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1
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Bottero M, Pessina G, Bason C, Vigo T, Uccelli A, Ferrara G. Nerve-Glial antigen 2: unmasking the enigmatic cellular identity in the central nervous system. Front Immunol 2024; 15:1393842. [PMID: 39136008 PMCID: PMC11317297 DOI: 10.3389/fimmu.2024.1393842] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Accepted: 07/05/2024] [Indexed: 08/15/2024] Open
Abstract
Chondroitin sulfate proteoglycans (CSPGs) are fundamental components of the extracellular matrix in the central nervous system (CNS). Among these, the Nerve-Glial antigen 2 (NG2) stands out as a transmembrane CSPG exclusively expressed in a different population of cells collectively termed NG2-expressing cells. These enigmatic cells, found throughout the developing and adult CNS, have been indicated with various names, including NG2 progenitor cells, polydendrocytes, synantocytes, NG2 cells, and NG2-Glia, but are more commonly referred to as oligodendrocyte progenitor cells. Characterized by high proliferation rates and unique morphology, NG2-expressing cells stand apart from neurons, astrocytes, and oligodendrocytes. Intriguingly, some NG2-expressing cells form functional glutamatergic synapses with neurons, challenging the long-held belief that only neurons possess the intricate machinery required for neurotransmission. In the CNS, the complexity surrounding NG2-expressing cells extends to their classification. Additionally, NG2 expression has been documented in pericytes and immune cells, suggesting a role in regulating brain innate immunity and neuro-immune crosstalk in homeostasis. Ongoing debates revolve around their heterogeneity, potential as progenitors for various cell types, responses to neuroinflammation, and the role of NG2. Therefore, this review aims to shed light on the enigma of NG2-expressing cells by delving into their structure, functions, and signaling pathways. We will critically evaluate the literature on NG2 expression across the CNS, and address the contentious issues surrounding their classification and roles in neuroinflammation and neurodegeneration. By unraveling the intricacies of NG2-expressing cells, we hope to pave the way for a more comprehensive understanding of their contributions to CNS health and during neurological disorders.
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Affiliation(s)
- Marta Bottero
- IRCCS Ospedale Policlinico San Martino, Genoa, Italy
| | - Giada Pessina
- Department of Experimental Medicine, University of Genoa, Genoa, Italy
| | | | - Tiziana Vigo
- IRCCS Ospedale Policlinico San Martino, Genoa, Italy
| | - Antonio Uccelli
- IRCCS Ospedale Policlinico San Martino, Genoa, Italy
- Department of Neurology, Rehabilitation, Ophthalmology, Genetics, Maternal and Child Health, University of Genoa, Genoa, Italy
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2
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Takeda-Okuda N, Yeon SJ, Matsumi Y, Matsuura Y, Hosaka YZ, Tamura JI. Quantitative, compositional, and immunohistochemical analyses of chondroitin sulfate, dermatan sulfate, and hyaluronan in internal organs of deer (Cervus nippon centralis and C. n. yesoensis) and cattle (Bos taurus). Int J Biol Macromol 2024; 261:129680. [PMID: 38281521 DOI: 10.1016/j.ijbiomac.2024.129680] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2023] [Revised: 01/14/2024] [Accepted: 01/21/2024] [Indexed: 01/30/2024]
Abstract
Chondroitin sulfate (CS) + dermatan sulfate (DS) and hyaluronan (HA) concentrations and the sulfation patterns of CS-DS in the cartilaginous tissues and alimentary canals of Honshu Sika deer, Hokkaido Sika deer, and cattle were investigated in the present study. CS + DS concentrations were high in cartilaginous tissues, namely, the trachea and scapular cartilage region (5- 12 g*), and low in the alimentary canal (~0.3 g*). HA concentrations were low in cartilaginous tissues and the alimentary canal (~0.2 g*). All tissues mainly contained A-type [HexAGalNAc(4-sulfate)] and C-type [HexAGalNAc(6-sulfate)] CS + DS. The ratios of A-type/C-type CS + DS were 1.2- 3.1 and 0.9- 16.4 in cartilaginous tissues and the alimentary canal, respectively. CS + DS predominantly comprised β-D-GlcA and α-L-IdoA in cartilaginous tissues and the alimentary canal, respectively. The alimentary canal characteristically contained up to 14 % highly sulfated E-type [HexAGalNAc(4,6-disulfate)] and D-type [HexA(2-sulfate)GalNAc(6-sulfate)] CS + DS. The specific distributions of CS and DS were immunohistochemically confirmed using CS + DS-specific antibodies. Although the omasum of cattle is more likely to have higher concentrations of CS + DS and HA, no significant species differences were observed in the concentrations or sulfation patterns of CS + DS among species for Honshu Sika deer, Hokkaido Sika deer, and cattle. (*per 100 g of defatted dry tissue).
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Affiliation(s)
- Naoko Takeda-Okuda
- Department of Agricultural, Life and Environmental Sciences, Faculty of Agriculture, Tottori University, Koyamacho-minami 4-101, Tottori 680-8553, Japan
| | - Su-Jung Yeon
- Department of Agricultural, Life and Environmental Sciences, Faculty of Agriculture, Tottori University, Koyamacho-minami 4-101, Tottori 680-8553, Japan
| | - Yoshiaki Matsumi
- Technical Department, Tottori University, Koyamacho-minami 4-101, Tottori, 680-8550, Japan
| | - Yoshinori Matsuura
- Technical Department, Tottori University, Koyamacho-minami 4-101, Tottori, 680-8550, Japan
| | - Yoshinao Z Hosaka
- Laboratory of Functional Anatomy, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
| | - Jun-Ichi Tamura
- Department of Agricultural, Life and Environmental Sciences, Faculty of Agriculture, Tottori University, Koyamacho-minami 4-101, Tottori 680-8553, Japan.
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3
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Buddle JE, Fagan RP. Pathogenicity and virulence of Clostridioides difficile. Virulence 2023; 14:2150452. [PMID: 36419222 DOI: 10.1080/21505594.2022.2150452] [Citation(s) in RCA: 49] [Impact Index Per Article: 24.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/18/2022] [Revised: 11/02/2022] [Accepted: 11/17/2022] [Indexed: 11/25/2022] Open
Abstract
Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhea, and is responsible for a spectrum of diseases characterized by high levels of recurrence, morbidity, and mortality. Treatment is complex, since antibiotics constitute both the main treatment and the major risk factor for infection. Worryingly, resistance to multiple antibiotics is becoming increasingly widespread, leading to the classification of this pathogen as an urgent threat to global health. As a consummate opportunist, C. difficile is well equipped for promoting disease, owing to its arsenal of virulence factors: transmission of this anaerobe is highly efficient due to the formation of robust endospores, and an array of adhesins promote gut colonization. C. difficile produces multiple toxins acting upon gut epithelia, resulting in manifestations typical of diarrheal disease, and severe inflammation in a subset of patients. This review focuses on such virulence factors, as well as the importance of antimicrobial resistance and genome plasticity in enabling pathogenesis and persistence of this important pathogen.
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Affiliation(s)
- Jessica E Buddle
- Molecular Microbiology, School of Biosciences, University of Sheffield, Sheffield, UK
| | - Robert P Fagan
- Molecular Microbiology, School of Biosciences, University of Sheffield, Sheffield, UK
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4
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Childress KO, Cencer CS, Tyska MJ, Lacy DB. Nectin-3 and shed forms of CSPG4 can serve as epithelial cell receptors for Clostridioides difficile TcdB. mBio 2023; 14:e0185723. [PMID: 37747247 PMCID: PMC10653914 DOI: 10.1128/mbio.01857-23] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2023] [Accepted: 07/26/2023] [Indexed: 09/26/2023] Open
Abstract
IMPORTANCE Toxin B (TcdB) is a major virulence factor of Clostridioides difficile, a Gram-positive pathogen that is a leading cause of hospital-acquired diarrhea. While previous studies have established that TcdB can engage multiple cell surface receptors in vitro, little is known about how these interactions promote disease and where these receptors localize on colonic tissue. Here, we used immunofluorescence microscopy to visualize Nectin-3 and CSPG4 on tissue, revealing unexpected localization of both receptors on colonic epithelial cells. We show that Nectin-3, which was previously characterized as an adherens junction protein, is also localized to the brush border of colonocytes. Staining for CSPG4 revealed that it is present along epithelial cell junctions, suggesting that it is shed by fibroblasts along the crypt-surface axis. Collectively, our study provides new insights into how TcdB can gain access to the receptors Nectin-3 and CSPG4 to intoxicate colonic epithelial cells.
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Affiliation(s)
- Kevin O. Childress
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Caroline S. Cencer
- Department of Cell and Development Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - Matthew J. Tyska
- Department of Cell and Development Biology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
| | - D. Borden Lacy
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
- Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, USA
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5
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Manion J, Musser MA, Kuziel GA, Liu M, Shepherd A, Wang S, Lee PG, Zhao L, Zhang J, Marreddy RKR, Goldsmith JD, Yuan K, Hurdle JG, Gerhard R, Jin R, Rakoff-Nahoum S, Rao M, Dong M. C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation. Nature 2023; 622:611-618. [PMID: 37699522 PMCID: PMC11188852 DOI: 10.1038/s41586-023-06607-2] [Citation(s) in RCA: 24] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2022] [Accepted: 09/05/2023] [Indexed: 09/14/2023]
Abstract
Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.
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Affiliation(s)
- John Manion
- Department of Urology, Boston Children's Hospital, Boston, MA, USA
- Department of Surgery, Harvard Medical School, Boston, MA, USA
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
| | - Melissa A Musser
- Division of Gastroenterology, Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA
| | - Gavin A Kuziel
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
- Division of Infectious Diseases, Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA
| | - Min Liu
- Department of Urology, Boston Children's Hospital, Boston, MA, USA
- Department of Surgery, Harvard Medical School, Boston, MA, USA
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
| | - Amy Shepherd
- Division of Gastroenterology, Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA
| | - Siyu Wang
- Department of Urology, Boston Children's Hospital, Boston, MA, USA
- Department of Surgery, Harvard Medical School, Boston, MA, USA
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
- Department of Colorectal Surgery, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
| | - Pyung-Gang Lee
- Department of Urology, Boston Children's Hospital, Boston, MA, USA
- Department of Surgery, Harvard Medical School, Boston, MA, USA
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
| | - Leo Zhao
- Department of Urology, Boston Children's Hospital, Boston, MA, USA
- Department of Surgery, Harvard Medical School, Boston, MA, USA
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
| | - Jie Zhang
- Department of Urology, Boston Children's Hospital, Boston, MA, USA
- Department of Surgery, Harvard Medical School, Boston, MA, USA
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
| | - Ravi K R Marreddy
- Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USA
| | | | - Ke Yuan
- Division of Pulmonary Medicine, Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA
| | - Julian G Hurdle
- Center for Infectious and Inflammatory Diseases, Institute of Biosciences and Technology, Texas A&M Health Science Center, Houston, TX, USA
| | - Ralf Gerhard
- Institute of Toxicology, Hannover Medical School, Hannover, Germany
| | - Rongsheng Jin
- Department of Physiology and Biophysics, School of Medicine, University of California Irvine, Irvine, CA, USA
| | - Seth Rakoff-Nahoum
- Department of Microbiology, Harvard Medical School, Boston, MA, USA
- Division of Gastroenterology, Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA
- Division of Infectious Diseases, Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA
| | - Meenakshi Rao
- Division of Gastroenterology, Department of Pediatrics, Boston Children's Hospital and Harvard Medical School, Boston, MA, USA.
| | - Min Dong
- Department of Urology, Boston Children's Hospital, Boston, MA, USA.
