1
|
Pan M, O'Flaherty S, Hibberd A, Gerdes S, Morovic W, Barrangou R. The curated Lactobacillus acidophilus NCFM genome provides insights into strain specificity and microevolution. BMC Genomics 2025; 26:1. [PMID: 39754036 PMCID: PMC11697832 DOI: 10.1186/s12864-024-11177-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2024] [Accepted: 12/20/2024] [Indexed: 01/07/2025] Open
Abstract
BACKGROUND The advent of next generation sequencing technologies has enabled a surge in the number of whole genome sequences in public databases, and our understanding of the composition and evolution of bacterial genomes. Besides model organisms and pathogens, some attention has been dedicated to industrial bacteria, notably members of the Lactobacillaceae family that are commonly studied and formulated as probiotic bacteria. Of particular interest is Lactobacillus acidophilus NCFM, an extensively studied strain that has been widely commercialized for decades and is being used for the delivery of vaccines and therapeutics. RESULTS Here, we revisit the L. acidophilus genome, which was sequenced twenty years ago, and determined the core and pan genomes of 114 publicly available L. acidophilus strains, spanning commercial isolates, academic strains and clones from the scientific literature. Results indicate a predictable high level of homogeneity within the species, but also reveal surprising mis-assemblies. Furthermore, by investigating twenty one available L. acidophilus NCFM-derived variants, we document overall genomic stability, with no observed genomic re-arrangement or inversions. CONCLUSION This study provides a comparative analysis of the currently available genomes for L. acidophilus and examines microevolution patterns for several strains derived from L. acidophilus NCFM, which revealed no to very few SNPs with strains sequenced at different points in time using different sequencing technologies and platforms. This re-affirms its suitability for industrial deployment as a probiotic and its use as an engineering chassis and delivery modality for novel biotherapeutics.
Collapse
Affiliation(s)
- Meichen Pan
- Department of Food, Bioprocessing, & Nutrition Sciences, North Carolina State University, Raleigh, NC, USA
| | - Sarah O'Flaherty
- Department of Food, Bioprocessing, & Nutrition Sciences, North Carolina State University, Raleigh, NC, USA
| | | | | | | | - Rodolphe Barrangou
- Department of Food, Bioprocessing, & Nutrition Sciences, North Carolina State University, Raleigh, NC, USA.
| |
Collapse
|
2
|
Xie Z, McAuliffe O, Jin YS, Miller MJ. Invited review: Genomic modifications of lactic acid bacteria and their applications in dairy fermentation. J Dairy Sci 2024; 107:8749-8764. [PMID: 38969005 DOI: 10.3168/jds.2024-24989] [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: 03/31/2024] [Accepted: 06/11/2024] [Indexed: 07/07/2024]
Abstract
Lactic acid bacteria (LAB) have a long history of safe use in milk fermentation and are generally recognized as health-promoting microorganisms when present in fermented foods. Lactic acid bacteria are also important components of the human intestinal microbiota and are widely used as probiotics. Considering their safe and health-beneficial properties, LAB are considered appropriate vehicles that can be genetically modified for food, industrial and pharmaceutical applications. Here, this review describes (1) the potential opportunities for application of genetically modified LAB strains in dairy fermentation and (2) the various genomic modification tools for LAB strains, such as random mutagenesis, adaptive laboratory evolution, conjugation, homologous recombination, recombineering, and CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein)-based genome engineering. Finally, this review also discusses the potential future developments of these genomic modification technologies and their applications in dairy fermentations.
Collapse
Affiliation(s)
- Zifan Xie
- Food Science and Human Nutrition, University of Illinois Urbana-Champaign, Urbana, IL 61801
| | - Olivia McAuliffe
- Teagasc Food Research Centre, Moorepark, Fermoy, Cork, Ireland P61 C996; School of Biological Sciences, Queen's University Belfast, Belfast, Northern Ireland BT9 5DL
| | - Yong-Su Jin
- Food Science and Human Nutrition, University of Illinois Urbana-Champaign, Urbana, IL 61801; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801
| | - Michael J Miller
- Food Science and Human Nutrition, University of Illinois Urbana-Champaign, Urbana, IL 61801; Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61801.
| |
Collapse
|
3
|
Zhu M, Frank MW, Radka CD, Jeanfavre S, Xu J, Tse MW, Pacheco JA, Kim JS, Pierce K, Deik A, Hussain FA, Elsherbini J, Hussain S, Xulu N, Khan N, Pillay V, Mitchell CM, Dong KL, Ndung'u T, Clish CB, Rock CO, Blainey PC, Bloom SM, Kwon DS. Vaginal Lactobacillus fatty acid response mechanisms reveal a metabolite-targeted strategy for bacterial vaginosis treatment. Cell 2024; 187:5413-5430.e29. [PMID: 39163861 PMCID: PMC11429459 DOI: 10.1016/j.cell.2024.07.029] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2023] [Revised: 05/15/2024] [Accepted: 07/18/2024] [Indexed: 08/22/2024]
Abstract
Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related lactobacilli, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the vaginal microbiota and enhances bacterial fitness by biochemically sequestering OA in a derivative form only ohyA-harboring organisms can exploit. OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro BV model, suggesting a metabolite-based treatment approach.
Collapse
Affiliation(s)
- Meilin Zhu
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA
| | - Matthew W Frank
- Department of Host-Microbe Interactions, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Christopher D Radka
- Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky, Lexington, KY, USA
| | | | - Jiawu Xu
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA
| | - Megan W Tse
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | | | - Jae Sun Kim
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA
| | - Kerry Pierce
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Amy Deik
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Fatima Aysha Hussain
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA
| | | | - Salina Hussain
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA
| | - Nondumiso Xulu
- HIV Pathogenesis Programme, The Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, South Africa
| | - Nasreen Khan
- HIV Pathogenesis Programme, The Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, South Africa
| | | | - Caroline M Mitchell
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Department of Obstetrics and Gynecology, Massachusetts General Hospital, Boston, MA, USA
| | - Krista L Dong
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Health Systems Trust, Durban, South Africa; Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA
| | - Thumbi Ndung'u
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; HIV Pathogenesis Programme, The Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, South Africa; Africa Health Research Institute, Durban, South Africa; Max Planck Institute for Infection Biology, Berlin, Germany; Division of Infection and Immunity, University College London, London, UK
| | - Clary B Clish
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Charles O Rock
- Department of Host-Microbe Interactions, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Paul C Blainey
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA; Broad Institute of MIT and Harvard, Cambridge, MA, USA; Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Cambridge, MA, USA.
| | - Seth M Bloom
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA.
| | - Douglas S Kwon
- Ragon Institute of MGH, MIT, and Harvard, Cambridge, MA, USA; Harvard Medical School, Boston, MA, USA; Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA.
| |
Collapse
|
4
|
Wang S, Liu Y, Guo H, Meng Y, Xiong W, Liu R, Yang C. Establishment of low-cost production platforms of polyhydroxyalkanoate bioplastics from Halomonas cupida J9. Biotechnol Bioeng 2024; 121:2106-2120. [PMID: 38587130 DOI: 10.1002/bit.28694] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2023] [Revised: 02/05/2024] [Accepted: 02/27/2024] [Indexed: 04/09/2024]
Abstract
Microbial production of polyhydroxyalkanoate (PHA) is greatly restricted by high production cost arising from high-temperature sterilization and expensive carbon sources. In this study, a low-cost PHA production platform was established from Halomonas cupida J9. First, a marker-less genome-editing system was developed in H. cupida J9. Subsequently, H. cupida J9 was engineered to efficiently utilize xylose for PHA biosynthesis by introducing a new xylose metabolism module and blocking xylonate production. The engineered strain J9UΔxylD-P8xylA has the highest PHA yield (2.81 g/L) obtained by Halomonas with xylose as the sole carbon source so far. This is the first report on the production of short- and medium-chain-length (SCL-co-MCL) PHA from xylose by Halomonas. Interestingly, J9UΔxylD-P8xylA was capable of efficiently utilizing glucose and xylose as co-carbon sources for PHA production. Furthermore, fed-batch fermentation of J9UΔxylD-P8xylA coupled to a glucose/xylose co-feeding strategy reached up to 12.57 g/L PHA in a 5-L bioreactor under open and unsterile condition. Utilization of corn straw hydrolysate as the carbon source by J9UΔxylD-P8xylA reached 7.0 g/L cell dry weight (CDW) and 2.45 g/L PHA in an open fermentation. In summary, unsterile production in combination with inexpensive feedstock highlights the potential of the engineered strain for the low-cost production of PHA from lignocellulose-rich agriculture waste.
Collapse
Affiliation(s)
- Siqi Wang
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China
| | - Yujie Liu
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China
| | - Hongfu Guo
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China
| | - Yan Meng
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China
| | - Weini Xiong
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China
| | - Ruihua Liu
- Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin, China
| | - Chao Yang
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin, China
| |
Collapse
|
5
|
Sagmeister T, Gubensäk N, Buhlheller C, Grininger C, Eder M, Ðordić A, Millán C, Medina A, Murcia PAS, Berni F, Hynönen U, Vejzović D, Damisch E, Kulminskaya N, Petrowitsch L, Oberer M, Palva A, Malanović N, Codée J, Keller W, Usón I, Pavkov-Keller T. The molecular architecture of Lactobacillus S-layer: Assembly and attachment to teichoic acids. Proc Natl Acad Sci U S A 2024; 121:e2401686121. [PMID: 38838019 PMCID: PMC11181022 DOI: 10.1073/pnas.2401686121] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2024] [Accepted: 04/26/2024] [Indexed: 06/07/2024] Open
Abstract
S-layers are crystalline arrays found on bacterial and archaeal cells. Lactobacillus is a diverse family of bacteria known especially for potential gut health benefits. This study focuses on the S-layer proteins from Lactobacillus acidophilus and Lactobacillus amylovorus common in the mammalian gut. Atomic resolution structures of Lactobacillus S-layer proteins SlpA and SlpX exhibit domain swapping, and the obtained assembly model of the main S-layer protein SlpA aligns well with prior electron microscopy and mutagenesis data. The S-layer's pore size suggests a protective role, with charged areas aiding adhesion. A highly similar domain organization and interaction network are observed across the Lactobacillus genus. Interaction studies revealed conserved binding areas specific for attachment to teichoic acids. The structure of the SlpA S-layer and the suggested incorporation of SlpX as well as its interaction with teichoic acids lay the foundation for deciphering its role in immune responses and for developing effective treatments for a variety of infectious and bacteria-mediated inflammation processes, opening opportunities for targeted engineering of the S-layer or lactobacilli bacteria in general.
