434 PMCID: PMC8043192 DOI: 10.1080/21505594.2021.1903199] [Citation(s) in RCA: 62] [Impact Index Per Article: 15.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
Bacteria of the genus Legionella are natural pathogens of amoebae that can cause a severe pneumonia in humans called Legionnaires’ Disease. Human disease results from inhalation of Legionella-contaminated aerosols and subsequent bacterial replication within alveolar macrophages. Legionella pathogenicity in humans has resulted from extensive co-evolution with diverse genera of amoebae. To replicate intracellularly, Legionella generates a replication-permissive compartment called the Legionella-containing vacuole (LCV) through the concerted action of hundreds of Dot/Icm-translocated effector proteins. In this review, we present a collective overview of Legionella pathogenicity including infection mechanisms, secretion systems, and translocated effector function. We also discuss innate and adaptive immune responses to L. pneumophila, the implications of Legionella genome diversity and future avenues for the field.
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Affiliation(s)
- Deepika Chauhan
- Division of Biology, Kansas State University, Manhattan, Kansas, USA
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Voss OH, Rahman MS. Rickettsia-host interaction: strategies of intracytosolic host colonization. Pathog Dis 2021; 79:ftab015. [PMID: 33705517 PMCID: PMC8023194 DOI: 10.1093/femspd/ftab015] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2020] [Accepted: 03/09/2021] [Indexed: 12/29/2022] Open
Abstract
Bacterial infection is a highly complex biological process involving a dynamic interaction between the invading microorganism and the host. Specifically, intracellular pathogens seize control over the host cellular processes including membrane dynamics, actin cytoskeleton, phosphoinositide metabolism, intracellular trafficking and immune defense mechanisms to promote their host colonization. To accomplish such challenging tasks, virulent bacteria deploy unique species-specific secreted effectors to evade and/or subvert cellular defense surveillance mechanisms to establish a replication niche. However, despite superficially similar infection strategies, diverse Rickettsia species utilize different effector repertoires to promote host colonization. This review will discuss our current understandings on how different Rickettsia species deploy their effector arsenal to manipulate host cellular processes to promote their intracytosolic life within the mammalian host.
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Affiliation(s)
- Oliver H Voss
- Department of Microbiology and Immunology, University of Maryland School of Medicine, HSF2, room 416, 20 Penn St, Baltimore, MD 21201, USA
| | - M Sayeedur Rahman
- Department of Microbiology and Immunology, University of Maryland School of Medicine, HSF2, room 416, 20 Penn St, Baltimore, MD 21201, USA
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Legionella hijacks the host Golgi-to-ER retrograde pathway for the association of Legionella-containing vacuole with the ER. PLoS Pathog 2021; 17:e1009437. [PMID: 33760868 PMCID: PMC8021152 DOI: 10.1371/journal.ppat.1009437] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Revised: 04/05/2021] [Accepted: 03/01/2021] [Indexed: 11/19/2022] Open
Abstract