- Department of Surgery, Harvard Medical School, Boston, MA, USA.
- Department of Microbiology, Harvard Medical School, Boston, MA, USA.
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6
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Fettucciari K, Dini F, Marconi P, Bassotti G. Role of the Alteration in Calcium Homeostasis in Cell Death Induced by Clostridioides difficile Toxin A and Toxin B. BIOLOGY 2023; 12:1117. [PMID: 37627001 PMCID: PMC10452684 DOI: 10.3390/biology12081117] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/11/2023] [Revised: 08/05/2023] [Accepted: 08/08/2023] [Indexed: 08/27/2023]
Abstract
Clostridioides difficile (C. difficile), responsible for 15-25% of gastrointestinal infections, causes health problems mainly due to the toxic activity of toxins A and B (Tcds). These are responsible for its clinical manifestations, including diarrhea, pseudomembranous colitis, toxic megacolon and death, with a mortality of 5-30% in primary infection, that increase following relapses. Studies on Tcd-induced cell death have highlighted a key role of caspases, calpains, and cathepsins, with involvement of mitochondria and reactive oxygen species (ROS) in a complex signaling pathway network. The complex response in the execution of various types of cell death (apoptosis, necrosis, pyroptosis and pyknosis) depends on the amount of Tcd, cell types, and Tcd receptors involved, and could have as initial/precocious event the alterations in calcium homeostasis. The entities, peculiarities and cell types involved in these alterations will decide the signaling pathways activated and cell death type. Calcium homeostasis alterations can be caused by calcium influx through calcium channel activation, transient intracellular calcium oscillations, and leakage of calcium from intracellular stores. These increases in cytoplasmic calcium have important effects on all calcium-regulated molecules, which may play a direct role in several cell death types and/or activate other cell death effectors, such as caspases, calpains, ROS and proapoptotic Bcl-2 family members. Furthermore, some support for the possible role of the calcium homeostasis alteration in Tcd-induced cell death originates from the similarity with cytotoxic effects that cause pore-forming toxins, based mainly on calcium influx through plasma membrane pores.
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Affiliation(s)
- Katia Fettucciari
- Biosciences & Medical Embryology Section, Department of Medicine and Surgery, University of Perugia, 06129 Perugia, Italy;
| | - Fabrizio Dini
- School of Biosciences and Veterinary Medicine, University of Camerino, 62024 Matelica, Italy;
| | - Pierfrancesco Marconi
- Biosciences & Medical Embryology Section, Department of Medicine and Surgery, University of Perugia, 06129 Perugia, Italy;
| | - Gabrio Bassotti
- Gastroenterology, Hepatology & Digestive Endoscopy Section, Department of Medicine and Surgery, University of Perugia, 06129 Perugia, Italy;
- Gastroenterology & Hepatology Unit, Santa Maria Della Misericordia Hospital, 06129 Perugia, Italy
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7
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Fettucciari K, Fruganti A, Stracci F, Spaterna A, Marconi P, Bassotti G. Clostridioides difficile Toxin B Induced Senescence: A New Pathologic Player for Colorectal Cancer? Int J Mol Sci 2023; 24:8155. [PMID: 37175861 PMCID: PMC10179142 DOI: 10.3390/ijms24098155] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Revised: 04/26/2023] [Accepted: 04/28/2023] [Indexed: 05/15/2023] Open
Abstract
Clostridioides difficile (C. difficile) is responsible for a high percentage of gastrointestinal infections and its pathological activity is due to toxins A and B. C. difficile infection (CDI) is increasing worldwide due to the unstoppable spread of C. difficile in the anthropized environment and the progressive human colonization. The ability of C. difficile toxin B to induce senescent cells and the direct correlation between CDI, irritable bowel syndrome (IBS), and inflammatory bowel diseases (IBD) could cause an accumulation of senescent cells with important functional consequences. Furthermore, these senescent cells characterized by long survival could push pre-neoplastic cells originating in the colon towards the complete neoplastic transformation in colorectal cancer (CRC) by the senescence-associated secretory phenotype (SASP). Pre-neoplastic cells could appear as a result of various pro-carcinogenic events, among which, are infections with bacteria that produce genotoxins that generate cells with high genetic instability. Therefore, subjects who develop IBS and/or IBD after CDI should be monitored, especially if they then have further CDI relapses, waiting for the availability of senolytic and anti-SASP therapies to resolve the pro-carcinogenic risk due to accumulation of senescent cells after CDI followed by IBS and/or IBD.
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Affiliation(s)
- Katia Fettucciari
- Biosciences & Medical Embryology Section, Department of Medicine and Surgery, University of Perugia, 06129 Perugia, Italy;
| | - Alessandro Fruganti
- School of Biosciences and Veterinary Medicine, University of Camerino, 62024 Matelica, Italy
| | - Fabrizio Stracci
- Public Health Section, Department of Medicine and Surgery, University of Perugia, 06129 Perugia, Italy;
| | - Andrea Spaterna
- School of Biosciences and Veterinary Medicine, University of Camerino, 62024 Matelica, Italy
| | - Pierfrancesco Marconi
- Biosciences & Medical Embryology Section, Department of Medicine and Surgery, University of Perugia, 06129 Perugia, Italy;
| | - Gabrio Bassotti
- Gastroenterology, Hepatology & Digestive Endoscopy Section, Department of Medicine and Surgery, University of Perugia, 06129 Perugia, Italy;
- Gastroenterology & Hepatology Unit, Santa Maria Della Misericordia Hospital, 06129 Perugia, Italy
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8
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Csukovich G, Kramer N, Pratscher B, Gotic I, Freund P, Hahn R, Himmler G, Brandt S, Burgener IA. Neutralising Effects of Different Antibodies on Clostridioides difficile Toxins TcdA and TcdB in a Translational Approach. Int J Mol Sci 2023; 24:ijms24043867. [PMID: 36835278 PMCID: PMC9962434 DOI: 10.3390/ijms24043867] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2022] [Revised: 01/30/2023] [Accepted: 02/13/2023] [Indexed: 02/17/2023] Open
Abstract
Given the high prevalence of intestinal disease in humans and animals, there is a strong need for clinically relevant models recapitulating gastrointestinal systems, ideally replacing in vivo models in accordance with the principles of the 3R. We established a canine organoid system and analysed the neutralising effects of recombinant versus natural antibodies on Clostridioides difficile toxins A and B in this in vitro system. Sulforhodamine B cytotoxicity assays in 2D and FITC-dextran barrier integrity assays on basal-out and apical-out organoids revealed that recombinant, but not natural antibodies, effectively neutralised C. difficile toxins. Our findings emphasise that canine intestinal organoids can be used to test different components and suggest that they can be further refined to also mirror complex interactions between the intestinal epithelium and other cells.
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Affiliation(s)
- Georg Csukovich
- Small Animal Internal Medicine, Department for Companion Animals and Horses, Vetmeduni, 1210 Vienna, Austria
| | - Nina Kramer
- Small Animal Internal Medicine, Department for Companion Animals and Horses, Vetmeduni, 1210 Vienna, Austria
| | - Barbara Pratscher
- Small Animal Internal Medicine, Department for Companion Animals and Horses, Vetmeduni, 1210 Vienna, Austria
| | | | - Patricia Freund
- Small Animal Internal Medicine, Department for Companion Animals and Horses, Vetmeduni, 1210 Vienna, Austria
| | - Rainer Hahn
- Department for Biotechnology, University of Natural Resources and Life Sciences Vienna, 1190 Vienna, Austria
| | | | - Sabine Brandt
- Research Group Oncology (RGO), Clinical Unit of Equine Surgery, Department for Companion Animals and Horses, Vetmeduni, 1210 Vienna, Austria
| | - Iwan Anton Burgener
- Small Animal Internal Medicine, Department for Companion Animals and Horses, Vetmeduni, 1210 Vienna, Austria
- Correspondence:
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9
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Chen P, Jin R. Receptor binding mechanisms of Clostridioides difficile toxin B and implications for therapeutics development. FEBS J 2023; 290:962-969. [PMID: 34862749 PMCID: PMC9344982 DOI: 10.1111/febs.16310] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2021] [Revised: 11/19/2021] [Accepted: 12/02/2021] [Indexed: 12/29/2022]
Abstract
Clostridioides difficile is classified as an urgent antibiotic resistance threat by the Centers for Disease Control and Prevention (CDC). C. difficile infection (CDI) is mainly caused by the C. difficile exotoxin TcdB, which invades host cells via receptor-mediated endocytosis. However, many natural variants of TcdB have been identified including some from the hypervirulent strains, which pose significant challenges for developing effective CDI therapies. Here, we review the recent research progress on the molecular mechanisms by which TcdB recognizes Frizzed proteins (FZDs) and chondroitin sulfate proteoglycan 4 (CSPG4) as two major host receptors. We suggest that the receptor-binding sites and several previously identified neutralizing epitopes on TcdB are ideal targets for the development of broad-spectrum inhibitors to protect against diverse TcdB variants.