Collapse
Affiliation(s)
- Theo Sagmeister
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
| | - Nina Gubensäk
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
| | | | | | - Markus Eder
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
| | - Anđela Ðordić
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
| | - Claudia Millán
- Structural Biology Unit, Institute of Molecular Biology of Barcelona, Spanish National Research Council, Barcelona08028, Spain
| | - Ana Medina
- Structural Biology Unit, Institute of Molecular Biology of Barcelona, Spanish National Research Council, Barcelona08028, Spain
| | - Pedro Alejandro Sánchez Murcia
- Laboratory of Computer-Aided Molecular Design, Division of Medicinal Chemistry, Otto-Loewi Research Center, Medical University of Graz, Graz, Austria8010
| | - Francesca Berni
- Department of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden2333, The Netherlands
| | - Ulla Hynönen
- Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, University of Helsinki, Helsinki00100, Finland
| | - Djenana Vejzović
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
| | - Elisabeth Damisch
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
| | | | - Lukas Petrowitsch
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
| | - Monika Oberer
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
- Field of Excellence BioHealth, University of Graz, Graz8010, Austria
- BioTechMed-Graz, University of Graz, Graz8010, Austria
| | - Airi Palva
- Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, University of Helsinki, Helsinki00100, Finland
| | - Nermina Malanović
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
- Field of Excellence BioHealth, University of Graz, Graz8010, Austria
- BioTechMed-Graz, University of Graz, Graz8010, Austria
| | - Jeroen Codée
- Department of Bio-Organic Synthesis, Leiden Institute of Chemistry, Leiden University, Leiden2333, The Netherlands
| | - Walter Keller
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
- Field of Excellence BioHealth, University of Graz, Graz8010, Austria
- BioTechMed-Graz, University of Graz, Graz8010, Austria
| | - Isabel Usón
- Structural Biology Unit, Institute of Molecular Biology of Barcelona, Spanish National Research Council, Barcelona08028, Spain
- Institució Catalana de Recerca i Estudis Avançats, Barcelona08003, Spain
| | - Tea Pavkov-Keller
- Institute of Molecular Biosciences, University of Graz, Graz, Austria8010
- Field of Excellence BioHealth, University of Graz, Graz8010, Austria
- BioTechMed-Graz, University of Graz, Graz8010, Austria
| |
Collapse
|
6
|
Han X, Chang L, Chen H, Zhao J, Tian F, Ross RP, Stanton C, van Sinderen D, Chen W, Yang B. Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve. Appl Environ Microbiol 2024; 90:e0207423. [PMID: 38319094 PMCID: PMC10952402 DOI: 10.1128/aem.02074-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2023] [Accepted: 01/14/2024] [Indexed: 02/07/2024] Open
Abstract
Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.
Collapse
Affiliation(s)
- Xiao Han
- State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China
- School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
| | - Lulu Chang
- State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China
- School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
| | - Haiqin Chen
- State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China
- School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
| | - Jianxin Zhao
- State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China
- School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
| | - Fengwei Tian
- State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China
- School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
- International Joint Research Center for Probiotics & Gut Health, Jiangnan University, Wuxi, Jiangsu, China
| | - R. Paul Ross
- International Joint Research Center for Probiotics & Gut Health, Jiangnan University, Wuxi, Jiangsu, China
- APC Microbiome Ireland, University College Cork, Cork, Ireland
| | - Catherine Stanton
- International Joint Research Center for Probiotics & Gut Health, Jiangnan University, Wuxi, Jiangsu, China
- Teagasc Food Research Centre, Moorepark, Fermoy, Cork, Ireland
| | | | - Wei Chen
- State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China
- School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
- National Engineering Research Center for Functional Food, Jiangnan University, Wuxi, Jiangsu, China
| | - Bo Yang
- State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi, Jiangsu, China
- School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, China
- International Joint Research Center for Probiotics & Gut Health, Jiangnan University, Wuxi, Jiangsu, China
| |
Collapse
|
7
|
Li F, Zhao H, Sui L, Yin F, Liu X, Guo G, Li J, Jiang Y, Cui W, Shan Z, Zhou H, Wang L, Qiao X, Tang L, Wang X, Li Y. Assessing immunogenicity of CRISPR-NCas9 engineered strain against porcine epidemic diarrhea virus. Appl Microbiol Biotechnol 2024; 108:248. [PMID: 38430229 PMCID: PMC10908614 DOI: 10.1007/s00253-023-12989-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2023] [Revised: 12/18/2023] [Accepted: 12/26/2023] [Indexed: 03/03/2024]
Abstract
Porcine epidemic diarrhea (PED) caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly infectious disease, resulting in substantial economic losses in the pig industry. Given that PEDV primarily infects the mucosal surfaces of the intestinal tract, it is crucial to improve the mucosal immunity to prevent viral invasion. Lactic acid bacteria (LAB) oral vaccines offer unique advantages and potential applications in combatting mucosal infectious diseases, making them an ideal approach for controlling PED outbreaks. However, traditional LAB oral vaccines use plasmids for exogenous protein expression and antibiotic genes as selection markers. Antibiotic genes can be diffused through transposition, transfer, or homologous recombination, resulting in the generation of drug-resistant strains. To overcome these issues, genome-editing technology has been developed to achieve gene expression in LAB genomes. In this study, we used the CRISPR-NCas9 system to integrate the PEDV S1 gene into the genome of alanine racemase-deficient Lactobacillus paracasei △Alr HLJ-27 (L. paracasei △Alr HLJ-27) at the thymidylate synthase (thyA) site, generating a strain, S1/△Alr HLJ-27. We conducted immunization assays in mice and piglets to evaluate the level of immune response and evaluated its protective effect against PEDV through challenge tests in piglets. Oral administration of the strain S1/△Alr HLJ-27 in mice and piglets elicited mucosal, humoral, and cellular immune responses. The strain also exhibited a certain level of resistance against PEDV infection in piglets. These results demonstrate the potential of S1/△Alr HLJ-27 as an oral vaccine candidate for PEDV control. KEY POINTS: • A strain S1/△Alr HLJ-27 was constructed as the candidate for an oral vaccine. • Immunogenicity response and challenge test was carried out to analyze the ability of the strain. • The strain S1/△Alr HLJ-27 could provide protection for piglets to a certain extent.
Collapse
Affiliation(s)
- Fengsai Li
- Hebei Key Laboratory of Preventive Veterinary Medicine, College of Animal Science and Technology, Hebei Normal University of Science and Technology, Qinhuangdao, 066004, China
| | - Haiyuan Zhao
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
| | - Ling Sui
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
| | - Fangjie Yin
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
| | - Xinzi Liu
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
| | - Guihai Guo
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
| | - Jiaxuan Li
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China
| | - Yanping Jiang
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China
| | - Wen Cui
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China
| | - Zhifu Shan
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China
| | - Han Zhou
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China
| | - Li Wang
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China
| | - Xinyuan Qiao
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China
| | - Lijie Tang
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China
| | - Xiaona Wang
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China.
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China.
| | - Yijing Li
- College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China.
- Heilongjiang Key Laboratory for Animal Disease Control and Pharmaceutical Development, Harbin, 150030, China.
| |
Collapse
|
8
|
Sundararaman A, Halami PM. Metabolic Engineering of Bifidobacterium sp. Using Genome Editing Techniques. GENOME EDITING IN BACTERIA (PART 1) 2024:88-105. [DOI: 10.2174/9789815165678124010008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
Abstract
The gut microbiome is significant in maintaining human health by
facilitating absorption and digestion in the intestine. Probiotics have diverse and
significant applications in the health sector, so probiotic strains require an
understanding of the genome-level organizations. Probiotics elucidate various
functional parameters that control their metabolic functions. Gut dysbiosis leads to
inflammatory bowel disease and other neurological disorders. The application of
probiotic bacteria to modulate the gut microbiota prevents diseases and has gained
large interest. In a recent decade, the development of modern tools in molecular
biology has led to the discovery of genome engineering. Synthetic biology approaches
provide information about diverse biosynthetic pathways and also facilitate novel
metabolic engineering approaches for probiotic strain improvement. The techniques
enable engineering probiotics with the desired functionalities to benefit human health.
This chapter describes the recent advances in probiotic strain improvement for
diagnostic and therapeutic applications via CRISPR-Cas tools. Also, the application of
probiotics, current challenges, and future perspectives in disease treatment are
discussed.