Legionella pneumophila (L. pneumophila) is a gram-negative bacterium that replicates in a compartment that resembles the host endoplasmic reticulum (ER). To create its replicative niche, L. pneumophila manipulates host membrane traffic and fusion machineries. Bacterial proteins called Legionella effectors are translocated into the host cytosol and play a crucial role in these processes. In an early stage of infection, Legionella subverts ER-derived vesicles (ERDVs) by manipulating GTPase Rab1 to facilitate remodeling of the Legionella-containing vacuole (LCV). Subsequently, the LCV associates with the ER in a mechanism that remains elusive. In this study, we show that L. pneumophila recruits GTPases Rab33B and Rab6A, which regulate vesicle trafficking from the Golgi to the ER, to the LCV to promote the association of LCV with the ER. We found that recruitment of Rab6A to the LCV depends on Rab33B. Legionella effector SidE family proteins, which phosphoribosyl-ubiquitinate Rab33B, were found to be necessary for the recruitment of Rab33B to the LCV. Immunoprecipitation experiments revealed that L. pneumophila facilitates the interaction of Rab6 with ER-resident SNAREs comprising syntaxin 18, p31, and BNIP1, but not tethering factors including NAG, RINT-1, and ZW10, which are normally required for syntaxin 18-mediated fusion of Golgi-derived vesicles with the ER. Our results identified a Rab33B-Rab6A cascade on the LCV and the interaction of Rab6 with ER-resident SNARE proteins for the association of LCV with the ER and disclosed the unidentified physiological role of SidE family proteins. Legionella pneumophila causes a sever pneumonia called Legionnaires’ disease and a threat of this disease has increased on a world-wide scale. As a feature of L. pneumophila, it secrets over 300 bacterial effectors to adapt and survive inside the host and many of effectors modify the host proteins in a unique manner. L. pneumophila is known to travel inside the host and final destination of this pathogens is the host ER. In the initial step of this travel, L. pneumophila subverts host early vesicular trafficking to remodel the membrane composition of Legionella-containing vacuole (LCV). Although this remodeling process has been well characterized, the molecular machinery of association of remodeled vacuoles with the ER is still obscure. This paper shows that the host GTPases Rab6A and Rab33B, both of which control Golgi-to-ER traffic, are recruited to the LCV in a cascade manner and are required for the association of LCVs with the ER through the interaction between Rab6A and ER-resident t-SNARE proteins. Of note, we demonstrate that a bacteria-specific Rab33B modification called phosphoribosyl-ubiquitination by Legionella effectors proteins of the SidE family is essential for the recruitment of Rab33B to the LCV.
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Li G, Liu H, Luo ZQ, Qiu J. Modulation of phagosome phosphoinositide dynamics by a Legionella phosphoinositide 3-kinase. EMBO Rep 2021; 22:e51163. [PMID: 33492731 DOI: 10.15252/embr.202051163] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2020] [Revised: 12/06/2020] [Accepted: 12/21/2020] [Indexed: 12/12/2022] Open
Abstract
The phagosome harboring the bacterial pathogen Legionella pneumophila is known to be enriched with phosphatidylinositol 4-phosphate (PtdIns4P), which is important for anchoring a subset of its virulence factors and potentially for signaling events implicated in the biogenesis of the Legionella-containing vacuole (LCV) that supports intracellular bacterial growth. Here we demonstrate that the effector MavQ is a phosphoinositide 3-kinase that specifically catalyzes the conversion of phosphatidylinositol (PtdIns) into PtdIns3P. The product of MavQ is subsequently phosphorylated by the effector LepB to yield PtdIns(3,4)P2, whose 3-phosphate is then removed by another effector SidF to generate PtdIns4P. We also show that MavQ is associated with the LCV and the ∆mavQ mutant displays phenotypes in the anchoring of a PtdIns4P-binding effector similar to those of ∆lepB or ∆sidF mutants. Our results establish a mechanism of de novo PtdIns4P biosynthesis by L. pneumophila via a catalysis axis comprised of MavQ, LepB, and SidF on the surface of its phagosome.