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Affiliation(s)
- Peng Chen
- Department of Physiology and Biophysics, University of California Irvine, Irvine, California, 92697, USA
| | - Rongsheng Jin
- Department of Physiology and Biophysics, University of California Irvine, Irvine, California, 92697, USA
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10
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Bassotti G, Fruganti A, Stracci F, Marconi P, Fettucciari K. Cytotoxic synergism of Clostridioides difficile toxin B with proinflammatory cytokines in subjects with inflammatory bowel diseases. World J Gastroenterol 2023; 29:582-596. [PMID: 36742168 PMCID: PMC9896618 DOI: 10.3748/wjg.v29.i4.582] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/15/2022] [Revised: 12/03/2022] [Accepted: 12/27/2022] [Indexed: 01/20/2023] Open
Abstract
Clostridioides difficile (C. difficile) is progressively colonizing humans and animals living with humans. During this process, hypervirulent strains and mutated toxin A and B of C. difficile (TcdA and TcdB) are originating and developing. While in healthy subjects colonization by C. difficile becomes a risk after the use of antibiotics that alter the microbiome, other categories of people are more susceptible to infection and at risk of relapse, such as those with inflammatory bowel disease (IBD). Recent in vitro studies suggest that this increased susceptibility could be due to the strong cytotoxic synergism between TcdB and proinflammatory cytokines the tumor necrosis factor-alpha and interferon-gamma (CKs). Therefore, in subjects with IBD the presence of an inflammatory state in the colon could be the driver that increases the susceptibility to C. difficile infection and its progression and relapses. TcdB is internalized in the cell via three receptors: chondroitin sulphate proteoglycan 4; poliovirus receptor-like 3; and Wnt receptor frizzled family. Chondroitin sulphate proteoglycan 4 and Wnt receptor frizzled family are involved in cell death by apoptosis or necrosis depending on the concentration of TcdB and cell types, while poliovirus receptor-like 3 induces only necrosis. It is possible that cytokines could also induce a greater expression of receptors for TcdB that are more involved in necrosis than in apoptosis. Therefore, in subjects with IBD there are the conditions: (1) For greater susceptibility to C. difficile infection, such as the inflammatory state, and abnormalities of the microbiome and of the immune system; (2) for the enhancement of the cytotoxic activity of TcdB +Cks; and (3) for a greater expression of TcdB receptors stimulated by cytokines that induce cell death by necrosis rather than apoptosis. The only therapeutic approach currently possible in IBD patients is monitoring of C. difficile colonization for interventions aimed at reducing tumor necrosis factor-alpha and interferon-gamma levels when the infection begins. The future perspective is to generate bacteriophages against C. difficile for targeted therapy.
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Affiliation(s)
- Gabrio Bassotti
- Department of Medicine and Surgery, Gastroenterology, Hepatology & Digestive Endoscopy Section University of Perugia Medical School, Piazza Lucio Severi, Perugia 06132, Italy, and Santa Maria della Misericordia Hospital, Gastroenterology & Hepatology Unit Perugia 06156, Italy
| | - Alessandro Fruganti
- School of Biosciences and Veterinary Medicine, University of Camerino, Matelica 62024, Italy
| | - Fabrizio Stracci
- Medicine and Surgery, Hygiene and Public Health Section, University of Perugia, Perugia 06123, Italy
| | - Pierfrancesco Marconi
- Medicine and Surgery, Biosciences & Medical Embryology Section, University of Perugia, Perugia 06132, Italy
| | - Katia Fettucciari
- Medicine and Surgery, Biosciences & Medical Embryology Section, University of Perugia, Perugia 06132, Italy
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11
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Luo J, Yang Q, Zhang X, Zhang Y, Wan L, Zhan X, Zhou Y, He L, Li D, Jin D, Zhen Y, Huang J, Li Y, Tao L. TFPI is a colonic crypt receptor for TcdB from hypervirulent clade 2 C. difficile. Cell 2022; 185:980-994.e15. [PMID: 35303428 DOI: 10.1016/j.cell.2022.02.010] [Citation(s) in RCA: 43] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Revised: 01/11/2022] [Accepted: 02/08/2022] [Indexed: 12/14/2022]
Abstract
The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.
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Affiliation(s)
- Jianhua Luo
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Qi Yang
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Xiaofeng Zhang
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Yuanyuan Zhang
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China; School of Food Science and Biotechnology Engineering, Zhejiang Gongshang University, Hangzhou, Zhejiang 310018, China
| | - Li Wan
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Xiechao Zhan
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Yao Zhou
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Liuqing He
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Danyang Li
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Dazhi Jin
- Center of Laboratory Medicine, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, Zhejiang 310014, China; School of Laboratory Medicine, Hangzhou Medical College, Hangzhou, Zhejiang 310014, China
| | - Ying Zhen
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Jing Huang
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China
| | - Yanyan Li
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China.
| | - Liang Tao
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang 310024, China; Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang 310024, China; Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang 310024, China.
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12
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Fettucciari K, Marconi P, Marchegiani A, Fruganti A, Spaterna A, Bassotti G. Invisible steps for a global endemy: molecular strategies adopted by Clostridioides difficile. Therap Adv Gastroenterol 2021; 14:17562848211032797. [PMID: 34413901 PMCID: PMC8369858 DOI: 10.1177/17562848211032797] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/07/2021] [Accepted: 06/26/2021] [Indexed: 02/06/2023] Open
Abstract
Clostridioides difficile infection (CDI) is on the rise worldwide and is associated with an increase in deaths and socio-health burden. C. difficile has become ubiquitous in anthropized environments because of the extreme resistance of its spores. Based on the epidemiological data and knowledge of molecular pathogenesis of C. difficile, it is possible to predict its progressive colonization of the human population for the following reasons: first, its global spread is unstoppable; second, the toxins (Tcds) produced by C. difficile, TcdA and TcdB, mainly cause cell death by apoptosis, but the surviving cells acquire a senescence state that favours persistence of C. difficile in the intestine; third, proinflammatory cytokines, tumour necrosis factor-α and interferon-γ, induced during CDI, enhance the cytotoxicity of Tcds and can increase the survival of senescent cells; fourth, Tcds block mobility and induce apoptosis in immune cells recruited at the infection site; and finally, after remission from primary infection or relapse, C. difficile causes functional abnormalities in the enteric glial cell (EGC) network that can result in irritable bowel syndrome, characterized by a latent inflammatory response that contributes to C. difficile survival and enhances the cytotoxic activity of low doses of TcdB, thus favouring further relapses. Since a 'global endemy' of C. difficile seems inevitable, it is necessary to develop an effective vaccine against Tcds for at-risk individuals, and to perform a prophylaxis/selective therapy with bacteriophages highly specific for C. difficile. We must be aware that CDI will become a global health problem in the forthcoming years, and we must be prepared to face this menace.
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Affiliation(s)
- Katia Fettucciari
- Biosciences & Medical Embryology Section, Department of Medicine and Surgery, University of Perugia, Medical School -Piazza Lucio Severi 1, Edificio B - IV piano; Sant’Andrea delle Fratte, Perugia, 06132, Italy
| | - Pierfrancesco Marconi
- Biosciences & Medical Embryology Section, Department of Medicine and Surgery, University of Perugia, Perugia, Italy
| | - Andrea Marchegiani
- School of Biosciences and Veterinary Medicine, University of Camerino, Macerata, Italy
| | - Alessandro Fruganti
- School of Biosciences and Veterinary Medicine, University of Camerino, Macerata, Italy
| | - Andrea Spaterna
- School of Biosciences and Veterinary Medicine, University of Camerino, Macerata, Italy
| | - Gabrio Bassotti
- Gastroenterology, Hepatology & Digestive Endoscopy Section, Department of Medicine and Surgery, University of Perugia, Perugia, Italy
- Gastroenterology & Hepatology Unit, Santa Maria della Misericordia Hospital, Perugia, Italy
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13
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Chen P, Zeng J, Liu Z, Thaker H, Wang S, Tian S, Zhang J, Tao L, Gutierrez CB, Xing L, Gerhard R, Huang L, Dong M, Jin R. Structural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection. Nat Commun 2021; 12:3748. [PMID: 34145250 PMCID: PMC8213806 DOI: 10.1038/s41467-021-23878-3] [Citation(s) in RCA: 42] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2021] [Accepted: 05/19/2021] [Indexed: 12/13/2022] Open
Abstract
C. difficile is a major cause of antibiotic-associated gastrointestinal infections. Two C. difficile exotoxins (TcdA and TcdB) are major virulence factors associated with these infections, and chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for TcdB, but its pathophysiological relevance and the molecular details that govern recognition remain unknown. Here, we determine the cryo-EM structure of a TcdB–CSPG4 complex, revealing a unique binding site spatially composed of multiple discontinuous regions across TcdB. Mutations that selectively disrupt CSPG4 binding reduce TcdB toxicity in mice, while CSPG4-knockout mice show reduced damage to colonic tissues during C. difficile infections. We further show that bezlotoxumab, the only FDA approved anti-TcdB antibody, blocks CSPG4 binding via an allosteric mechanism, but it displays low neutralizing potency on many TcdB variants from epidemic hypervirulent strains due to sequence variations in its epitopes. In contrast, a CSPG4-mimicking decoy neutralizes major TcdB variants, suggesting a strategy to develop broad-spectrum therapeutics against TcdB. Chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for C. difficile toxin B (TcdB) during C. difficile infections (CDIs). Here, the cryo-EM structure of a TcdB–CSPG4 complex and CDI mouse models offer insights into CSPG4 role in CDIs and suggest a therapeutic strategy targeting TcdB.
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Affiliation(s)
- Peng Chen
- Department of Physiology and Biophysics, University of California, Irvine, CA, USA
| | - Ji Zeng
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.,Department of Microbiology, Harvard Medical School, Boston, MA, USA.,Department of Surgery, Harvard Medical School, Boston, MA, USA
| | - Zheng Liu
- Department of Physiology and Biophysics, University of California, Irvine, CA, USA
| | - Hatim Thaker
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.,Department of Microbiology, Harvard Medical School, Boston, MA, USA.,Department of Surgery, Harvard Medical School, Boston, MA, USA
| | - Siyu Wang
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.,Department of Microbiology, Harvard Medical School, Boston, MA, USA.,Department of Surgery, Harvard Medical School, Boston, MA, USA.,Department of Gastrointestinal, Colorectal and Anal Surgery, China-Japan Union Hospital of Jilin University, Changchun, China
| | - Songhai Tian
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.,Department of Microbiology, Harvard Medical School, Boston, MA, USA.,Department of Surgery, Harvard Medical School, Boston, MA, USA
| | - Jie Zhang
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA.,Department of Microbiology, Harvard Medical School, Boston, MA, USA.,Department of Surgery, Harvard Medical School, Boston, MA, USA
| | - Liang Tao
- Center for Infectious Disease Research, Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, Zhejiang, China.,Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China
| | - Craig B Gutierrez
- Department of Physiology and Biophysics, University of California, Irvine, CA, USA
| | - Li Xing
- UC Irvine Materials Research Institute (IMRI), University of California, Irvine, CA, USA
| | - Ralf Gerhard
- Institute of Toxicology, Hannover Medical School, Hannover, Germany
| | - Lan Huang
- Department of Physiology and Biophysics, University of California, Irvine, CA, USA
| | - Min Dong
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. .,Department of Microbiology, Harvard Medical School, Boston, MA, USA. .,Department of Surgery, Harvard Medical School, Boston, MA, USA.
| | - Rongsheng Jin
- Department of Physiology and Biophysics, University of California, Irvine, CA, USA.