Collapse
Affiliation(s)
- Aravind Sundararaman
- Department of Microbiology and Fermentation Technology, CSIR- Central Food Technological
Research Institute, Mysuru-570020, India
| | - Prakash M. Halami
- Department of Microbiology and Fermentation Technology, CSIR- Central Food Technological
Research Institute, Mysuru-570020, India
| |
Collapse
|
9
|
Dorosky RJ, Schreier JE, Lola SL, Sava RL, Coryell MP, Akue A, KuKuruga M, Carlson PE, Dreher-Lesnick SM, Stibitz S. Nanobodies as potential tools for microbiological testing of live biotherapeutic products. AMB Express 2024; 14:9. [PMID: 38245586 PMCID: PMC10799837 DOI: 10.1186/s13568-023-01659-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2023] [Accepted: 12/23/2023] [Indexed: 01/22/2024] Open
Abstract
Nanobodies are highly specific binding domains derived from naturally occurring single chain camelid antibodies. Live biotherapeutic products (LBPs) are biological products containing preparations of live organisms, such as Lactobacillus, that are intended for use as drugs, i.e. to address a specific disease or condition. Demonstrating potency of multi-strain LBPs can be challenging. The approach investigated here is to use strain-specific nanobody reagents in LBP potency assays. Llamas were immunized with radiation-killed Lactobacillus jensenii or L. crispatus whole cell preparations. A nanobody phage-display library was constructed and panned against bacterial preparations to identify nanobodies specific for each species. Nanobody-encoding DNA sequences were subcloned and the nanobodies were expressed, purified, and characterized. Colony immunoblots and flow cytometry showed that binding by Lj75 and Lj94 nanobodies were limited to a subset of L. jensenii strains while binding by Lc38 and Lc58 nanobodies were limited to L. crispatus strains. Mass spectrometry was used to demonstrate that Lj75 specifically bound a peptidase of L. jensenii, and that Lc58 bound an S-layer protein of L. crispatus. The utility of fluorescent nanobodies in evaluating multi-strain LBP potency assays was assessed by evaluating a L. crispatus and L. jensenii mixture by fluorescence microscopy, flow cytometry, and colony immunoblots. Our results showed that the fluorescent nanobody labelling enabled differentiation and quantitation of the strains in mixture by these methods. Development of these nanobody reagents represents a potential advance in LBP testing, informing the advancement of future LBP potency assays and, thereby, facilitation of clinical investigation of LBPs.
Collapse
Affiliation(s)
- Robert J Dorosky
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA.
| | - Jeremy E Schreier
- Department of Marine Sciences, University of Georgia, Athens, GA, USA
| | - Stephanie L Lola
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA
| | - Rosa L Sava
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA
| | - Michael P Coryell
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA
| | - Adovi Akue
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA
| | - Mark KuKuruga
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA
| | - Paul E Carlson
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA
| | - Sheila M Dreher-Lesnick
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA
| | - Scott Stibitz
- Office of Vaccines Research and Review, Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD, USA
| |
Collapse
|
10
|
Zhu M, Frank MW, Radka CD, Jeanfavre S, Tse MW, Pacheco JA, Pierce K, Deik A, Xu J, Hussain S, Hussain FA, Xulu N, Khan N, Pillay V, Dong KL, Ndung’u T, Clish CB, Rock CO, Blainey PC, Bloom SM, Kwon DS. Vaginal Lactobacillus fatty acid response mechanisms reveal a novel strategy for bacterial vaginosis treatment. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.30.573720. [PMID: 38234804 PMCID: PMC10793477 DOI: 10.1101/2023.12.30.573720] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/19/2024]
Abstract
Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related species, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the human vaginal microbiota and sequesters OA in a derivative form that only ohyA-harboring organisms can exploit. Finally, OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro model of BV, suggesting a novel approach for treatment.
Collapse
Affiliation(s)
- Meilin Zhu
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
| | - Matthew W. Frank
- Department of Host-Microbe Interactions, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
| | - Christopher D. Radka
- Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky
| | | | - Megan W. Tse
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | | | - Kerry Pierce
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Amy Deik
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
| | - Jiawu Xu
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
| | - Salina Hussain
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
| | - Fatima Aysha Hussain
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
- Harvard Medical School, Boston, MA, USA
| | - Nondumiso Xulu
- HIV Pathogenesis Programme (HPP), The Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, South Africa
| | - Nasreen Khan
- HIV Pathogenesis Programme (HPP), The Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, South Africa
| | | | - Krista L. Dong
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
- Health Systems Trust, Durban, South Africa
- Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| | - Thumbi Ndung’u
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
- HIV Pathogenesis Programme (HPP), The Doris Duke Medical Research Institute, University of KwaZulu-Natal, Durban, South Africa
- Africa Health Research Institute (AHRI), Durban, South Africa
- Max Planck Institute for Infection Biology, Berlin, Germany
- Division of Infection and Immunity, University College London, London, UK
| | | | - Charles O. Rock
- Department of Host-Microbe Interactions, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA
- passed away on September 22, 2023
| | - Paul C. Blainey
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute of MIT and Harvard, Cambridge, MA, USA
- Koch Institute for Integrative Cancer Research at Massachusetts Institute of Technology, Cambridge, MA, USA
| | - Seth M. Bloom
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
- Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| | - Douglas S. Kwon
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
- Division of Infectious Diseases, Massachusetts General Hospital, Boston, MA, USA
- Harvard Medical School, Boston, MA, USA
| |
Collapse
|
11
|
Gajbhiye S, Gonzales ED, Toso DB, Kirk NA, Hickey WJ. Identification of NpdA as the protein forming the surface layer in Paracidovorax citrulli and evidence of its occurrence as a surface layer protein in diverse genera of the Betaproteobacteria and Gammaproteobacteria. Access Microbiol 2023; 5:000685.v3. [PMID: 38188235 PMCID: PMC10765051 DOI: 10.1099/acmi.0.000685.v3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2023] [Accepted: 11/13/2023] [Indexed: 01/09/2024] Open
Abstract
The phytopathogen Paracidovorax citrulli possesses an ortholog of a newly identified surface layer protein (SLP) termed NpdA but has not been reported to produce a surface layer (S-layer). This study had two objectives. First, to determine if P. citrulli formed an NpdA-based S-layer and, if so, assess the effects of S-layer formation on virulence, production of nanostructures termed nanopods, and other phenotypes. Second, to establish the distribution of npdA orthologs throughout the Pseudomonadota and examine selected candidate cultures for physical evidence of S-layer formation. Formation of an NpdA-based S-layer by P. citrulli AAC00-1 was confirmed by gene deletion mutagenesis (ΔnpdA), proteomics, and cryo-electron microscopy. There were no significant differences between the wild-type and mutant in virulence assays with detached watermelon fruit. Nanopods contiguous with S-layers of multiple biofilm cells were visualized by transmission electron microscopy. Orthologs of npdA were identified in 62 Betaproteobacteria species and 49 Gammaproteobacteria species. In phylogenetic analyses, NpdA orthologs largely segregated into distinct groups. Cryo-electron microscopy imaging revealed an NpdA-like S-layer in all but one of the 16 additional cultures examined. We conclude that NpdA represents a new family of SLP, forming an S-layer in P. citrulli and other Pseudomonadota. While the S-layer did not contribute to virulence in watermelon fruit, a potential role of the P. citrulli S-layer in another dimension of pathogenesis cannot be ruled out. Lastly, formation of cell-bridging nanopods in biofilms is a new property of S-layers; it remains to be determined if nanopods can mediate intercellular movement of materials.
Collapse
Affiliation(s)
- Shabda Gajbhiye
- Department of Bacteriology, University of Wisconsin, Madison, Wisconsin, USA
| | - Erin D Gonzales
- Department of Soil Science, University of Wisconsin, Madison, Wisconsin, USA
| | - Daniel B Toso
- Department of Soil Science, University of Wisconsin, Madison, Wisconsin, USA
- Present address: California Institute for Quantitative Biosciences, University of California, Berkeley, California, USA
| | - Natalie A Kirk
- Department of Soil Science, University of Wisconsin, Madison, Wisconsin, USA
- Present address: Department of Art and Art History, University of Utah, Salt Lake City, Utah, USA
| | - William J Hickey
- Department of Soil Science, University of Wisconsin, Madison, Wisconsin, USA
| |
Collapse
|
12
|
Gilfillan D, Vilander AC, Pan M, Goh YJ, O’Flaherty S, Feng N, Fox BE, Lang C, Greenberg HB, Abdo Z, Barrangou R, Dean GA. Lactobacillus acidophilus Expressing Murine Rotavirus VP8 and Mucosal Adjuvants Induce Virus-Specific Immune Responses. Vaccines (Basel) 2023; 11:1774. [PMID: 38140179 PMCID: PMC10747613 DOI: 10.3390/vaccines11121774] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Revised: 11/14/2023] [Accepted: 11/22/2023] [Indexed: 12/24/2023] Open
Abstract
Rotavirus diarrhea-associated illness remains a major cause of global death in children under five, attributable in part to discrepancies in vaccine performance between high- and low-middle-income countries. Next-generation probiotic vaccines could help bridge this efficacy gap. We developed a novel recombinant Lactobacillus acidophilus (rLA) vaccine expressing rotavirus antigens of the VP8* domain from the rotavirus EDIM VP4 capsid protein along with the adjuvants FimH and FliC. The upp-based counterselective gene-replacement system was used to chromosomally integrate FimH, VP8Pep (10 amino acid epitope), and VP8-1 (206 amino acid protein) into the L. acidophilus genome, with FliC expressed from a plasmid. VP8 antigen and adjuvant expression were confirmed by flow cytometry and Western blot. Rotavirus naïve adult BALB/cJ mice were orally immunized followed by murine rotavirus strain ECWT viral challenge. Antirotavirus serum IgG and antigen-specific antibody-secreting cell responses were detected in rLA-vaccinated mice. A day after the oral rotavirus challenge, fecal antigen shedding was significantly decreased in the rLA group. These results indicate that novel rLA constructs expressing VP8 can be successfully constructed and used to generate modest homotypic protection from rotavirus challenge in an adult murine model, indicating the potential for a probiotic next-generation vaccine construct against human rotavirus.