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Affiliation(s)
- Gen Li
- Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Hongtao Liu
- Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Zhao-Qing Luo
- Purdue Institute for Inflammation, Immunology and Infectious Disease and Department of Biological Sciences, Purdue University, West Lafayette, IN, USA
| | - Jiazhang Qiu
- Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
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Herrera A, Satchell KJF. Cross-Kingdom Activation of Vibrio Toxins by ADP-Ribosylation Factor Family GTPases. J Bacteriol 2020; 202:e00278-20. [PMID: 32900828 PMCID: PMC7685564 DOI: 10.1128/jb.00278-20] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Pathogenic Vibrio species use many different approaches to subvert, attack, and undermine the host response. The toxins they produce are often responsible for the devastating effects associated with their diseases. These toxins target a variety of host proteins, which leads to deleterious effects, including dissolution of cell organelle integrity and inhibition of protein secretion. Becoming increasingly prevalent as cofactors for Vibrio toxins are proteins of the small GTPase families. ADP-ribosylation factor small GTPases (ARFs) in particular are emerging as a common host cofactor necessary for full activation of Vibrio toxins. While ARFs are not the direct target of Vibrio cholerae cholera toxin (CT), ARF binding is required for its optimal activity as an ADP-ribosyltransferase. The makes caterpillars floppy (MCF)-like and the domain X (DmX) effectors of the Vibrio vulnificus multifunctional autoprocessing repeats-in-toxin (MARTX) toxin also both require ARFs to initiate autoprocessing and activation as independent effectors. ARFs are ubiquitously expressed in eukaryotes and are key regulators of many cellular processes, and as such they are ideal cofactors for Vibrio pathogens that infect many host species. In this review, we cover in detail the known Vibrio toxins that use ARFs as cross-kingdom activators to both stimulate and optimize their activity. We further discuss how these contrast to toxins and effectors from other bacterial species that coactivate, stimulate, or directly modify host ARFs as their mechanisms of action.
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Affiliation(s)
- Alfa Herrera
- Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
| | - Karla J F Satchell
- Department of Microbiology-Immunology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA
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42
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Liu S, Luo J, Zhen X, Qiu J, Ouyang S, Luo ZQ. Interplay between bacterial deubiquitinase and ubiquitin E3 ligase regulates ubiquitin dynamics on Legionella phagosomes. eLife 2020; 9:58114. [PMID: 33136002 PMCID: PMC7669269 DOI: 10.7554/elife.58114] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2020] [Accepted: 11/01/2020] [Indexed: 12/12/2022] Open
Abstract
Legionella pneumophila extensively modulates the host ubiquitin network to create the Legionella-containing vacuole (LCV) for its replication. Many of its virulence factors function as ubiquitin ligases or deubiquitinases (DUBs). Here, we identify Lem27 as a DUB that displays a preference for diubiquitin formed by K6, K11, or K48. Lem27 is associated with the LCV where it regulates Rab10 ubiquitination in concert with SidC and SdcA, two bacterial E3 ubiquitin ligases. Structural analysis of the complex formed by an active fragment of Lem27 and the substrate-based suicide inhibitor ubiquitin-propargylamide (PA) reveals that it harbors a fold resembling those in the OTU1 DUB subfamily with a Cys-His catalytic dyad and that it recognizes ubiquitin via extensive hydrogen bonding at six contact sites. Our results establish Lem27 as a DUB that functions to regulate protein ubiquitination on L. pneumophila phagosomes by counteracting the activity of bacterial ubiquitin E3 ligases.
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Affiliation(s)
- Shuxin Liu
- Department of Respiratory Medicine and Center of Infection and Immunity, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital, Jilin University, Changchun, China
| | - Jiwei Luo
- The Key Laboratory of Innate Immune Biology of Fujian Province, Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China.,Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, China
| | - Xiangkai Zhen
- The Key Laboratory of Innate Immune Biology of Fujian Province, Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China.,Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, China
| | - Jiazhang Qiu
- Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
| | - Songying Ouyang
- The Key Laboratory of Innate Immune Biology of Fujian Province, Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, China.,Laboratory for Marine Biology and Biotechnology, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao, China
| | - Zhao-Qing Luo
- Department of Respiratory Medicine and Center of Infection and Immunity, Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, The First Hospital, Jilin University, Changchun, China.,Department of Biological Sciences, Purdue University, West Lafayette, United States
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Abstract
Through coevolution with host cells, microorganisms have acquired mechanisms to avoid the detection by the host surveillance system and to use the cell's supplies to establish themselves. Indeed, certain pathogens have evolved proteins that imitate specific eukaryotic cell proteins, allowing them to manipulate host pathways, a phenomenon termed molecular mimicry. Bacterial "eukaryotic-like proteins" are a remarkable example of molecular mimicry. They are defined as proteins that strongly resemble eukaryotic proteins or that carry domains that are predominantly present in eukaryotes and that are generally absent from prokaryotes. The widest diversity of eukaryotic-like proteins known to date can be found in members of the bacterial genus Legionella, some of which cause a severe pneumonia in humans. The characterization of a number of these proteins shed light on their importance during infection. The subsequent identification of eukaryotic-like genes in the genomes of other amoeba-associated bacteria and bacterial symbionts suggested that eukaryotic-like proteins are a common means of bacterial evasion and communication, shaped by the continuous interactions between bacteria and their protozoan hosts. In this review, we discuss the concept of molecular mimicry using Legionella as an example and show that eukaryotic-like proteins effectively manipulate host cell pathways. The study of the function and evolution of such proteins is an exciting field of research that is leading us toward a better understanding of the complex world of bacterium-host interactions. Ultimately, this knowledge will teach us how host pathways are manipulated and how infections may possibly be tackled.