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14
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Abstract
Large clostridial toxins (LCTs) are a family of bacterial exotoxins that infiltrate and destroy target cells. Members of the LCT family include Clostridioides difficile toxins TcdA and TcdB, Paeniclostridium sordellii toxins TcsL and TcsH, Clostridium novyi toxin TcnA, and Clostridium perfringens toxin TpeL. Since the 19th century, LCT-secreting bacteria have been isolated from the blood, organs, and wounds of diseased individuals, and LCTs have been implicated as the primary virulence factors in a variety of infections, including C. difficile infection and some cases of wound-associated gas gangrene. Clostridia express and secrete LCTs in response to various physiological signals. LCTs invade host cells by binding specific cell surface receptors, ultimately leading to internalization into acidified vesicles. Acidic pH promotes conformational changes within LCTs, which culminates in translocation of the N-terminal glycosyltransferase and cysteine protease domain across the endosomal membrane and into the cytosol, leading first to cytopathic effects and later to cytotoxic effects. The focus of this review is on the role of LCTs in infection and disease, the mechanism of LCT intoxication, with emphasis on recent structural work and toxin subtyping analysis, and the genomic discovery and characterization of LCT homologues. We provide a comprehensive review of these topics and offer our perspective on emerging questions and future research directions for this enigmatic family of toxins.
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15
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Tamura Y, Takata K, Eguchi A, Maeda M, Kataoka Y. Age-related changes in NG2-expressing telocytes of rat stomach. PLoS One 2021; 16:e0249729. [PMID: 33822814 PMCID: PMC8023479 DOI: 10.1371/journal.pone.0249729] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Accepted: 03/23/2021] [Indexed: 11/18/2022] Open
Abstract
NG2 immunoreactive cells (NG2 cells) are found in the brain and peripheral tissues including the skin, intestinal tracts, and bladder. In a previous study, we observed the presence of NG2 cells in the stomach using bioluminescence imaging techniques in NG2-firefly luciferase (fLuc) transgenic (Tg) rats. Here, we aimed to identify and characterize NG2 cells in the adult rat stomach. Immunohistochemical studies showed that NG2 cells were mainly present in the lamina propria and most of the cells were gastric telocytes, co-expressing CD34, and platelet-derived growth factor receptor alpha (PDGFRα), with a small oval-shaped cell body and extremely long and thin cellular prolongations. In the rat stomach, NG2-expressing telocytes comprised two subpopulations: NG2+/CD34+/PDGFRα+ and NG2+/CD34+/PDGFRα-. Furthermore, we showed that the expression of NG2 gene in the aged rat stomach decreased relative to that of the young rat stomach and the decline of NG2 expression in aged rats was mainly observed in NG2+/CD34+/PDGFRα+ telocytes. These findings suggested age-related alterations in NG2+/CD34+/PDGFRα+ telocytes of rat stomach.
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Affiliation(s)
- Yasuhisa Tamura
- Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Chuo-ku, Kobe, Japan
- Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Chuo-ku, Kobe, Japan
| | - Kumi Takata
- Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Chuo-ku, Kobe, Japan
- Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Chuo-ku, Kobe, Japan
| | - Asami Eguchi
- Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Chuo-ku, Kobe, Japan
- Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Chuo-ku, Kobe, Japan
| | - Mitsuyo Maeda
- Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Chuo-ku, Kobe, Japan
- Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Chuo-ku, Kobe, Japan
| | - Yosky Kataoka
- Laboratory for Cellular Function Imaging, RIKEN Center for Biosystems Dynamics Research, Chuo-ku, Kobe, Japan
- Multi-Modal Microstructure Analysis Unit, RIKEN-JEOL Collaboration Center, Chuo-ku, Kobe, Japan
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16
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Pan Z, Zhang Y, Luo J, Li D, Zhou Y, He L, Yang Q, Dong M, Tao L. Functional analyses of epidemic Clostridioides difficile toxin B variants reveal their divergence in utilizing receptors and inducing pathology. PLoS Pathog 2021; 17:e1009197. [PMID: 33507919 PMCID: PMC7842947 DOI: 10.1371/journal.ppat.1009197] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2020] [Accepted: 11/30/2020] [Indexed: 02/06/2023] Open
Abstract
Clostridioides difficile toxin B (TcdB) is a key virulence factor that causes C. difficile associated diseases (CDAD) including diarrhea and pseudomembranous colitis. TcdB can be divided into multiple subtypes/variants based on their sequence variations, of which four (TcdB1-4) are dominant types found in major epidemic isolates. Here, we find that these variants are highly diverse in their receptor preference: TcdB1 uses two known receptors CSPG4 and Frizzled (FZD) proteins, TcdB2 selectively uses CSPG4, TcdB3 prefers to use FZDs, whereas TcdB4 uses neither CSPG4 nor FZDs. By creating chimeric toxins and systematically switching residues between TcdB1 and TcdB3, we determine that regions in the N-terminal cysteine protease domain (CPD) are involved in CSPG4-recognition. We further evaluate the pathological effects induced by TcdB1-4 with a mouse intrarectal installation model. TcdB1 leads to the most severe overall symptoms, followed by TcdB2 and TcdB3. When comparing the TcdB2 and TcdB3, TcdB2 causes stronger oedema while TcdB3 induces severer inflammatory cell infiltration. These findings together demonstrate divergence in the receptor preference and further lead to colonic pathology for predominant TcdB subtypes. Clostridioides difficile is a major cause of nosocomial and community-associated gastrointestinal infections. The bacterium produces three exotoxins including TcdA, TcdB, and CDT, of which TcdB is known as a key virulence factor causing the diseases. Since C. difficile was first linked to antibiotic-associated infections in 1978, a large number of clinically relevant strains were characterized and many of them were found to harbor some variant forms of TcdB. In this study, we examined four predominant TcdB variants from epidemic C. difficile strains. We found that these variants are highly diverse in preference to the known receptors, CSPG4 and Frizzled proteins. By conducting a systematically designed mutagenesis study, we determined that TcdB interacts with CSPG4 via regions across multiple domains. We also found that TcdB variants could induce distinguishable pathological phenotypes in a mouse model, suggesting C. difficile strains harboring divergent TcdB variants might exhibit different disease progression. Our study provides new insights into the toxicology and pathology of C. difficile toxin variants.
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Affiliation(s)
- Zhenrui Pan
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Yuanyuan Zhang
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Jianhua Luo
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Danyang Li
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Yao Zhou
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Liuqing He
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Qi Yang
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
| | - Min Dong
- Department of Urology, Boston Children’s Hospital, Boston, Massechusetts, United States of America
- Department of Surgery and Department of Microbiology, Harvard Medical School, Boston, Massechusetts, United States of America
- * E-mail: (MD); (LT)
| | - Liang Tao
- Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China
- Center for Infectious Disease Research, Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China
- Institute of Basic Medical Sciences, Westlake Institute for Advanced Study, Hangzhou, China
- * E-mail: (MD); (LT)
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17
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Salinas-Marín R, Villanueva-Cabello TM, Martínez-Duncker I. Biology of Proteoglycans and Associated Glycosaminoglycans. COMPREHENSIVE GLYCOSCIENCE 2021:63-102. [DOI: 10.1016/b978-0-12-819475-1.00065-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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18
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Danz HR, Lee S, Chapman-Bonofiglio SP, Ginese M, Beamer G, Girouard DJ, Tzipori S. The Impact of Actotoxumab Treatment of Gnotobiotic Piglets Infected With Different Clostridium difficile Isogenic Mutants. J Infect Dis 2020; 221:276-284. [PMID: 31495879 DOI: 10.1093/infdis/jiz459] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2019] [Accepted: 09/06/2019] [Indexed: 11/14/2022] Open
Abstract
Nosocomial infections with Clostridium difficile are on the rise in the Unites States, attributed to emergence of antibiotic-resistant and hypervirulent strains associated with greater likelihood of recurrent infections. In addition to antibiotics, treatment with Merck anti-toxin B (TcdB) antibody bezlotoxumab is reported to reduce recurrent infections. However, treatment with anti-toxin A (TcdA) antibody actotoxumab was associated with dramatically increased disease severity and mortality rates in humans and gnotobiotic piglets. Using isogenic mutants of C. difficile strain NAPI/BI/027 deficient in TcdA (A-B+) or TcdB (A+B-), and the wild type, we investigated how and why treatment of infected animals with anti-TcdA dramatically increased disease severity. Contrary to the hypothesis, among piglets treated with anti-TcdA, those with A+B- infection were disease free, in contrast to the disease enhancement seen in those with wild-type or A-B+ infection. It seems that the lack of TcdA, through either deletion or neutralization with anti-TcdA, reduces a competitive pressure, allowing TcdB to freely exert its profound effect, leading to increased mucosal injury and disease severity.
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Affiliation(s)
- Hillary R Danz
- Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts, USA
| | - Sangun Lee
- Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts, USA
| | - Susan P Chapman-Bonofiglio
- Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts, USA
| | - Melanie Ginese
- Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts, USA
| | - Gillian Beamer
- Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts, USA
| | - Donald J Girouard
- Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts, USA
| | - Saul Tzipori
- Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine at Tufts University, North Grafton, Massachusetts, USA
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19
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Identification of the role of toxin B in the virulence of Clostridioides difficile based on integrated bioinformatics analyses. Int Microbiol 2020; 23:575-587. [PMID: 32388701 DOI: 10.1007/s10123-020-00128-y] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Revised: 03/03/2020] [Accepted: 04/13/2020] [Indexed: 02/08/2023]
Abstract
PURPOSE Clostridioides difficile toxin B (TcdB) plays a critical role in C. difficile infection (CDI), a common and costly healthcare-associated disease. The aim of the current study was to explore the intracellular and potent systemic effects of TcdB on human colon epithelial cells utilizing Gene Expression Omnibus and bioinformatic methods. METHODS Two datasets (GSE63880 and GSE29008) were collected to extract data components of mRNA of TcdB-treated human colon epithelial cells; "limma" package of "R" software was used to screen the differential genes, and "pheatmap" package was applied to construct heat maps for the differential genes; Metascape website was utilized for protein-protein interaction network and Molecular Complex Detection analysis, and Genome Ontology (GO) was used to analyze the selected differential genes. Quantitative real-time PCR (qRT-PCR) and Western blot were performed to validate the expression of hub genes. RESULTS GO terms involved in DNA replication and cell cycle were identified significantly enriched in TcdB-treated human colon epithelial cells. Moreover, the decreased expression of DNA replication-related genes, MCM complex, and CDC45 in C. difficile (TcdA-/TcdB+)-infected Caco-2 cells were validated via qRT-PCR and Western blot assays. CONCLUSIONS In conclusion, the integrated analysis of different gene expression datasets allowed us to identify a set of genes and GO terms underlying the mechanisms of CDI induced by TcdB. It would aid in understanding of the molecular mechanisms underlying TcdB-exposed colon epithelial cells and provide the basis for developing diagnosis biomarkers, treatment, and prevention strategies.