Collapse
Affiliation(s)
- Darby Gilfillan
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; (D.G.); (A.C.V.); (B.E.F.); (C.L.); (Z.A.)
| | - Allison C. Vilander
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; (D.G.); (A.C.V.); (B.E.F.); (C.L.); (Z.A.)
| | - Meichen Pan
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA; (M.P.); (Y.J.G.); (S.O.); (R.B.)
| | - Yong Jun Goh
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA; (M.P.); (Y.J.G.); (S.O.); (R.B.)
| | - Sarah O’Flaherty
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA; (M.P.); (Y.J.G.); (S.O.); (R.B.)
| | - Ningguo Feng
- Departments of Medicine and Microbiology and Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA (H.B.G.)
- VA Palo Alto Health Care System, Department of Veterans Affairs, Palo Alto, CA 94304, USA
| | - Bridget E. Fox
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; (D.G.); (A.C.V.); (B.E.F.); (C.L.); (Z.A.)
| | - Callie Lang
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; (D.G.); (A.C.V.); (B.E.F.); (C.L.); (Z.A.)
| | - Harry B. Greenberg
- Departments of Medicine and Microbiology and Immunology, School of Medicine, Stanford University, Stanford, CA 94305, USA (H.B.G.)
- VA Palo Alto Health Care System, Department of Veterans Affairs, Palo Alto, CA 94304, USA
| | - Zaid Abdo
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; (D.G.); (A.C.V.); (B.E.F.); (C.L.); (Z.A.)
| | - Rodolphe Barrangou
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA; (M.P.); (Y.J.G.); (S.O.); (R.B.)
| | - Gregg A. Dean
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; (D.G.); (A.C.V.); (B.E.F.); (C.L.); (Z.A.)
| |
Collapse
|
13
|
Recent advances in genetic tools for engineering probiotic lactic acid bacteria. Biosci Rep 2023; 43:232386. [PMID: 36597861 PMCID: PMC9842951 DOI: 10.1042/bsr20211299] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2022] [Revised: 12/19/2022] [Accepted: 01/03/2023] [Indexed: 01/05/2023] Open
Abstract
Synthetic biology has grown exponentially in the last few years, with a variety of biological applications. One of the emerging applications of synthetic biology is to exploit the link between microorganisms, biologics, and human health. To exploit this link, it is critical to select effective synthetic biology tools for use in appropriate microorganisms that would address unmet needs in human health through the development of new game-changing applications and by complementing existing technological capabilities. Lactic acid bacteria (LAB) are considered appropriate chassis organisms that can be genetically engineered for therapeutic and industrial applications. Here, we have reviewed comprehensively various synthetic biology techniques for engineering probiotic LAB strains, such as clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated genome editing, homologous recombination, and recombineering. In addition, we also discussed heterologous protein expression systems used in engineering probiotic LAB. By combining computational biology with genetic engineering, there is a lot of potential to develop next-generation synthetic LAB with capabilities to address bottlenecks in industrial scale-up and complex biologics production. Recently, we started working on Lactochassis project where we aim to develop next generation synthetic LAB for biomedical application.
Collapse
|
14
|
LeBlanc N, Charles TC. Bacterial genome reductions: Tools, applications, and challenges. Front Genome Ed 2022; 4:957289. [PMID: 36120530 PMCID: PMC9473318 DOI: 10.3389/fgeed.2022.957289] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2022] [Accepted: 07/29/2022] [Indexed: 11/16/2022] Open
Abstract
Bacterial cells are widely used to produce value-added products due to their versatility, ease of manipulation, and the abundance of genome engineering tools. However, the efficiency of producing these desired biomolecules is often hindered by the cells’ own metabolism, genetic instability, and the toxicity of the product. To overcome these challenges, genome reductions have been performed, making strains with the potential of serving as chassis for downstream applications. Here we review the current technologies that enable the design and construction of such reduced-genome bacteria as well as the challenges that limit their assembly and applicability. While genomic reductions have shown improvement of many cellular characteristics, a major challenge still exists in constructing these cells efficiently and rapidly. Computational tools have been created in attempts at minimizing the time needed to design these organisms, but gaps still exist in modelling these reductions in silico. Genomic reductions are a promising avenue for improving the production of value-added products, constructing chassis cells, and for uncovering cellular function but are currently limited by their time-consuming construction methods. With improvements to and the creation of novel genome editing tools and in silico models, these approaches could be combined to expedite this process and create more streamlined and efficient cell factories.
Collapse
Affiliation(s)
- Nicole LeBlanc
- Department of Biology, University of Waterloo, Waterloo, ON, Canada
- *Correspondence: Nicole LeBlanc,
| | - Trevor C. Charles
- Department of Biology, University of Waterloo, Waterloo, ON, Canada
- Metagenom Bio Life Science Inc., Waterloo, ON, Canada
| |
Collapse
|
15
|
Adácsi C, Kovács S, Pócsi I, Pusztahelyi T. Elimination of Deoxynivalenol, Aflatoxin B1, and Zearalenone by Gram-Positive Microbes (Firmicutes). Toxins (Basel) 2022; 14:toxins14090591. [PMID: 36136529 PMCID: PMC9501497 DOI: 10.3390/toxins14090591] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2022] [Revised: 08/21/2022] [Accepted: 08/24/2022] [Indexed: 11/16/2022] Open
Abstract
Mycotoxin contaminations in the feed and food chain are common. Either directly or indirectly, mycotoxins enter the human body through the consumption of food of plant and animal origin. Bacteria with a high mycotoxin elimination capability can reduce mycotoxin contamination in feed and food. Four Gram-positive endospore-forming bacteria (Bacillus thuringiensis AMK10/1, Lysinibacillus boronitolerans AMK9/1, Lysinibacillus fusiformis AMK10/2, and Rummeliibacillus suwonensis AMK9/2) were isolated from fermented forages and tested for their deoxynivalenol (DON), aflatoxin B1 (AFB1), and zearalenone (ZEA) elimination potentials. Notably, the contribution of bacterial cell wall fractions to the observed outstanding ZEA elimination rates was demonstrated; however, the ZEA elimination differed considerably within the tested group of Gram-positive bacteria. It is worth noting that the purified cell wall of L. boronitolerans AMK9/1, L. fusiformis AMK10/2 and B. thuringiensis AMK10/1 were highly efficient in eliminating ZEA and the teichoic acid fractions of B. thuringiensis AMK10/1, and L. fusiformis AMK10/2 could also be successfully used in ZEA binding. The ZEA elimination capacity of viable R. suwonensis AMK9/2 cells was outstanding (40%). Meanwhile, R. suwonensis AMK9/2 and L. boronitolerans AMK9/1 cells produced significant esterase activities, and ZEA elimination of the cell wall fractions of that species did not correlate with esterase activity. DON and AFB1 binding capabilities of the tested bacterial cells and their cell wall fractions were low, except for B. thuringiensis AMK10/1, where the observed high 64% AFB1 elimination could be linked to the surface layer (S-layer) fraction of the cell wall.
Collapse
Affiliation(s)
- Cintia Adácsi
- Doctoral School of Nutrition and Food Sciences, University of Debrecen, Böszörményi Str. 138, H-4032 Debrecen, Hungary
| | - Szilvia Kovács
- Central Laboratory of Agricultural and Food Products, Faculty of Agricultural and Food Sciences and Environmental Management, University of Debrecen, Böszörményi Str. 138, H-4032 Debrecen, Hungary
| | - István Pócsi
- Department of Molecular Biotechnology and Microbiology, Institute of Biotechnology, Faculty of Science and Technology, University of Debrecen, Egyetem Tér 1, H-4032 Debrecen, Hungary
| | - Tünde Pusztahelyi
- Central Laboratory of Agricultural and Food Products, Faculty of Agricultural and Food Sciences and Environmental Management, University of Debrecen, Böszörményi Str. 138, H-4032 Debrecen, Hungary
- Correspondence: ; Tel.: +36-20-210-9491
| |
Collapse
|
16
|
Development of a Markerless Deletion Mutagenesis System in Nitrate-Reducing Bacterium Rhodanobacter denitrificans. Appl Environ Microbiol 2022; 88:e0040122. [PMID: 35737807 PMCID: PMC9317963 DOI: 10.1128/aem.00401-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Rhodanobacter has been found as the dominant genus in aquifers contaminated with high concentrations of nitrate and uranium in Oak Ridge, TN, USA. The in situ stimulation of denitrification has been proposed as a potential method to remediate nitrate and uranium contamination. Among the Rhodanobacter species, Rhodanobacter denitrificans strains have been reported to be capable of denitrification and contain abundant metal resistance genes. However, due to the lack of a mutagenesis system in these strains, our understanding of the mechanisms underlying low-pH resistance and the ability to dominate in the contaminated environment remains limited. Here, we developed an in-frame markerless deletion system in two R. denitrificans strains. First, we optimized the growth conditions, tested antibiotic resistance, and determined appropriate transformation parameters in 10 Rhodanobacter strains. We then deleted the upp gene, which encodes uracil phosphoribosyltransferase, in R. denitrificans strains FW104-R3 and FW104-R5. The resulting strains were designated R3_Δupp and R5_Δupp and used as host strains for mutagenesis with 5-fluorouracil (5-FU) resistance as the counterselection marker to generate markerless deletion mutants. To test the developed protocol, the narG gene encoding nitrate reductase was knocked out in the R3_Δupp and R5_Δupp host strains. As expected, the narG mutants could not grow in anoxic medium with nitrate as the electron acceptor. Overall, these results show that the in-frame markerless deletion system is effective in two R. denitrificans strains, which will allow for future functional genomic studies in these strains furthering our understanding of the metabolic and resistance mechanisms present in Rhodanobacter species. IMPORTANCE Rhodanobacter denitrificans is capable of denitrification and is also resistant to toxic heavy metals and low pH. Accordingly, the presence of Rhodanobacter species at a particular environmental site is considered an indicator of nitrate and uranium contamination. These characteristics suggest its future potential application in bioremediation of nitrate or concurrent nitrate and uranium contamination in groundwater ecosystems. Due to the lack of genetic tools in this organism, the mechanisms of low-pH and heavy metal resistance in R. denitrificans strains remain elusive, which impedes its use in bioremediation strategies. Here, we developed a genome editing method in two R. denitrificans strains. This work marks a crucial step in developing Rhodanobacter as a model for studying the diverse mechanisms of low-pH and heavy metal resistance associated with denitrification.