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Swart AL, Gomez-Valero L, Buchrieser C, Hilbi H. Evolution and function of bacterial RCC1 repeat effectors. Cell Microbiol 2020; 22:e13246. [PMID: 32720355 DOI: 10.1111/cmi.13246] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Revised: 07/10/2020] [Accepted: 07/11/2020] [Indexed: 01/02/2023]
Abstract
Intracellular bacterial pathogens harbour genes, the closest homologues of which are found in eukaryotes. Regulator of chromosome condensation 1 (RCC1) repeat proteins are phylogenetically widespread and implicated in protein-protein interactions, such as the activation of the small GTPase Ran by its cognate guanine nucleotide exchange factor, RCC1. Legionella pneumophila and Coxiella burnetii, the causative agents of Legionnaires' disease and Q fever, respectively, harbour RCC1 repeat coding genes. Legionella pneumophila secretes the RCC1 repeat 'effector' proteins LegG1, PpgA and PieG into eukaryotic host cells, where they promote the activation of the pleiotropic small GTPase Ran, microtubule stabilisation, pathogen vacuole motility and intracellular bacterial growth as well as host cell migration. The RCC1 repeat effectors localise to the pathogen vacuole or the host plasma membrane and target distinct components of the Ran GTPase cycle, including Ran modulators and the small GTPase itself. Coxiella burnetii translocates the RCC1 repeat effector NopA into host cells, where the protein localises to nucleoli. NopA binds to Ran GTPase and promotes the nuclear accumulation of Ran(GTP), thus pertubing the import of the transcription factor NF-κB and innate immune signalling. Hence, divergent evolution of bacterial RCC1 repeat effectors defines the range of Ran GTPase cycle targets and likely allows fine-tuning of Ran GTPase activation by the pathogens at different cellular sites.
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Affiliation(s)
- Anna Leoni Swart
- Institute of Medical Microbiology, Faculty of Medicine, University of Zurich, Zürich, Switzerland
| | - Laura Gomez-Valero
- Institut Pasteur, Unité de Biologie des Bactéries Intracellulaires, Paris, France.,CNRS UMR 3525, Paris, France
| | - Carmen Buchrieser
- Institut Pasteur, Unité de Biologie des Bactéries Intracellulaires, Paris, France.,CNRS UMR 3525, Paris, France
| | - Hubert Hilbi
- Institute of Medical Microbiology, Faculty of Medicine, University of Zurich, Zürich, Switzerland
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45
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Li P, Vassiliadis D, Ong SY, Bennett-Wood V, Sugimoto C, Yamagishi J, Hartland EL, Pasricha S. Legionella pneumophila Infection Rewires the Acanthamoeba castellanii Transcriptome, Highlighting a Class of Sirtuin Genes. Front Cell Infect Microbiol 2020; 10:428. [PMID: 32974218 PMCID: PMC7468528 DOI: 10.3389/fcimb.2020.00428] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2020] [Accepted: 07/14/2020] [Indexed: 12/13/2022] Open
Abstract
Legionella pneumophila is an environmental bacterium that has evolved to survive predation by soil and water amoebae such as Acanthamoeba castellanii, and this has inadvertently led to the ability of L. pneumophila to survive and replicate in human cells. L. pneumophila causes Legionnaire's Disease, with human exposure occurring via the inhalation of water aerosols containing both amoebae and the bacteria. These aerosols originate from aquatic biofilms found in artifical water sources, such as air-conditioning cooling towers and humidifiers. In these man-made environments, A. castellanii supports L. pneumophila intracellular replication, thereby promoting persistence and dissemination of the bacteria and providing protection from external stress. Despite this close evolutionary relationship, very little is known about how A. castellanii responds to L. pneumophila infection. In this study, we examined the global transcriptional response of A. castellanii to L. pneumophila infection. We compared A. castellanii infected with wild type L. pneumophila to A. castellanii infected with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We showed that A. castellanii underwent clear morphological and transcriptional rewiring over the course of L. pneumophila infection. Through improved annotation of the A. castellanii genome, we determined that these transcriptional changes primarily involved biological processes utilizing small GTPases, including cellular transport, signaling, metabolism and replication. In addition, a number of sirtuin-encoding genes in A. castellanii were found to be conserved and upregulated during L. pneumophila infection. Silencing of sirtuin gene, sir6f (ACA1_153540) resulted in the inhibition of A. castellanii cell proliferation during infection and reduced L. pneumophila replication. Overall our findings identified several biological pathways in amoebae that may support L. pneumophila replication and A. castellanii proliferation in environmental conditions.