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20
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Schöttelndreier D, Seeger K, Grassl GA, Winny MR, Lindner R, Genth H. Expression and (Lacking) Internalization of the Cell Surface Receptors of Clostridioides difficile Toxin B. Front Microbiol 2018; 9:1483. [PMID: 30022975 PMCID: PMC6039548 DOI: 10.3389/fmicb.2018.01483] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2018] [Accepted: 06/14/2018] [Indexed: 12/30/2022] Open
Abstract
Toxin-producing strains of Clostridioides difficile and Clostridium perfringens cause infections of the gastrointestinal tract in humans and ruminants, with the toxins being major virulence factors, essential for the infection, and responsible for the onset of severe symptoms. C. difficile toxin A (TcdA) and toxin B (TcdB), and the large cytotoxin (TpeL) from C. perfringens are single chain bacterial protein toxins with an AB-like toxin structure. The C-terminal delivery domain mediates cell entry of the N-terminal glycosyltransferase domain by receptor-mediated endocytosis. Several cell surface proteins have been proposed to serve as toxin receptors, including chondroitin-sulfate proteoglycan 4 (CSPG4), poliovirus receptor-like 3 (PVRL3), and frizzled-1/2/7 (FZD1/2/7) for TcdB and LDL-receptor-related protein-1 (LRP1) for TpeL. The expression of the TcdB receptors was investigated in human intestinal organoids (HIOs) and in cultured cell lines. HIOs from four human donors exhibited a comparable profile of receptor expression, with PVRL3, LRP1, and FZD7 being expressed and CSPG4 and FZD2 not being expressed. In human epithelial Caco-2 cells and HT29 cells as well as in immortalized murine fibroblasts, either receptor FZD2/7, CSPG4, PVRL3, and LRP1 was expressed. The question whether the toxins take advantage of the normal turnover of their receptors (i.e., constitutive endocytosis and recycling) from the cell surface or whether the toxins activity induce the internalization of their receptors has not yet been addressed. For the analysis of receptor internalization, temperature-induced uptake of biotinylated toxin receptors into immortalized mouse embryonic fibroblasts (MEFs) and Caco-2 cells was exploited. Solely LRP1 exhibited constitutive endocytosis from the plasma membrane to the endosome, which might be abused by TpeL (and possibly TcdB as well) for cell entry. Furthermore, internalization of CSPG4, PVRL3, FZD2, and FZD7 was observed neither in MEFs nor in Caco-2 cells. FZD2/7, CSPG4, and PVRL3 did thus exhibit no constitutive recycling. The presence of TcdB and the p38 activation induced by anisomycin were not able to induce or enhance CSPG4 or PVRL3 uptake in MEFs. In conclusion, FZD2/7, CSPG4, and PVRL3 seem to serve as cell surface binding receptors rather than internalizing receptors of TcdB.
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Affiliation(s)
| | - Katrin Seeger
- Institute of Medical Microbiology and Hospital Epidemiology and DZIF Partner Site Hannover-Braunschweig, Hannover Medical School, Hanover, Germany
| | - Guntram A Grassl
- Institute of Medical Microbiology and Hospital Epidemiology and DZIF Partner Site Hannover-Braunschweig, Hannover Medical School, Hanover, Germany
| | - Markus R Winny
- Department of General, Visceral and Transplantation Surgery, Hannover Medical School, Hanover, Germany
| | - Robert Lindner
- Neuroanatomy and Cell Biology, Hannover Medical School, Hanover, Germany
| | - Harald Genth
- Institute for Toxicology, Hannover Medical School, Hanover, Germany
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21
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Chen P, Tao L, Wang T, Zhang J, He A, Lam KH, Liu Z, He X, Perry K, Dong M, Jin R. Structural basis for recognition of frizzled proteins by Clostridium difficile toxin B. Science 2018; 360:664-669. [PMID: 29748286 PMCID: PMC6231499 DOI: 10.1126/science.aar1999] [Citation(s) in RCA: 101] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2017] [Accepted: 04/04/2018] [Indexed: 12/12/2022]
Abstract
Clostridium difficile infection is the most common cause of antibiotic-associated diarrhea in developed countries. The major virulence factor, C. difficile toxin B (TcdB), targets colonic epithelia by binding to the frizzled (FZD) family of Wnt receptors, but how TcdB recognizes FZDs is unclear. Here, we present the crystal structure of a TcdB fragment in complex with the cysteine-rich domain of human FZD2 at 2.5-angstrom resolution, which reveals an endogenous FZD-bound fatty acid acting as a co-receptor for TcdB binding. This lipid occupies the binding site for Wnt-adducted palmitoleic acid in FZDs. TcdB binding locks the lipid in place, preventing Wnt from engaging FZDs and signaling. Our findings establish a central role of fatty acids in FZD-mediated TcdB pathogenesis and suggest strategies to modulate Wnt signaling.
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Affiliation(s)
- Peng Chen
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA
| | - Liang Tao
- Department of Urology, Boston Children's Hospital, Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, MA, USA
| | - Tianyu Wang
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA
| | - Jie Zhang
- Department of Urology, Boston Children's Hospital, Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, MA, USA
| | - Aina He
- Department of Urology, Boston Children's Hospital, Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, MA, USA
- Department of Oncology, Affiliated Sixth People's Hospital, Shanghai Jiaotong University, No. 600, Yishan Road, 200233 Shanghai, PRC
| | - Kwok-Ho Lam
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA
| | - Zheng Liu
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA
| | - Xi He
- F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, MA, USA
| | - Kay Perry
- NE-CAT and Department of Chemistry and Chemical Biology, Cornell University, Argonne National Laboratory, Argonne, IL, USA
| | - Min Dong
- Department of Urology, Boston Children's Hospital, Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, MA, USA.
| | - Rongsheng Jin
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA.
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22
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Tamura Y, Takata K, Eguchi A, Kataoka Y. In vivo monitoring of hair cycle stages via bioluminescence imaging of hair follicle NG2 cells. Sci Rep 2018; 8:393. [PMID: 29321681 PMCID: PMC5762894 DOI: 10.1038/s41598-017-18763-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2017] [Accepted: 12/15/2017] [Indexed: 12/17/2022] Open
Abstract
Hair growth occurs periodically in a cycle that consists of three different phases: growth, regression, and resting. The length of each phase is regulated by both intrinsic and extrinsic factors throughout life, and influenced by physiological and pathological conditions. Elongation of the resting phase and shortening of the growth phase occur during physiological ageing and in baldness, respectively. In vivo discrimination of each phase of the hair cycle can be used to research for regeneration of hair follicles as well as to evaluate the efficacy of hair regrowth treatments in the same individual. Here we show that NG2+ epithelial cells in the hair follicles encompass bulge stem cells, and that the number of hair follicle NG2 cells underwent dramatic changes during the hair cycle. Transgenic rats with expression of firefly luciferase gene in NG2 cells were generated to monitor the hair cycle in vivo. Hair follicle NG2 cells were clearly visualized via bioluminescence imaging to study each phase of the hair cycle in the rats, from infancy to old age.
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Affiliation(s)
- Yasuhisa Tamura
- Cellular Function Imaging Team, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan. .,Multi-Modal Microstructure Analysis Unit, RIKEN CLST-JEOL Collaboration Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan.
| | - Kumi Takata
- Cellular Function Imaging Team, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan
| | - Asami Eguchi
- Cellular Function Imaging Team, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan.,Multi-Modal Microstructure Analysis Unit, RIKEN CLST-JEOL Collaboration Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan
| | - Yosky Kataoka
- Cellular Function Imaging Team, RIKEN Center for Life Science Technologies, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan.,Multi-Modal Microstructure Analysis Unit, RIKEN CLST-JEOL Collaboration Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan
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23
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Chandrasekaran R, Lacy DB. The role of toxins in Clostridium difficile infection. FEMS Microbiol Rev 2017; 41:723-750. [PMID: 29048477 PMCID: PMC5812492 DOI: 10.1093/femsre/fux048] [Citation(s) in RCA: 232] [Impact Index Per Article: 29.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2017] [Accepted: 10/10/2017] [Indexed: 02/06/2023] Open
Abstract
Clostridium difficile is a bacterial pathogen that is the leading cause of nosocomial antibiotic-associated diarrhea and pseudomembranous colitis worldwide. The incidence, severity, mortality and healthcare costs associated with C. difficile infection (CDI) are rising, making C. difficile a major threat to public health. Traditional treatments for CDI involve use of antibiotics such as metronidazole and vancomycin, but disease recurrence occurs in about 30% of patients, highlighting the need for new therapies. The pathogenesis of C. difficile is primarily mediated by the actions of two large clostridial glucosylating toxins, toxin A (TcdA) and toxin B (TcdB). Some strains produce a third toxin, the binary toxin C. difficile transferase, which can also contribute to C. difficile virulence and disease. These toxins act on the colonic epithelium and immune cells and induce a complex cascade of cellular events that result in fluid secretion, inflammation and tissue damage, which are the hallmark features of the disease. In this review, we summarize our current understanding of the structure and mechanism of action of the C. difficile toxins and their role in disease.
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Affiliation(s)
- Ramyavardhanee Chandrasekaran
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
| | - D. Borden Lacy
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
- The Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN 37232, USA
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24
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Gupta P, Zhang Z, Sugiman-Marangos SN, Tam J, Raman S, Julien JP, Kroh HK, Lacy DB, Murgolo N, Bekkari K, Therien AG, Hernandez LD, Melnyk RA. Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants. J Biol Chem 2017; 292:17290-17301. [PMID: 28842504 DOI: 10.1074/jbc.m117.806687] [Citation(s) in RCA: 47] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2017] [Revised: 08/11/2017] [Indexed: 12/11/2022] Open
Abstract
Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.