Collapse
|
17
|
Li X, Fan X, Shi Z, Xu J, Cao Y, Zhang T, Pan D. AI-2E Family Transporter Protein in Lactobacillus acidophilus Exhibits AI-2 Exporter Activity and Relate With Intestinal Juice Resistance of the Strain. Front Microbiol 2022; 13:908145. [PMID: 35633722 PMCID: PMC9134010 DOI: 10.3389/fmicb.2022.908145] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Accepted: 04/12/2022] [Indexed: 11/18/2022] Open
Abstract
The function of the autoinducer-2 exporters (AI-2E) family transporter protein of Lactobacillus acidophilus is still unclear. The phylogenetic analysis was used to analyze the relationship between the AI-2E protein of the L. acidophilus CICC 6074 strain and other AI-2E family members. Escherichia coli KNabc strain was used to verify whether the protein has Na+ (Li+)/H+ antiporter activity. The AI-2E protein overexpression strain was constructed by using the pMG36e expression vector, and the overexpression efficiency was determined by real-time quantitative PCR. The vitality and AI-2 activity of L. acidophilus CICC 6074 strains were determined. The results showed that the AI-2E protein of Lactobacillus formed a single branch on the phylogenetic tree and was closer to the AI-2E family members whose function was AI-2 exporter group I. The expression of AI-2E protein in the E. coli KNabc strain did not recover the resistance of the bacteria to the saline environment. Overexpression of AI-2E protein in L. acidophilus CICC 6074 could promote the AI-2 secretion of L. acidophilus CICC 6074 strain and enhance their survival ability in intestinal juice.
Collapse
Affiliation(s)
- Xiefei Li
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Ningbo University, Ningbo, China
| | - Xiankang Fan
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Ningbo University, Ningbo, China
| | - Zihang Shi
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Ningbo University, Ningbo, China
| | - Jue Xu
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Ningbo University, Ningbo, China
| | - Yingying Cao
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Ningbo University, Ningbo, China
| | - Tao Zhang
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Ningbo University, Ningbo, China
| | - Daodong Pan
- State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Ningbo University, Ningbo, China
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Ningbo University, Ningbo, China
- *Correspondence: Daodong Pan
| |
Collapse
|
18
|
|
19
|
Kim DS, Park Y, Choi JW, Park SH, Cho ML, Kwok SK. Lactobacillus acidophilus Supplementation Exerts a Synergistic Effect on Tacrolimus Efficacy by Modulating Th17/Treg Balance in Lupus-Prone Mice via the SIGNR3 Pathway. Front Immunol 2021; 12:696074. [PMID: 34956169 PMCID: PMC8704231 DOI: 10.3389/fimmu.2021.696074] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2021] [Accepted: 11/19/2021] [Indexed: 11/13/2022] Open
Abstract
ObjectiveTacrolimus (Tac) is an immunosuppressant used in the treatment of systemic lupus erythematosus (SLE); however, it induces T cell subset imbalances by reducing regulatory T (Treg) cells. Lactobacillus acidophilus (LA) is reported to have therapeutic efficacy in immune-mediated diseases via T cell regulation.MethodsThis study investigated whether a combination therapy of LA and Tac improves the therapeutic efficacy of Tac by modulating T cell subset populations in an animal model of SLE. Eight-week-old MRL/lpr mice were orally administered with 5 mg/kg of Tac and/or 50 mg/kg of LA daily for 8 weeks. Cecal microbiota compositions, serum autoantibodies levels, the degree of proteinuria, histological changes in the kidney, and populations of various T cell subsets in the spleen were analyzed.ResultsMice presented with significant gut dysbiosis, which were subsequently recovered by the combination treatment of Tac and LA. Double negative T cells in the peripheral blood and spleens of MRL/lpr mice were significantly decreased by the combination therapy. The combination treatment reduced serum levels of anti-dsDNA antibodies and Immunoglobulin G2a, and renal pathology scores were also markedly alleviated. The combination therapy induced Treg cells and decreased T helper 17 (Th17) cells both in vitro and in vivo. In vitro treatment with LA induced the production of indoleamine-2,3-dioxygenase, programmed death-ligand 1, and interleukin-10 via the specific intracellular adhesion molecule-3 grabbing non-integrin homolog-related 3 receptor signals.ConclusionThe present findings indicate that LA augments the therapeutic effect of Tac and modulates Th17/Treg balance in a murine model of SLE.
Collapse
Affiliation(s)
- Da Som Kim
- The Rheumatism Research Center, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- Laboratory of Translational ImmunoMedicine, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- Department of Biomedicine & Health Sciences, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Youngjae Park
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Jeong-Won Choi
- The Rheumatism Research Center, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- Laboratory of Translational ImmunoMedicine, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Sung-Hwan Park
- The Rheumatism Research Center, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea
| | - Mi-La Cho
- The Rheumatism Research Center, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- Laboratory of Translational ImmunoMedicine, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- Department of Medical Lifescience, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- *Correspondence: Mi-La Cho, ; Seung-Ki Kwok,
| | - Seung-Ki Kwok
- The Rheumatism Research Center, Catholic Research Institute of Medical Science, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- Division of Rheumatology, Department of Internal Medicine, Seoul St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul, South Korea
- *Correspondence: Mi-La Cho, ; Seung-Ki Kwok,
| |
Collapse
|
20
|
Li Q, Zhang J, Yang J, Jiang Y, Yang S. Recent progress on n-butanol production by lactic acid bacteria. World J Microbiol Biotechnol 2021; 37:205. [PMID: 34698975 DOI: 10.1007/s11274-021-03173-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2021] [Accepted: 10/13/2021] [Indexed: 11/26/2022]
Abstract
n-Butanol is an essential chemical intermediate produced through microbial fermentation. However, its toxicity to microbial cells has limited its production to a great extent. The anaerobe lactic acid bacteria (LAB) are the most resistant to n-butanol, so it should be the first choice for improving n-butanol production. The present article aims to review the following aspects of n-butanol production by LAB: (1) the tolerance of LAB to n-butanol, including its tolerance level and potential tolerance mechanisms; (2) genome editing tools in the n-butanol-resistant LAB; (3) methods of LAB modification for n-butanol production and the production levels after modification. This review will provide a theoretical basis for further research on n-butanol production by LAB.
Collapse
Affiliation(s)
- Qi Li
- College of Life Sciences, Sichuan Normal University, Chengdu, 610101, China
- Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China
| | - Jieze Zhang
- Department of Chemistry, University of Southern California, Los Angeles, CA, 90089, USA
| | - Junjie Yang
- Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China
| | - Yu Jiang
- Huzhou Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China
- Shanghai Taoyusheng Biotechnology Company Ltd, Shanghai, 200032, China
| | - Sheng Yang
- Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai, 200032, China.
- Huzhou Center of Industrial Biotechnology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Huzhou, 313000, China.
| |
Collapse
|
21
|
Dorau R, Liu J, Solem C, Jensen PR. Metabolic Engineering of Lactic Acid Bacteria. Metab Eng 2021. [DOI: 10.1002/9783527823468.ch15] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
|
22
|
Lactobacillus bile salt hydrolase substrate specificity governs bacterial fitness and host colonization. Proc Natl Acad Sci U S A 2021; 118:2017709118. [PMID: 33526676 PMCID: PMC8017965 DOI: 10.1073/pnas.2017709118] [Citation(s) in RCA: 91] [Impact Index Per Article: 22.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
The transformation of bile acids (BAs) by the gut microbiota is increasingly recognized as an important factor shaping host health. The prerequisite step of BA metabolism is carried out by bile salt hydrolases (BSHs), which are encoded by select gut and probiotic bacteria. Despite their prevalence, the utility of harboring a bsh is unclear. Here, we investigate the role of BSHs encoded by Lactobacillus acidophilus and Lactobacillus gasseri. We show that BA type and BSH substrate preferences affect in vitro and in vivo growth of both species. These findings contribute to a mechanistic understanding of bacterial survival in various BA-rich niches and inform future efforts to leverage BSHs as a therapeutic tool for manipulating the gut microbiota. Primary bile acids (BAs) are a collection of host-synthesized metabolites that shape physiology and metabolism. BAs transit the gastrointestinal tract and are subjected to a variety of chemical transformations encoded by indigenous bacteria. The resulting microbiota-derived BA pool is a mediator of host–microbiota interactions. Bacterial bile salt hydrolases (BSHs) cleave the conjugated glycine or taurine from BAs, an essential upstream step for the production of deconjugated and secondary BAs. Probiotic lactobacilli harbor a considerable number and diversity of BSHs; however, their contribution to Lactobacillus fitness and colonization remains poorly understood. Here, we define and compare the functions of multiple BSHs encoded by Lactobacillus acidophilus and Lactobacillus gasseri. Our genetic and biochemical characterization of lactobacilli BSHs lend to a model of Lactobacillus adaptation to the gut. These findings deviate from previous notions that BSHs generally promote colonization and detoxify bile. Rather, we show that BSH enzymatic preferences and the intrinsic chemical features of various BAs determine the toxicity of these molecules during Lactobacillus growth. BSHs were able to alter the Lactobacillus transcriptome in a BA-dependent manner. Finally, BSHs were able to dictate differences in bacterial competition in vitro and in vivo, defining their impact on BSH-encoding bacteria within the greater gastrointestinal tract ecosystem. This work emphasizes the importance of considering the enzymatic preferences of BSHs alongside the conjugated/deconjugated BA–bacterial interaction. These results deepen our understanding of the BA–microbiome axis and provide a framework to engineer lactobacilli with improved bile resistance and use probiotics as BA-altering therapeutics.