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Affiliation(s)
- Pengfei Li
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.,Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
| | - Dane Vassiliadis
- Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, VIC, Australia
| | - Sze Ying Ong
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia
| | - Vicki Bennett-Wood
- Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
| | - Chihiro Sugimoto
- Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, Sapporo, Japan.,Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Junya Yamagishi
- Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, Sapporo, Japan.,Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan
| | - Elizabeth L Hartland
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.,Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.,Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan.,Department of Molecular and Translational Science, Monash University, Clayton, VIC, Australia
| | - Shivani Pasricha
- Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC, Australia.,Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
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46
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Kim H, Kubori T, Yamazaki K, Kwak MJ, Park SY, Nagai H, Vogel JP, Oh BH. Structural basis for effector protein recognition by the Dot/Icm Type IVB coupling protein complex. Nat Commun 2020; 11:2623. [PMID: 32457311 PMCID: PMC7251119 DOI: 10.1038/s41467-020-16397-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2019] [Accepted: 04/27/2020] [Indexed: 01/25/2023] Open
Abstract
The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is extremely versatile, translocating ~300 effector proteins into host cells. This specialized secretion system employs the Dot/Icm type IVB coupling protein (T4CP) complex, which includes IcmS, IcmW and LvgA, that are known to selectively assist the export of a subclass of effectors. Herein, the crystal structure of a four-subunit T4CP subcomplex bound to the effector protein VpdB reveals an interaction between LvgA and a linear motif in the C-terminus of VpdB. The same binding interface of LvgA also interacts with the C-terminal region of three additional effectors, SidH, SetA and PieA. Mutational analyses identified a FxxxLxxxK binding motif that is shared by VpdB and SidH, but not by SetA and PieA, showing that LvgA recognizes more than one type of binding motif. Together, this work provides a structural basis for how the Dot/Icm T4CP complex recognizes effectors, and highlights the multiple substrate-binding specificities of its adaptor subunit.
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Affiliation(s)
- Hyunmin Kim
- Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea
| | - Tomoko Kubori
- Department of Microbiology, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu, 501-1194, Japan
| | - Kohei Yamazaki
- Department of Microbiology, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu, 501-1194, Japan.,Veterinary Public Health, Kitasato University, Higashi 23-35-1, Towada, Aomori, 034-8628, Japan
| | - Mi-Jeong Kwak
- Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea.,CKD Research Institute, Yongin, Gyeonggi, 16995, Republic of Korea
| | - Suk-Youl Park
- Pohang Accelerator Laboratory, POSTECH, Pohang, Gyeongbuk, 37673, Republic of Korea
| | - Hiroki Nagai
- Department of Microbiology, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu, 501-1194, Japan
| | - Joseph P Vogel
- Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, 63110, USA
| | - Byung-Ha Oh
- Department of Biological Sciences, KAIST Institute for the Biocentury, Korea Advanced Institute of Science and Technology, Daejeon, 34141, Republic of Korea.