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Affiliation(s)
- Pulkit Gupta
- From Merck & Co., Inc., Kenilworth, New Jersey 07033
| | - Zhifen Zhang
- Molecular Medicine, The Hospital for Sick Children, Ontario M5G 0A4, Canada.,the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and
| | | | - John Tam
- Molecular Medicine, The Hospital for Sick Children, Ontario M5G 0A4, Canada
| | - Swetha Raman
- Molecular Medicine, The Hospital for Sick Children, Ontario M5G 0A4, Canada
| | - Jean-Phillipe Julien
- Molecular Medicine, The Hospital for Sick Children, Ontario M5G 0A4, Canada.,the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and
| | - Heather K Kroh
- the Departments of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37212
| | - D Borden Lacy
- the Departments of Pathology, Microbiology, and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37212
| | | | | | | | | | - Roman A Melnyk
- Molecular Medicine, The Hospital for Sick Children, Ontario M5G 0A4, Canada, .,the Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada, and
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25
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Amino Acid Differences in the 1753-to-1851 Region of TcdB Influence Variations in TcdB1 and TcdB2 Cell Entry. mSphere 2017; 2:mSphere00268-17. [PMID: 28776043 PMCID: PMC5541160 DOI: 10.1128/msphere.00268-17] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2017] [Accepted: 07/13/2017] [Indexed: 01/24/2023] Open
Abstract
Clostridium difficile TcdB2 enters cells with a higher efficiency than TcdB1 and exhibits an overall higher level of toxicity. However, the TcdB2-specific sequences that account for more efficient cell entry have not been reported. In this study, we examined the contribution of carboxy-terminal sequence differences to TcdB activity by comparing the binding, uptake, and endosomal localization of TcdB1 and TcdB2 or selected recombinant fragments of these proteins. Our findings suggest that sequence differences in the amino acid 1753 to 1851 region proximal to the combined repetitive oligopeptide domain (CROP) support enhanced uptake of TcdB2 and localization of toxin in acidified endosomes. In the absence of this region, the CROP domains of both forms of the toxin exhibited similar levels of cell interaction, while the addition of amino acids 1753 to 1851 greatly increased toxin binding by only TcdB2. Moreover, the amino acid 1753 to 2366 fragment of TcdB2, but not TcdB1, accumulated to detectable levels in acidified endosomes. Unexpectedly, we discovered an unusual relationship between endocytosis and the efficiency of cell binding for TcdB1 and TcdB2 wherein inhibition of endocytosis by a chemical inhibitor or incubation at a low temperature resulted in a dramatic reduction in cell binding. These findings provide information on sequence variations that may contribute to differences in TcdB1 and TcdB2 toxicity and reveal a heretofore unknown connection between endocytosis and cell binding for this toxin. IMPORTANCE TcdB is a major virulence factor produced by Clostridium difficile, a leading cause of antibiotic-associated diarrhea. Hypervirulent strains of C. difficile encode a variant of TcdB (TcdB2) that is more toxic than toxin derived from historical strains (TcdB1). Though TcdB1 and TcdB2 exhibit 92% overall identity, a 99-amino-acid region previously associated with cell entry and spanning amino acids 1753 to 1851 has only 77% sequence identity. Results from the present study indicate that the substantial sequence variation in this region could contribute to the differences in cell entry between TcdB1 and TcdB2 and possibly explain TcdB2's heightened toxicity. Finally, during the course of these studies, an unusual aspect of TcdB cell entry was discovered wherein cell binding appeared to depend on endocytosis. These findings provide insight into TcdB's variant forms and their mechanisms of cell entry.
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26
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Orrell KE, Zhang Z, Sugiman-Marangos SN, Melnyk RA. Clostridium difficile toxins A and B: Receptors, pores, and translocation into cells. Crit Rev Biochem Mol Biol 2017; 52:461-473. [DOI: 10.1080/10409238.2017.1325831] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Affiliation(s)
- Kathleen E. Orrell
- Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ON, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | - Zhifen Zhang
- Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ON, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
| | | | - Roman A. Melnyk
- Molecular Medicine Program, The Hospital for Sick Children Research Institute, Toronto, ON, Canada
- Department of Biochemistry, University of Toronto, Toronto, ON, Canada
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27
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Tao L, Zhang J, Meraner P, Tovaglieri A, Wu X, Gerhard R, Zhang X, Stallcup WB, Miao J, He X, Hurdle JG, Breault DT, Brass AL, Dong M. Frizzled proteins are colonic epithelial receptors for C. difficile toxin B. Nature 2016; 538:350-355. [PMID: 27680706 PMCID: PMC5519134 DOI: 10.1038/nature19799] [Citation(s) in RCA: 217] [Impact Index Per Article: 24.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2016] [Accepted: 08/26/2016] [Indexed: 02/06/2023]
Abstract
Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR-Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7-knockout mice, combined with knockdown of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7-knockout mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.
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Affiliation(s)
- Liang Tao
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.,Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Jie Zhang
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.,Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Paul Meraner
- Department of Microbiology and Physiological Systems (MaPS), University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
| | - Alessio Tovaglieri
- Division of Endocrinology, Boston Children's Hospital, Boston, Massachusetts 02115, USA
| | - Xiaoqian Wu
- Center for Infectious and Inflammatory Diseases, Texas A &M Health Science Center, Houston, Texas 77030, USA
| | - Ralf Gerhard
- Institute of Toxicology, Hannover Medical School, 30625 Hannover, Germany
| | - Xinjun Zhang
- The F. M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.,Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - William B Stallcup
- Tumor Microenvironment and Cancer Immunology Program, Sanford-Burnham Prebys Medical Discovery Institute, Cancer Center, La Jolla, California 92037, USA
| | - Ji Miao
- Division of Endocrinology, Boston Children's Hospital, Boston, Massachusetts 02115, USA.,Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Xi He
- The F. M. Kirby Neurobiology Center, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.,Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115, USA
| | - Julian G Hurdle
- Center for Infectious and Inflammatory Diseases, Texas A &M Health Science Center, Houston, Texas 77030, USA
| | - David T Breault
- Division of Endocrinology, Boston Children's Hospital, Boston, Massachusetts 02115, USA.,Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.,Harvard Stem Cell Institute, Cambridge, Massachusetts 02138, USA
| | - Abraham L Brass
- Department of Microbiology and Physiological Systems (MaPS), University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.,Gastroenterology Division, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA
| | - Min Dong
- Department of Urology, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.,Department of Microbiology and Immunobiology and Department of Surgery, Harvard Medical School, Boston, Massachusetts 02115, USA
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28
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Abstract
Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota.
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Affiliation(s)
- Wiep Klaas Smits
- Section Experimental Bacteriology, Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
| | - Dena Lyras
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Victoria, Australia
| | - D. Borden Lacy
- Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, and The Veterans Affairs Tennessee Valley Healthcare System, Nashville Tennessee, USA
| | - Mark H. Wilcox
- Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK
| | - Ed J. Kuijper
- Section Experimental Bacteriology, Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
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29
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Gerhard R. Receptors and Binding Structures for Clostridium difficile Toxins A and B. Curr Top Microbiol Immunol 2016; 406:79-96. [PMID: 27380268 DOI: 10.1007/82_2016_17] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Two characteristics of toxins A and B from C. difficile (TcdA, TcdB) are important for the understanding of the pathogenic effect of these homologous toxins. First, these toxins are huge single-chain but multidomain proteins that display their action intracellularly within the cytosol of host cells. And second, albeit various cell types highly differ in their sensitivity toward these toxins, no toxin-resistant cell type has been described yet. Investigation of receptor-mediated uptake of these toxins is very ambitious. It demands discrimination between cell surface binding, interaction with more than one functional receptor responsible for uptake as well as other functional receptors that recognize bacterial pathogens and are not necessarily related with endocytosis. The current understanding of a complex uptake process is that TcdB interacts with at least two facultative receptors that mediate entry into host cells by redundant endocytotic pathways. Although both homologous toxins do obviously not share the same receptors, this principle of redundant binding domains found for TcdB does also account for TcdA.
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Affiliation(s)
- Ralf Gerhard
- Institut für Toxikologie, Medizinische Hochschule, Hannover, Germany.
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30
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She ZG, Chang Y, Pang HB, Han W, Chen HZ, Smith JW, Stallcup WB. NG2 Proteoglycan Ablation Reduces Foam Cell Formation and Atherogenesis via Decreased Low-Density Lipoprotein Retention by Synthetic Smooth Muscle Cells. Arterioscler Thromb Vasc Biol 2016; 36:49-59. [PMID: 26543095 PMCID: PMC4690796 DOI: 10.1161/atvbaha.115.306074] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2015] [Accepted: 10/24/2015] [Indexed: 12/30/2022]
Abstract
OBJECTIVES Obesity and hyperlipidemia are critical risk factors for atherosclerosis. Because ablation of NG2 proteoglycan in mice leads to hyperlipidemia and obesity, we investigated the impact of NG2 ablation on atherosclerosis in apoE null mice. APPROACH AND RESULTS Immunostaining indicates that NG2 expression in plaque, primarily by synthetic smooth muscle cells, increases during atherogenesis. NG2 ablation unexpectedly results in decreased (30%) plaque development, despite aggravated obesity and hyperlipidemia. Mechanistic studies reveal that NG2-positive plaque synthetic smooth muscle cells in culture can sequester low-density lipoprotein to enhance foam-cell formation, processes in which NG2 itself plays direct roles. In agreement with these observations, low-density lipoprotein retention and lipid accumulation in the NG2/ApoE knockout aorta is 30% less than that seen in the control aorta. CONCLUSIONS These results indicate that synthetic smooth muscle cell-dependent low-density lipoprotein retention and foam cell formation outweigh obesity and hyperlipidemia in promoting mouse atherogenesis. Our study sheds new light on the role of synthetic smooth muscle cells during atherogenesis. Blocking plaque NG2 or altering synthetic smooth muscle cells function may be promising therapeutic strategies for atherosclerosis.