Collapse
|
23
|
Zhao Y, Che Y, Zhang F, Wang J, Gao W, Zhang T, Yang C. Development of an efficient pathway construction strategy for rapid evolution of the biodegradation capacity of Pseudomonas putida KT2440 and its application in bioremediation. THE SCIENCE OF THE TOTAL ENVIRONMENT 2021; 761:143239. [PMID: 33158512 DOI: 10.1016/j.scitotenv.2020.143239] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/25/2020] [Revised: 10/11/2020] [Accepted: 10/16/2020] [Indexed: 06/11/2023]
Abstract
In this work, we developed an efficient pathway construction strategy, consisting of DNA assembler-assisted pathway assembly and counterselection system-based chromosomal integration, for the rapid and efficient integration of synthetic biodegradation pathways into the chromosome of Pseudomonas putida KT2440. Using this strategy, we created a novel degrader capable of complete mineralization of γ-hexachlorocyclohexane (γ-HCH) and 1,2,3-trichloropropane (TCP) by integrating γ-HCH and TCP biodegradation pathways into the chromosome of P. putida KT2440. Furthermore, the chromosomal integration efficiencies of γ-HCH and TCP biodegradation pathways were improved to 50% and 41.6% in P. putida KT2440, respectively, by the inactivation of a type I DNA restriction-modification system. The currently developed pathway construction strategy coupled with the mutant KTUΔhsdRMS will facilitate implantation of heterologous catabolic pathways into the chromosome for rapid evolution of the biodegradation capacity of P. putida. More importantly, the successful removal of γ-HCH (10 mg/kg soil) and TCP (0.2 mM) from soil and wastewater within 14 days, respectively, highlighted the potential of the novel degrader for in situ bioremediation of γ-HCH- and TCP-contaminated sites. Moreover, chromosomal integration of gfp made the degrader to be monitored easily during bioremediation. In the future, this strategy can be expanded to a broad range of bacterial species for widespread applications in bioremediation.
Collapse
Affiliation(s)
- Yuxin Zhao
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - You Che
- Environmental Microbiome Engineering and Biotechnology Laboratory, The University of Hong Kong, Hong Kong
| | - Fang Zhang
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Jiacheng Wang
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China
| | - Weixia Gao
- MOE Key Laboratory of Industrial Fermentation Microbiology, College of Life Sciences, Tianjin University of Science and Technology, Tianjin, China
| | - Tong Zhang
- Environmental Microbiome Engineering and Biotechnology Laboratory, The University of Hong Kong, Hong Kong
| | - Chao Yang
- Key Laboratory of Molecular Microbiology and Technology for Ministry of Education, College of Life Sciences, Nankai University, Tianjin 300071, China.
| |
Collapse
|
24
|
Portable CRISPR-Cas9 N System for Flexible Genome Engineering in Lactobacillus acidophilus, Lactobacillus gasseri, and Lactobacillus paracasei. Appl Environ Microbiol 2021; 87:AEM.02669-20. [PMID: 33397707 DOI: 10.1128/aem.02669-20] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2020] [Accepted: 12/10/2020] [Indexed: 12/18/2022] Open
Abstract
Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplement industries, and specific strains, such as Lactobacillus acidophilus NCFM, have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR (clustered regularly interspaced palindromic repeats)-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9 N under the regulation of a Lactobacillus promoter and a cloning region for a customized single guide RNA (sgRNA) and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS, and ltaS), yielding 35 to 100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted an mCherry fluorescent-protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate a concurrent single-base substitution and gene deletion in Lactobacillus gasseri ATCC 33323 and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered with by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli and open new avenues for the engineering of health-promoting lactic acid bacteria.IMPORTANCE This work describes the development of a lactobacillus CRISPR-based editing system for genome manipulations in three Lactobacillus species belonging to the lactic acid bacteria (LAB), which are commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiotas and for their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.
Collapse
|
25
|
Hartz P, Gehl M, König L, Bernhardt R, Hannemann F. Development and application of a highly efficient CRISPR-Cas9 system for genome engineering in Bacillus megaterium. J Biotechnol 2021; 329:170-179. [PMID: 33600891 DOI: 10.1016/j.jbiotec.2021.02.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Revised: 01/11/2021] [Accepted: 02/10/2021] [Indexed: 12/26/2022]
Abstract
Bacillus megaterium has become increasingly important for the biotechnological production of valuable compounds of industrial and pharmaceutical importance. Despite recent advances in rational strain design of B. megaterium, these studies have been largely impaired by the lack of molecular tools that are not state-of-the-art for comprehensive genome engineering approaches. In the current work, we describe the adaptation of the CRISPR-Cas9 vector pJOE8999 to enable efficient genome editing in B. megaterium. Crucial modifications comprise the exchange of promoter elements and associated ribosomal binding sites as well as the implementation of a 5-fluorouracil based counterselection system to facilitate proper plasmid curing. In addition, the functionality and performance of the new CRISPR-Cas9 vector pMOE was successfully evaluated by chromosomal disruption studies of the endogenous β-galactosidase gene (BMD_2126) and demonstrated an outstanding efficiency of 100 % based on combinatorial pheno- and genotype analyses. Furthermore, pMOE was applied for the genomic deletion of a steroid esterase gene (BMD_2256) that was identified among several other candidates as the gene encoding the esterase, which prevented accumulation of pharmaceutically important glucocorticoid esters. Recombinant expression of the bacterial chloramphenicol acetyltransferase 1 gene (cat1) in the resulting esterase deficient B. megaterium strain ultimately yielded C21-acetylated as well as novel C21-esterified derivates of cortisone.
Collapse
Affiliation(s)
- Philip Hartz
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany
| | - Manuel Gehl
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany; Present address: Microbial Protein Structure Group, Max Planck Institute for Terrestrial Microbiology, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany
| | - Lisa König
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany
| | - Rita Bernhardt
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany
| | - Frank Hannemann
- Department of Biochemistry, Saarland University, Campus Building B2.2, 66123 Saarbrücken, Germany.
| |
Collapse
|
26
|
Wakai T, Kano C, Karsens H, Kok J, Yamamoto N. Functional role of surface layer proteins of Lactobacillus acidophilus L-92 in stress tolerance and binding to host cell proteins. BIOSCIENCE OF MICROBIOTA FOOD AND HEALTH 2021; 40:33-42. [PMID: 33520567 PMCID: PMC7817507 DOI: 10.12938/bmfh.2020-005] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/29/2020] [Accepted: 08/08/2020] [Indexed: 02/02/2023]
Abstract
Lactobacillus acidophilus surface layer proteins (SLPs) self-assemble
into a monolayer that is non-covalently bound to the outer surface of the cells. There
they are in direct contact with the environment, environmental stressors and gut
components of the host in which the organism resides. The role of L.
acidophilus SLPs is not entirely understood, although SLPs seem to be essential
for bacterial growth. We constructed three L. acidophilus L-92 strains,
each expressing a mutant of the most abundant SLP, SlpA. Each carried a 12-amino acid
c-myc epitope substitution at a different position in the protein. A strain was also
obtained that expressed the SlpA paralog SlpB from an originally silent
slpB gene. All four strains behaved differently with respect to growth
under various stress conditions, such as the presence of salt, ox gall or ethanol,
suggesting that SlpA affects stress tolerance in L. acidophilus L-92.
Also, the four mutants showed differential in vitro binding ability to
human host cell proteins such as uromodulin or dendritic cell (DC)-specific intercellular
adhesion molecule-3 grabbing non-integrin (DC-SIGN). Furthermore, co-culture of murine
immature DCs with a mutant strain expressing one of the recombinant SlpA proteins changed
the concentrations of the cytokines IL-10 and IL-12. Our data suggest that SlpA and SlpB
of L. acidophilus participate in bacterial stress tolerance and binding
to uromodulin or DC-SIGN, possibly leading to effective immune-modification.