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47
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Hostile Takeover: Hijacking of Endoplasmic Reticulum Function by T4SS and T3SS Effectors Creates a Niche for Intracellular Pathogens. Microbiol Spectr 2020; 7. [PMID: 31198132 DOI: 10.1128/microbiolspec.psib-0027-2019] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
After entering a cell, intracellular pathogens must evade destruction and generate a niche for intracellular replication. A strategy shared by multiple intracellular pathogens is the deployment of type III secretion system (T3SS)- and type IV secretion system (T4SS)-injected proteins (effectors) that subvert cellular functions. A subset of these effectors targets activities of the host cell's endoplasmic reticulum (ER). Effectors are now appreciated to interfere with the ER in multiple ways, including capture of secretory vesicles, tethering of pathogen vacuoles to the ER, and manipulation of ER-based autophagy initiation and the unfolded-protein response. These strategies enable pathogens to generate a niche with access to cellular nutrients and to evade the host cell's defenses.
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48
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Swart AL, Hilbi H. Phosphoinositides and the Fate of Legionella in Phagocytes. Front Immunol 2020; 11:25. [PMID: 32117224 PMCID: PMC7025538 DOI: 10.3389/fimmu.2020.00025] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2019] [Accepted: 01/08/2020] [Indexed: 01/28/2023] Open
Abstract
Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires' disease. The environmental bacterium replicates in free-living amoebae as well as in lung macrophages in a distinct compartment, the Legionella-containing vacuole (LCV). The LCV communicates with a number of cellular vesicle trafficking pathways and is formed by a plethora of secreted bacterial effector proteins, which target host cell proteins and lipids. Phosphoinositide (PI) lipids are pivotal determinants of organelle identity, membrane dynamics and vesicle trafficking. Accordingly, eukaryotic cells tightly regulate the production, turnover, interconversion, and localization of PI lipids. L. pneumophila modulates the PI pattern in infected cells for its own benefit by (i) recruiting PI-decorated vesicles, (ii) producing effectors acting as PI interactors, phosphatases, kinases or phospholipases, and (iii) subverting host PI metabolizing enzymes. The PI conversion from PtdIns(3)P to PtdIns(4)P represents a decisive step during LCV maturation. In this review, we summarize recent progress on elucidating the strategies, by which L. pneumophila subverts host PI lipids to promote LCV formation and intracellular replication.
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Affiliation(s)
- A Leoni Swart
- Faculty of Medicine, Institute of Medical Microbiology, University of Zürich, Zurich, Switzerland
| | - Hubert Hilbi
- Faculty of Medicine, Institute of Medical Microbiology, University of Zürich, Zurich, Switzerland
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49
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Mondino S, Schmidt S, Rolando M, Escoll P, Gomez-Valero L, Buchrieser C. Legionnaires’ Disease: State of the Art Knowledge of Pathogenesis Mechanisms of Legionella. ANNUAL REVIEW OF PATHOLOGY-MECHANISMS OF DISEASE 2020; 15:439-466. [DOI: 10.1146/annurev-pathmechdis-012419-032742] [Citation(s) in RCA: 72] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Legionella species are environmental gram-negative bacteria able to cause a severe form of pneumonia in humans known as Legionnaires’ disease. Since the identification of Legionella pneumophila in 1977, four decades of research on Legionella biology and Legionnaires’ disease have brought important insights into the biology of the bacteria and the molecular mechanisms that these intracellular pathogens use to cause disease in humans. Nowadays, Legionella species constitute a remarkable model of bacterial adaptation, with a genus genome shaped by their close coevolution with amoebae and an ability to exploit many hosts and signaling pathways through the secretion of a myriad of effector proteins, many of which have a eukaryotic origin. This review aims to discuss current knowledge of Legionella infection mechanisms and future research directions to be taken that might answer the many remaining open questions. This research will without a doubt be a terrific scientific journey worth taking.