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MESH Headings
- Animals
- Antigens/genetics
- Aorta/metabolism
- Aorta/pathology
- Aortic Diseases/genetics
- Aortic Diseases/metabolism
- Aortic Diseases/pathology
- Aortic Diseases/prevention & control
- Apolipoproteins E/deficiency
- Apolipoproteins E/genetics
- Atherosclerosis/genetics
- Atherosclerosis/metabolism
- Atherosclerosis/pathology
- Atherosclerosis/prevention & control
- Cells, Cultured
- Diet, High-Fat
- Disease Models, Animal
- Female
- Foam Cells/metabolism
- Foam Cells/pathology
- Hyperlipidemias/genetics
- Hyperlipidemias/metabolism
- Lipoproteins, LDL/metabolism
- Male
- Mice, Inbred C57BL
- Mice, Knockout
- Muscle, Smooth, Vascular/metabolism
- Muscle, Smooth, Vascular/pathology
- Myocytes, Smooth Muscle/metabolism
- Myocytes, Smooth Muscle/pathology
- Obesity/genetics
- Obesity/metabolism
- Plaque, Atherosclerotic
- Proteoglycans/deficiency
- Proteoglycans/genetics
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Affiliation(s)
- Zhi-Gang She
- From the Tumor Microenvironment and Cancer Immunology Program, Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (Z.-G.S., Y.C., H.-B.P., W.H., J.W.S., W.B.S.); and Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing, Republic of China (H.-Z.C.).
| | - Yunchao Chang
- From the Tumor Microenvironment and Cancer Immunology Program, Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (Z.-G.S., Y.C., H.-B.P., W.H., J.W.S., W.B.S.); and Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing, Republic of China (H.-Z.C.)
| | - Hong-Bo Pang
- From the Tumor Microenvironment and Cancer Immunology Program, Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (Z.-G.S., Y.C., H.-B.P., W.H., J.W.S., W.B.S.); and Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing, Republic of China (H.-Z.C.)
| | - Wenlong Han
- From the Tumor Microenvironment and Cancer Immunology Program, Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (Z.-G.S., Y.C., H.-B.P., W.H., J.W.S., W.B.S.); and Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing, Republic of China (H.-Z.C.)
| | - Hou-Zao Chen
- From the Tumor Microenvironment and Cancer Immunology Program, Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (Z.-G.S., Y.C., H.-B.P., W.H., J.W.S., W.B.S.); and Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing, Republic of China (H.-Z.C.)
| | - Jeffrey W Smith
- From the Tumor Microenvironment and Cancer Immunology Program, Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (Z.-G.S., Y.C., H.-B.P., W.H., J.W.S., W.B.S.); and Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing, Republic of China (H.-Z.C.)
| | - William B Stallcup
- From the Tumor Microenvironment and Cancer Immunology Program, Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA (Z.-G.S., Y.C., H.-B.P., W.H., J.W.S., W.B.S.); and Department of Biochemistry and Molecular Biology, State Key Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Beijing, Republic of China (H.-Z.C.)
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Bernier-Latmani J, Cisarovsky C, Demir CS, Bruand M, Jaquet M, Davanture S, Ragusa S, Siegert S, Dormond O, Benedito R, Radtke F, Luther SA, Petrova TV. DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport. J Clin Invest 2015; 125:4572-86. [PMID: 26529256 DOI: 10.1172/jci82045] [Citation(s) in RCA: 140] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2015] [Accepted: 09/30/2015] [Indexed: 02/06/2023] Open
Abstract
The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature.
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Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity. Proc Natl Acad Sci U S A 2015; 112:7073-8. [PMID: 26038560 DOI: 10.1073/pnas.1500791112] [Citation(s) in RCA: 129] [Impact Index Per Article: 12.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection.
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Yuan P, Zhang H, Cai C, Zhu S, Zhou Y, Yang X, He R, Li C, Guo S, Li S, Huang T, Perez-Cordon G, Feng H, Wei W. Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B. Cell Res 2014; 25:157-68. [PMID: 25547119 PMCID: PMC4650570 DOI: 10.1038/cr.2014.169] [Citation(s) in RCA: 134] [Impact Index Per Article: 12.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2014] [Revised: 11/06/2014] [Accepted: 11/19/2014] [Indexed: 02/06/2023] Open
Abstract
As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.
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Affiliation(s)
- Pengfei Yuan
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Hongmin Zhang
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Changzu Cai
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Shiyou Zhu
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Yuexin Zhou
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Xiaozhou Yang
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Ruina He
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Chan Li
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Shengjie Guo
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
| | - Shan Li
- School of Bioscience and Biotechnology, South China University of Technology, Guangzhou, Guangdong 510006, China
| | - Tuxiong Huang
- Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland 21201, USA
| | - Gregorio Perez-Cordon
- Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland 21201, USA
| | - Hanping Feng
- Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland 21201, USA
| | - Wensheng Wei
- Biodynamic Optical Imaging Center (BIOPIC), State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871, China
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Marriott S, Baskir RS, Gaskill C, Menon S, Carrier EJ, Williams J, Talati M, Helm K, Alford CE, Kropski JA, Loyd J, Wheeler L, Johnson J, Austin E, Nozik-Grayck E, Meyrick B, West JD, Klemm DJ, Majka SM. ABCG2pos lung mesenchymal stem cells are a novel pericyte subpopulation that contributes to fibrotic remodeling. Am J Physiol Cell Physiol 2014; 307:C684-98. [PMID: 25122876 PMCID: PMC4200000 DOI: 10.1152/ajpcell.00114.2014] [Citation(s) in RCA: 67] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2014] [Accepted: 08/05/2014] [Indexed: 01/13/2023]
Abstract
Genesis of myofibroblasts is obligatory for the development of pathology in many adult lung diseases. Adult lung tissue contains a population of perivascular ABCG2(pos) mesenchymal stem cells (MSC) that are precursors of myofibroblasts and distinct from NG2 pericytes. We hypothesized that these MSC participate in deleterious remodeling associated with pulmonary fibrosis (PF) and associated hypertension (PH). To test this hypothesis, resident lung MSC were quantified in lung samples from control subjects and PF patients. ABCG2(pos) cell numbers were decreased in human PF and interstitial lung disease compared with control samples. Genetic labeling of lung MSC in mice enabled determination of terminal lineage and localization of ABCG2 cells following intratracheal administration of bleomycin to elicit fibrotic lung injury. Fourteen days following bleomycin injury enhanced green fluorescent protein (eGFP)-labeled lung MSC-derived cells were increased in number and localized to interstitial areas of fibrotic and microvessel remodeling. Finally, gene expression analysis was evaluated to define the response of MSC to bleomycin injury in vivo using ABCG2(pos) MSC isolated during the inflammatory phase postinjury and in vitro bleomycin or transforming growth factor-β1 (TGF-β1)-treated cells. MSC responded to bleomycin treatment in vivo with a profibrotic gene program that was not recapitulated in vitro with bleomycin treatment. However, TGF-β1 treatment induced the appearance of a profibrotic myofibroblast phenotype in vitro. Additionally, when exposed to the profibrotic stimulus, TGF-β1, ABCG2, and NG2 pericytes demonstrated distinct responses. Our data highlight ABCG2(pos) lung MSC as a novel cell population that contributes to detrimental myofibroblast-mediated remodeling during PF.
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Affiliation(s)
- Shennea Marriott
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse
| | - Rubin S Baskir
- Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennesse
| | - Christa Gaskill
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse
| | - Swapna Menon
- Pulmonary Vascular Research Institute Kochi and AnalyzeDat Consulting Services, Kerala, India
| | - Erica J Carrier
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse
| | - Janice Williams
- Vanderbilt Ingram Cancer Center, Electron Microscopy-Cell Imaging Shared Resource, Vanderbilt University, Nashville, Tennessee
| | - Megha Talati
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse
| | - Karen Helm
- Cancer Center Flow Cytometry Shared Resource, University of Colorado, Aurora, Colorado
| | - Catherine E Alford
- Department of Pathology and Laboratory Medicine, Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee
| | - Jonathan A Kropski
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse
| | - James Loyd
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse
| | - Lisa Wheeler
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse
| | - Joyce Johnson
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee
| | - Eric Austin
- Department of Pediatrics, Vanderbilt University, Nashville, Tennessee
| | - Eva Nozik-Grayck
- Department of Pediatrics or Medicine, Pulmonary and Critical Care Medicine, Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado, Aurora, Colorado; and
| | - Barbara Meyrick
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse
| | - James D West
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse; Vanderbilt Pulmonary Circulation Center, Vanderbilt University, Nashville, Tennessee
| | - Dwight J Klemm
- Department of Pediatrics or Medicine, Pulmonary and Critical Care Medicine, Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado, Aurora, Colorado; and
| | - Susan M Majka
- Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University, Nashville, Tennesse; Vanderbilt Center for Stem Cell Biology, Vanderbilt University, Nashville, Tennessee; Vanderbilt Pulmonary Circulation Center, Vanderbilt University, Nashville, Tennessee; Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, Tennessee; Department of Cell and Developmental Biology, Vanderbilt University, Nashville, Tennesse;
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Kennedy‐Lydon TM, Crawford C, Wildman SSP, Peppiatt‐Wildman CM. Renal pericytes: regulators of medullary blood flow. Acta Physiol (Oxf) 2013; 207:212-25. [PMID: 23126245 PMCID: PMC3561688 DOI: 10.1111/apha.12026] [Citation(s) in RCA: 63] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2012] [Revised: 07/03/2012] [Accepted: 09/27/2012] [Indexed: 01/29/2023]
Abstract
Regulation of medullary blood flow (MBF) is essential in maintaining normal kidney function. Blood flow to the medulla is supplied by the descending vasa recta (DVR), which arise from the efferent arterioles of juxtamedullary glomeruli. DVR are composed of a continuous endothelium, intercalated with smooth muscle-like cells called pericytes. Pericytes have been shown to alter the diameter of isolated and in situ DVR in response to vasoactive stimuli that are transmitted via a network of autocrine and paracrine signalling pathways. Vasoactive stimuli can be released by neighbouring tubular epithelial, endothelial, red blood cells and neuronal cells in response to changes in NaCl transport and oxygen tension. The experimentally described sensitivity of pericytes to these stimuli strongly suggests their leading role in the phenomenon of MBF autoregulation. Because the debate on autoregulation of MBF fervently continues, we discuss the evidence favouring a physiological role for pericytes in the regulation of MBF and describe their potential role in tubulo-vascular cross-talk in this region of the kidney. Our review also considers current methods used to explore pericyte activity and function in the renal medulla.