Collapse
Affiliation(s)
- Taketo Wakai
- Core Technology Laboratories, Asahi Quality and Innovations, Ltd., 5-11-10 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa, Japan
| | - Chie Kano
- Core Technology Laboratories, Asahi Quality and Innovations, Ltd., 5-11-10 Fuchinobe, Chuo-ku, Sagamihara-shi, Kanagawa, Japan
| | - Harma Karsens
- Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Linnaeusborg, Nijenborgh 7, Groningen, The Netherlands
| | - Jan Kok
- Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Linnaeusborg, Nijenborgh 7, Groningen, The Netherlands
| | - Naoyuki Yamamoto
- School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa, Japan
| |
Collapse
|
27
|
In Vivo Transcriptome of Lactobacillus acidophilus and Colonization Impact on Murine Host Intestinal Gene Expression. mBio 2021; 12:mBio.03399-20. [PMID: 33500337 PMCID: PMC7858073 DOI: 10.1128/mbio.03399-20] [Citation(s) in RCA: 18] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Lactobacillus acidophilus NCFM is a probiotic strain commonly used in dairy products and dietary supplements. Postgenome in vitro studies of NCFM thus far have linked potential key genotypes to its probiotic-relevant attributes, including gut survival, prebiotic utilization, host interactions, and immunomodulatory activities. To corroborate and extend beyond previous in vivo and in vitro functional studies, we employed a dual RNA sequencing (RNA-seq) transcriptomic approach to identify genes potentially driving the gut fitness and activities of L. acidophilus NCFM in vivo, and in parallel, examine the ileal transcriptional response of its murine hosts during monocolonization. Spatial expression profiling of NCFM from the ileum through the colon revealed a set of 134 core genes that were consistently overexpressed during gut transit. These in vivo core genes are predominantly involved in the metabolism of carbohydrates, amino acids, and nucleotides, along with mucus-binding proteins and adhesion factors, confirming their functionally important roles in nutrient acquisition and gut retention. Functional characterization of the highly expressed major S-layer-encoding gene established its indispensable role as a cell shape determinant and maintenance of cell surface integrity, essential for viability and probiotic attributes. Host colonization by L. acidophilus resulted in significant downregulation of several proinflammatory cytokines and tight junction proteins. Genes related to redox signaling, mucin glycosylation, and circadian rhythm modulation were induced, suggesting impacts on intestinal development and immune functions. Metagenomic analysis of NCFM populations postcolonization demonstrated the genomic stability of L. acidophilus as a gut transient and further established its safety as a probiotic and biotherapeutic delivery platform.IMPORTANCE To date, our basis for comprehending the probiotic mechanisms of Lactobacillus acidophilus, one of the most widely consumed probiotic microbes, was largely limited to in vitro functional genomic studies. Using a germfree murine colonization model, in vivo-based transcriptional studies provided the first view of how L. acidophilus survives in the mammalian gut environment, including gene expression patterns linked to survival, efficient nutrient acquisition, stress adaptation, and host interactions. Examination of the host ileal transcriptional response, the primary effector site of L. acidophilus, has also shed light into the mechanistic roles of this probiotic microbe in promoting anti-inflammatory responses, maintaining intestinal epithelial homeostasis and modulation of the circadian-metabolic axis in its host.
Collapse
|
28
|
Marcos-Fernández R, Ruiz L, Blanco-Míguez A, Margolles A, Sánchez B. Precision modification of the human gut microbiota targeting surface-associated proteins. Sci Rep 2021; 11:1270. [PMID: 33446697 PMCID: PMC7809461 DOI: 10.1038/s41598-020-80187-3] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2020] [Accepted: 12/14/2020] [Indexed: 01/29/2023] Open
Abstract
This work describes a new procedure that allows the targeted modification of the human gut microbiota by using antibodies raised against bacterial surface-associated proteins specific to the microorganism of interest. To this end, a polyclonal antibody recognising the surface-associated protein Surface Layer Protein A of Lactobacillus acidophilus DSM20079T was developed. By conjugating this antibody with fluorescent probes and magnetic particles, we were able to specifically identify this bacterium both in a synthetic, and in real gut microbiotas by means of a flow cytometry approach. Further, we demonstrated the applicability of this antibody to deplete complex human gut microbiotas from L. acidophilus in a single step. L. acidophilus was found to interact with other bacteria both in synthetic and in real microbiotas, as reflected by its concomitant depletion together with other species. Further optimization of the procedure including a trypsin step enabled to achieve the selective and complete isolation of this species. Depleting a single species from a gut microbiota, using antibodies recognizing specific cell surface elements of the target organism, will open up novel ways to tackle research on the specific immunomodulatory and metabolic contributions of a bacterium of interest in the context of a complex human gut microbiota, including the investigation into therapeutic applications by adding/depleting a key bacterium. This represents the first work in which an antibody/flow-cytometry based application enabled the targeted edition of human gut microbiotas, and represents the basis for the design of precision microbiome-based therapies.
Collapse
Affiliation(s)
- Raquel Marcos-Fernández
- Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias-Consejo Superior de Investigaciones Científicas (IPLA-CSIC), Paseo Río Linares s/n, 33300, Villaviciosa, Asturias, Spain
- Functionality and Ecology of Beneficial Microbes (MicroHealth) Group, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Asturias, Spain
| | - Lorena Ruiz
- Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias-Consejo Superior de Investigaciones Científicas (IPLA-CSIC), Paseo Río Linares s/n, 33300, Villaviciosa, Asturias, Spain
- Functionality and Ecology of Beneficial Microbes (MicroHealth) Group, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Asturias, Spain
| | - Aitor Blanco-Míguez
- Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias-Consejo Superior de Investigaciones Científicas (IPLA-CSIC), Paseo Río Linares s/n, 33300, Villaviciosa, Asturias, Spain
- Functionality and Ecology of Beneficial Microbes (MicroHealth) Group, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Asturias, Spain
| | - Abelardo Margolles
- Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias-Consejo Superior de Investigaciones Científicas (IPLA-CSIC), Paseo Río Linares s/n, 33300, Villaviciosa, Asturias, Spain.
- Functionality and Ecology of Beneficial Microbes (MicroHealth) Group, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Asturias, Spain.
| | - Borja Sánchez
- Department of Microbiology and Biochemistry of Dairy Products, Instituto de Productos Lácteos de Asturias-Consejo Superior de Investigaciones Científicas (IPLA-CSIC), Paseo Río Linares s/n, 33300, Villaviciosa, Asturias, Spain.
- Functionality and Ecology of Beneficial Microbes (MicroHealth) Group, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Asturias, Spain.
| |
Collapse
|
29
|
Zuo F, Marcotte H. Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing. Curr Opin Biotechnol 2021; 70:75-82. [PMID: 33445135 DOI: 10.1016/j.copbio.2020.12.015] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2020] [Revised: 12/02/2020] [Accepted: 12/17/2020] [Indexed: 12/01/2022]
Abstract
Typical traditional probiotics lactobacilli and bifidobacteria are gaining great interest to be developed as living diagnostics and therapeutics for improving human health. However, the mechanistic basis underlying their inherent health beneficial property remain incompletely understood which can slow down the translational pipeline in the functional food and pharmaceutical field. Efficient genome editing will advance the understanding of the molecular mechanism of the probiotics' physiological properties and their interaction with the host and the host microbiota, thereby further promote the development of next-generation designer probiotics with improved robustness and tailored functionalities. With the expansion of genome editing strategies such as CRISPR-Cas-based tools and IPSD assisted genome engineering as well as other synthetic biology technologies, the research and application of these health-promoting bacteria for the food and pharmaceutical industry will be further enhanced.
Collapse
Affiliation(s)
- Fanglei Zuo
- Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden; Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm SE-106 91, Sweden.
| | - Harold Marcotte
- Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden
| |
Collapse
|
30
|
Fan X, Zhang Y, Zhao F, Liu Y, Zhao Y, Wang S, Liu R, Yang C. Genome reduction enhances production of polyhydroxyalkanoate and alginate oligosaccharide in Pseudomonas mendocina. Int J Biol Macromol 2020; 163:2023-2031. [DOI: 10.1016/j.ijbiomac.2020.09.067] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2020] [Revised: 08/11/2020] [Accepted: 09/10/2020] [Indexed: 12/15/2022]
|
31
|
CRISPR-Cas-mediated gene editing in lactic acid bacteria. Mol Biol Rep 2020; 47:8133-8144. [PMID: 32926267 DOI: 10.1007/s11033-020-05820-w] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2020] [Accepted: 09/05/2020] [Indexed: 12/12/2022]
Abstract
The high efficiency, convenience and diversity of clustered regular interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are driving a technological revolution in the gene editing of lactic acid bacteria (LAB). Cas-RNA cassettes have been adopted as tools to perform gene deletion, insertion and point mutation in several species of LAB. In this article, we describe the basic mechanisms of the CRISPR-Cas system, and the current gene editing methods available, focusing on the CRISPR-Cas models developed for LAB. We also compare the different types of CRISPR-Cas-based genomic manipulations classified according to the different Cas proteins and the type of recombineering, and discuss the rapidly evolving landscape of CRISPR-Cas application in LAB.
Collapse
|
32
|
Rocha-Mendoza D, Kosmerl E, Miyagusuku-Cruzado G, Giusti MM, Jiménez-Flores R, García-Cano I. Growth of lactic acid bacteria in milk phospholipids enhances their adhesion to Caco-2 cells. J Dairy Sci 2020; 103:7707-7718. [DOI: 10.3168/jds.2020-18271] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2020] [Accepted: 05/03/2020] [Indexed: 01/09/2023]
|
33
|
Klotz C, Goh YJ, O'Flaherty S, Barrangou R. S-layer associated proteins contribute to the adhesive and immunomodulatory properties of Lactobacillus acidophilus NCFM. BMC Microbiol 2020; 20:248. [PMID: 32787778 PMCID: PMC7425073 DOI: 10.1186/s12866-020-01908-2] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2019] [Accepted: 07/16/2020] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND Surface layers (S-layers) are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost layer of many bacterial cell envelopes. Within the Lactobacillus genus, S-layer presence is frequently associated with probiotic-relevant properties such as improved adherence to host epithelial cells and modulation of the immune response. However, recent studies have demonstrated that certain S-layer functions may be supplemented by a novel subset of proteins embedded within its lattice, termed S-layer associated proteins (SLAPs). In the following study, four Lactobacillus acidophilus NCFM SLAPs (LBA0046, LBA0864, LBA1426, and LBA1539) were selected for in silico and phenotypic assessment. RESULTS Despite lacking any sequence similarity or catalytic domains that may indicate function, the genes encoding the four proteins of interest were shown to be unique to S-layer-forming, host-adapted lactobacilli species. Likewise, their corresponding deletion mutants exhibited broad, host-relevant phenotypes including decreased inflammatory profiles and reduced adherence to Caco-2 intestinal cells, extracellular matrices, and mucin in vitro. CONCLUSIONS Overall, the data presented in this study collectively links several previously uncharacterized extracellular proteins to roles in the underlying host adaptive mechanisms of L. acidophilus.