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Affiliation(s)
- Sonia Mondino
- Institut Pasteur, Biologie des Bactéries Intracellulaires, CNRS UMR 3525, 75015 Paris, France;, , , , ,
| | - Silke Schmidt
- Institut Pasteur, Biologie des Bactéries Intracellulaires, CNRS UMR 3525, 75015 Paris, France;, , , , ,
- Sorbonne Université, Collège doctoral, 75005 Paris, France
| | - Monica Rolando
- Institut Pasteur, Biologie des Bactéries Intracellulaires, CNRS UMR 3525, 75015 Paris, France;, , , , ,
| | - Pedro Escoll
- Institut Pasteur, Biologie des Bactéries Intracellulaires, CNRS UMR 3525, 75015 Paris, France;, , , , ,
| | - Laura Gomez-Valero
- Institut Pasteur, Biologie des Bactéries Intracellulaires, CNRS UMR 3525, 75015 Paris, France;, , , , ,
| | - Carmen Buchrieser
- Institut Pasteur, Biologie des Bactéries Intracellulaires, CNRS UMR 3525, 75015 Paris, France;, , , , ,
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50
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Christenson ET, Isaac DT, Yoshida K, Lipo E, Kim JS, Ghirlando R, Isberg RR, Banerjee A. The iron-regulated vacuolar Legionella pneumophila MavN protein is a transition-metal transporter. Proc Natl Acad Sci U S A 2019; 116:17775-17785. [PMID: 31431530 PMCID: PMC6731752 DOI: 10.1073/pnas.1902806116] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/20/2023] Open
Abstract
Legionella pneumophila causes a potentially fatal form of pneumonia by replicating within macrophages in the Legionella-containing vacuole (LCV). Bacterial survival and proliferation within the LCV rely on hundreds of secreted effector proteins comprising high functional redundancy. The vacuolar membrane-localized MavN, hypothesized to support iron transport, is unique among effectors because loss-of-function mutations result in severe intracellular growth defects. We show here an iron starvation response by L. pneumophila after infection of macrophages that was prematurely induced in the absence of MavN, consistent with MavN granting access to limiting cellular iron stores. MavN cysteine accessibilities to a membrane-impermeant label were determined during macrophage infections, revealing a topological pattern supporting multipass membrane transporter models. Mutations to several highly conserved residues that can take part in metal recognition and transport resulted in defective intracellular growth. Purified MavN and mutant derivatives were directly tested for transporter activity after heterologous purification and liposome reconstitution. Proteoliposomes harboring MavN exhibited robust transport of Fe2+, with the severity of defect of most mutants closely mimicking the magnitude of defects during intracellular growth. Surprisingly, MavN was equivalently proficient at transporting Fe2+, Mn2+, Co2+, or Zn2+ Consequently, flooding infected cells with either Mn2+ or Zn2+ allowed collaboration with iron to enhance intracellular growth of L. pneumophila ΔmavN strains, indicating a clear role for MavN in transporting each of these ions. These findings reveal that MavN is a transition-metal-ion transporter that plays a critical role in response to iron limitation during Legionella infection.
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Affiliation(s)
- Eric T Christenson
- Unit on Structural and Chemical Biology of Membrane Proteins, Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
| | - Dervla T Isaac
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111
| | - Karin Yoshida
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111
| | - Erion Lipo
- Program in Genetics, Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111
| | - Jin-Sik Kim
- Unit on Structural and Chemical Biology of Membrane Proteins, Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
| | - Rodolfo Ghirlando
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Ralph R Isberg
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111;
| | - Anirban Banerjee
- Unit on Structural and Chemical Biology of Membrane Proteins, Cell Biology and Neurobiology Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892;
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