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Affiliation(s)
| | - C. Crawford
- Medway School of Pharmacy The Universities of Kent and Greenwich at Medway Kent UK
| | - S. S. P. Wildman
- Medway School of Pharmacy The Universities of Kent and Greenwich at Medway Kent UK
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Chang Y, She ZG, Sakimura K, Roberts A, Kucharova K, Rowitch DH, Stallcup WB. Ablation of NG2 proteoglycan leads to deficits in brown fat function and to adult onset obesity. PLoS One 2012; 7:e30637. [PMID: 22295099 PMCID: PMC3266271 DOI: 10.1371/journal.pone.0030637] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/05/2011] [Accepted: 12/20/2011] [Indexed: 01/25/2023] Open
Abstract
Obesity is a major health problem worldwide. We are studying the causes and effects of obesity in C57Bl/6 mice following genetic ablation of NG2, a chondroitin sulfate proteoglycan widely expressed in progenitor cells and also in adipocytes. Although global NG2 ablation delays early postnatal adipogenesis in mouse skin, adult NG2 null mice are paradoxically heavier than wild-type mice, exhibiting larger white fat deposits. This adult onset obesity is not due to NG2-dependent effects on CNS function, since specific ablation of NG2 in oligodendrocyte progenitors yields the opposite phenotype; i.e. abnormally lean mice. Metabolic analysis reveals that, while activity and food intake are unchanged in global NG2 null mice, O(2) consumption and CO(2) production are decreased, suggesting a decrease in energy expenditure. Since brown fat plays important roles in regulating energy expenditure, we have investigated brown fat function via cold challenge and high fat diet feeding, both of which induce the adaptive thermogenesis that normally occurs in brown fat. In both tests, body temperatures in NG2 null mice are reduced compared to wild-type mice, indicating a deficit in brown fat function in the absence of NG2. In addition, adipogenesis in NG2 null brown pre-adipocytes is dramatically impaired compared to wild-type counterparts. Moreover, mRNA levels for PR domain containing 16 (PRDM16) and peroxisome proliferator-activated receptor γ coactivator (PGC)1-α, proteins important for brown adipocyte differentiation, are decreased in NG2 null brown fat deposits in vivo and NG2 null brown pre-adipocytes in vitro. Altogether, these results indicate that brown fat dysfunction in NG2 null mice results from deficits in the recruitment and/or development of brown pre-adipocytes. As a consequence, obesity in NG2 null mice may occur due to disruptions in brown fat-dependent energy homeostasis, with resulting effects on lipid storage in white adipocytes.
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Affiliation(s)
- Yunchao Chang
- Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.
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37
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Mifflin RC, Pinchuk IV, Saada JI, Powell DW. Intestinal myofibroblasts: targets for stem cell therapy. Am J Physiol Gastrointest Liver Physiol 2011; 300:G684-96. [PMID: 21252048 PMCID: PMC3094146 DOI: 10.1152/ajpgi.00474.2010] [Citation(s) in RCA: 114] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The subepithelial intestinal myofibroblast is an important cell orchestrating many diverse functions in the intestine and is involved in growth and repair, tumorigenesis, inflammation, and fibrosis. The myofibroblast is but one of several α-smooth muscle actin-positive (α-SMA(+)) mesenchymal cells present within the intestinal lamina propria, including vascular pericytes, bone marrow-derived stem cells (mesenchymal stem cells or hematopoietic stem cells), muscularis mucosae, and the lymphatic pericytes (colon) and organized smooth muscle (small intestine) associated with the lymphatic lacteals. These other mesenchymal cells perform many of the functions previously attributed to subepithelial myofibroblasts. This review discusses the definition of a myofibroblast and reconsiders whether the α-SMA(+) subepithelial cells in the intestine are myofibroblasts or other types of mesenchymal cells, i.e., pericytes. Current information about specific, or not so specific, molecular markers of lamina propria mesenchymal cells is reviewed, as well as the origins of intestinal myofibroblasts and pericytes in the intestinal lamina propria and their replenishment after injury. Current concepts and research on stem cell therapy for intestinal inflammation are summarized. Information about the stem cell origin of intestinal stromal cells may inform future stem cell therapies to treat human inflammatory bowel disease (IBD).
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Affiliation(s)
| | | | | | - D. W. Powell
- Departments of 1Internal Medicine and ,2Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas
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Abstract
The lack of effective conventional therapies for the treatment of advanced stage melanoma has stimulated interest in the development of novel strategies for the management of patients with malignant melanoma. Among them, immunotherapy has attracted much attention because of the potential role played by immunological events in the clinical course of melanoma. For many years, T cell-based immunotherapy has been emphasized in part because of the disappointing results of the monoclonal antibody (mAb)-based clinical trials conducted in the early 1980s and in part because of the postulated major role played by T cells in tumor growth control. More recently, mAb-based therapies have gained in popularity given their clinical and commercial success for a variety of malignant diseases. As a result, there has been increased interest in identifying and characterizing antibody-defined melanoma antigens. Among them, the chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA) or melanoma chondroitin sulfate proteoglycan (MCSP), has attracted much attention in recent years because of the growing experimental evidence that it fulfills two requirements for immunotherapy to be therapeutically effective: (1) targeting of cancer stem cells (CSC) and (2) development of combinatorial therapies to counteract the escape mechanisms driven by the genetic instability of tumor cells. With this in mind, in this chapter, we have reviewed recent information related to the distribution of CSPG4 on various types of tumors, including CSC, its expression on pericytes in the tumor microenvironment, its recognition by T cells, its role in cell biology as well as the potential mechanisms underlying the ability of CSPG4-specific immunity to control malignant cell growth.
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Abstract
The mesenchymal elements of the intestinal lamina propria reviewed here are the myofibroblasts, fibroblasts, mural cells (pericytes) of the vasculature, bone marrow-derived stromal stem cells, smooth muscle of the muscularis mucosae, and smooth muscle surrounding the lymphatic lacteals. These cells share similar marker molecules, origins, and coordinated biological functions previously ascribed solely to subepithelial myofibroblasts. We review the functional anatomy of intestinal mesenchymal cells and describe what is known about their origin in the embryo and their replacement in adults. As part of their putative role in intestinal mucosal morphogenesis, we consider the intestinal stem cell niche. Lastly, we review emerging information about myofibroblasts as nonprofessional immune cells that may be important as an alarm system for the gut and as a participant in peripheral immune tolerance.
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Affiliation(s)
- D.W. Powell
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-0764
- Department of Neuroscience and Cell Biology, University of Texas Medical Branch, Galveston, Texas 77555-0764
| | - I.V. Pinchuk
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-0764
| | - J.I. Saada
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-0764
| | - Xin Chen
- Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94143
| | - R.C. Mifflin
- Department of Internal Medicine, University of Texas Medical Branch, Galveston, Texas 77555-0764
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Boido M, Rupa R, Garbossa D, Fontanella M, Ducati A, Vercelli A. Embryonic and adult stem cells promote raphespinal axon outgrowth and improve functional outcome following spinal hemisection in mice. Eur J Neurosci 2009; 30:833-46. [PMID: 19712091 DOI: 10.1111/j.1460-9568.2009.06879.x] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
Abstract
Spinal cord injury (SCI) often results in permanent neurological deficits below the injury site. Serotonergic raphespinal projections promote functional recovery after SCI, but spontaneous regeneration of most severed axons is limited by the glial cyst and scar that form at the lesion site. Stem cell (SC) transplantation offers a promising approach for inducing regeneration through the damaged area. Here we compare the effects of transplantation of embryonic neural precursors (NPs) or adult mesenchymal SCs, both of which are potential candidates for SC therapy. The spinal cord was hemisected at the L2 neuromer in adult mice. Two weeks post-injury, we transplanted neural precursors or mesenchymal SCs into the cord, caudal to the hemisection. Injured mice without a graft served as controls. Mice were tested for functional recovery on a battery of motor tasks, then killed and analysed for survival of grafted cells, for effects of engraftment on the local cellular environment and for the sprouting of serotonergic axons. Both types of SCs survived and were integrated into the host tissue, but only the NPs expressed neuronal markers. All transplanted animals displayed an increased number of serotonin-positive fibres caudal to the hemisection, compared with untreated mice. And both cell types led to improved motor performance. These results point to a therapeutic potential for such cell grafting.
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Affiliation(s)
- Marina Boido
- Department of Anatomy, Pharmacology and Forensic Medicine, Torino, Italy.
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Kadoya K, Fukushi JI, Matsumoto Y, Yamaguchi Y, Stallcup WB. NG2 proteoglycan expression in mouse skin: altered postnatal skin development in the NG2 null mouse. J Histochem Cytochem 2007; 56:295-303. [PMID: 18040080 DOI: 10.1369/jhc.7a7349.2007] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
In early postnatal mouse skin, the NG2 proteoglycan is expressed in the subcutis, the dermis, the outer root sheath of hair follicles, and the basal keratinocyte layer of the epidermis. With further development, NG2 is most prominently expressed by stem cells in the hair follicle bulge region, as also observed in adult human skin. During telogen and anagen phases of the adult hair cycle, NG2 is also found in stem cell populations that reside in dermal papillae and the outer root sheaths of hair follicles. Ablation of NG2 produces alterations in both the epidermis and subcutis layers of neonatal skin. Compared with wild type, the NG2 null epidermis does not achieve its full thickness due to reduced proliferation of basal keratinocytes that serve as the stem cell population in this layer. Thickening of the subcutis is also delayed in NG2 null skin due to deficiencies in the adipocyte population.
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Affiliation(s)
- Kuniko Kadoya
- Burnham Institute for Medical Research, La Jolla, California, USA
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42
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Abstract
The progress in discerning the structure and function of cells and tissues in health and disease has been achieved to a large extent by the continued development of new reagents for histochemistry, the improvement of existing techniques and new imaging techniques. This review will highlight some advancements made in these fields.
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Taatjes DJ, Zuber C, Roth J. The histochemistry and cell biology vade mecum: a review of 2005–2006. Histochem Cell Biol 2006; 126:743-88. [PMID: 17149649 DOI: 10.1007/s00418-006-0253-7] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/03/2006] [Indexed: 02/07/2023]
Abstract
The procurement of new knowledge and understanding in the ever expanding discipline of cell biology continues to advance at a breakneck pace. The progress in discerning the physiology of cells and tissues in health and disease has been driven to a large extent by the continued development of new probes and imaging techniques. The recent introduction of semi-conductor quantum dots as stable, specific markers for both fluorescence light microscopy and electron microscopy, as well as a virtual treasure-trove of new fluorescent proteins, has in conjunction with newly introduced spectral imaging systems, opened vistas into the seemingly unlimited possibilities for experimental design. Although it oftentimes proves difficult to predict what the future will hold with respect to advances in disciplines such as cell biology and histochemistry, it is facile to look back on what has already occurred. In this spirit, this review will highlight some advancements made in these areas in the past 2 years.
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Affiliation(s)
- Douglas J Taatjes
- Department of Pathology, Microscopy Imaging Center, College of Medicine, University of Vermont, Burlington, VT 05405, USA.
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