Collapse
Affiliation(s)
- Courtney Klotz
- Genomic Sciences Graduate Program North Carolina State University, Raleigh, NC, USA.,Department of Food, Bioprocessing & Nutrition Sciences, North Carolina State University, Raleigh, NC, USA
| | - Yong Jun Goh
- Department of Food, Bioprocessing & Nutrition Sciences, North Carolina State University, Raleigh, NC, USA
| | - Sarah O'Flaherty
- Department of Food, Bioprocessing & Nutrition Sciences, North Carolina State University, Raleigh, NC, USA
| | - Rodolphe Barrangou
- Genomic Sciences Graduate Program North Carolina State University, Raleigh, NC, USA. .,Department of Food, Bioprocessing & Nutrition Sciences, North Carolina State University, Raleigh, NC, USA.
| |
Collapse
|
34
|
Cañez C, Selle K, Goh YJ, Barrangou R. Outcomes and characterization of chromosomal self-targeting by native CRISPR-Cas systems in Streptococcus thermophilus. FEMS Microbiol Lett 2020; 366:5488433. [PMID: 31077282 DOI: 10.1093/femsle/fnz105] [Citation(s) in RCA: 32] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2019] [Accepted: 05/09/2019] [Indexed: 12/21/2022] Open
Abstract
CRISPR-Cas systems provide adaptive immunity against phages in prokaryotes via DNA-encoded, RNA-mediated, nuclease-dependent targeting and cleavage. Due to inefficient and relatively limited DNA repair pathways in bacteria, CRISPR-Cas systems can be repurposed for lethal DNA targeting that selects for sequence variants. In this study, the relative killing efficiencies of endogenous Type I and Type II CRISPR-Cas systems in the model organism Streptococcus thermophilus DGCC7710 were assessed. Additionally, the genetic and phenotypic outcomes of chromosomal targeting by plasmid-programmed Type I-E or Type II-A systems were analyzed. Efficient killing was observed using both systems, in a dose-dependent manner when delivering 0.4-400 ng of plasmid DNA. Targeted PCR screening and genome sequencing were used to determine the genetic basis enabling survival, showing that evasion of Type I-E self-targeting was primarily the result of low-frequency defective plasmids that excised the targeting spacer. The most notable genotype recovered from Type II-A targeting of genomic locus, lacZ, was a 34 kb-deletion derived from homologous recombination (HR) between identical conserved sequences in two separate galE coding regions, resulting in 2% loss of the genome. Collectively, these results suggest that HR contributes to the plasticity and remodeling of bacterial genomes, leading to evasion of genome targeting by CRISPR-Cas systems.
Collapse
Affiliation(s)
- Cassandra Cañez
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.,Functional Genomics Graduate Program, North Carolina State University, Raleigh, NC 27695, USA
| | - Kurt Selle
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.,Functional Genomics Graduate Program, North Carolina State University, Raleigh, NC 27695, USA
| | - Yong Jun Goh
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA
| | - Rodolphe Barrangou
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695, USA.,Functional Genomics Graduate Program, North Carolina State University, Raleigh, NC 27695, USA
| |
Collapse
|
35
|
Klotz C, Goh YJ, O'Flaherty S, Johnson B, Barrangou R. Deletion of S-Layer Associated Ig-Like Domain Protein Disrupts the Lactobacillus acidophilus Cell Surface. Front Microbiol 2020; 11:345. [PMID: 32256464 PMCID: PMC7090030 DOI: 10.3389/fmicb.2020.00345] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Accepted: 02/17/2020] [Indexed: 01/18/2023] Open
Abstract
Bacterial surface-layers (S-layers) are crystalline arrays of repeating proteinaceous subunits that coat the exterior of many cell envelopes. S-layers have demonstrated diverse functions in growth and survival, maintenance of cell integrity, and mediation of host interactions. Additionally, S-layers can act as scaffolds for the outward display of auxiliary proteins and glycoproteins. These non-covalently bound S-layer associated proteins (SLAPs) have characterized roles in cell division, adherence to intestinal cells, and modulation of the host immune response. Recently, IgdA (LBA0695), a Lactobacillus acidophilus SLAP that possesses a Group 3 immunoglobulin (Ig)-like domain and GW (Gly-Tryp) dipeptide surface anchor, was recognized for its high conservation among S-layer-forming lactobacilli, constitutive expression, and surface localization. These findings prompted its selection for examination within the present study. Although IgdA and corresponding orthologs were shown to be unique to host-adapted lactobacilli, the Ig domain itself was specific to vertebrate-adapted species suggesting a role in vertebrate adaptation. Using a counterselective gene replacement system, igdA was deleted from the L. acidophilus NCFM chromosome. The resultant mutant, NCK2532, exhibited a visibly disrupted cell surface which likely contributed to its higher salt sensitivity, severely reduced adhesive capacity, and altered immunogenicity profile. Transcriptomic analyses revealed the induction of several stress response genes and secondary surface proteins. Due to the broad impact of IgdA on the cellular physiology and probiotic attributes of L. acidophilus, identification of similar proteins in alternative bacterial species may help pinpoint next-generation host-adapted probiotic candidates.
Collapse
Affiliation(s)
- Courtney Klotz
- Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC, United States.,Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States
| | - Yong Jun Goh
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States
| | - Sarah O'Flaherty
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States
| | - Brant Johnson
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States.,Microbiology Graduate Program, North Carolina State University, Raleigh, NC, United States
| | - Rodolphe Barrangou
- Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC, United States.,Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States.,Microbiology Graduate Program, North Carolina State University, Raleigh, NC, United States
| |
Collapse
|
36
|
Plavec TV, Berlec A. Safety Aspects of Genetically Modified Lactic Acid Bacteria. Microorganisms 2020; 8:E297. [PMID: 32098042 PMCID: PMC7074969 DOI: 10.3390/microorganisms8020297] [Citation(s) in RCA: 72] [Impact Index Per Article: 14.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2020] [Revised: 02/11/2020] [Accepted: 02/17/2020] [Indexed: 02/07/2023] Open
Abstract
Lactic acid bacteria (LAB) have a long history of use in the food industry. Some species are part of the normal human microbiota and have beneficial properties for human health. Their long-standing use and considerable biotechnological potential have led to the development of various systems for their engineering. Together with novel approaches such as CRISPR-Cas, the established systems for engineering now allow significant improvements to LAB strains. Nevertheless, genetically modified LAB (GM-LAB) still encounter disapproval and are under extensive regulatory requirements. This review presents data on the prospects for LAB to obtain 'generally recognized as safe' (GRAS) status. Genetic modification of LAB is discussed, together with problems that can arise from their engineering, including their dissemination into the environment and the spread of antibiotic resistance markers. Possible solutions that would allow the use of GM-LAB are described, such as biocontainment, alternative selection markers, and use of homologous DNA. The use of GM-LAB as cell factories in closed systems that prevent their environmental release is the least problematic aspect, and this is also discussed.
Collapse
Affiliation(s)
- Tina Vida Plavec
- Department of Biotechnology, Jožef Stefan Institute, SI-1000 Ljubljana, Slovenia;
- Faculty of Pharmacy, University of Ljubljana, SI-1000 Ljubljana, Slovenia
| | - Aleš Berlec
- Department of Biotechnology, Jožef Stefan Institute, SI-1000 Ljubljana, Slovenia;
- Faculty of Pharmacy, University of Ljubljana, SI-1000 Ljubljana, Slovenia
| |
Collapse
|
37
|
Wang XL, Dai SY, Wang QJ, Xu HN, Shi HQ, Kang YB, Zha DM. Efficient markerless gene deletions in Pseudomonas protegens Pf-5 using a upp-based counterselective system. Biotechnol Lett 2019; 42:277-285. [DOI: 10.1007/s10529-019-02772-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2019] [Accepted: 11/21/2019] [Indexed: 02/01/2023]
|
38
|
Wang T, Li Y, Li J, Zhang D, Cai N, Zhao G, Ma H, Shang C, Ma Q, Xu Q, Chen N. An update of the suicide plasmid-mediated genome editing system in Corynebacterium glutamicum. Microb Biotechnol 2019; 12:907-919. [PMID: 31180185 PMCID: PMC6680612 DOI: 10.1111/1751-7915.13444] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2019] [Revised: 05/12/2019] [Accepted: 05/16/2019] [Indexed: 12/21/2022] Open
Abstract
Corynebacterium glutamicum is an important industrial microorganism, but the availability of tools for its genetic modification has lagged compared to other model microorganisms such as Escherichia coli. Despite great progress in CRISPR-based technologies, the most feasible genome editing method in C. glutamicum is suicide plasmid-mediated, the editing efficiency of which is low due to high false-positive rates of sacB counter selection, and the requirement for tedious two-round selection and verification of rare double-cross-over events. In this study, an rpsL mutant conferring streptomycin resistance was harnessed for counter selection, significantly increasing the positive selection rate. More importantly, with the aid of high selection efficiencies through the use of antibiotics, namely kanamycin and streptomycin, the two-step verification strategy can be simplified to just one-step verification of the final edited strain. As proof of concept, a 2.5-kb DNA fragment comprising aroGfbr pheAfbr expressing cassettes was integrated into the genome of C. glutamicum, with an efficiency of 20% out of the theoretical 50%. The resulting strain produced 110 mg l-1 l-tyrosine in shake-flask fermentation. This updated suicide plasmid-mediated genome editing system will greatly facilitate genetic manipulations including single nucleotide mutation, gene deletion and gene insertion in C. glutamicum and can be easily applied to other microbes.
Collapse
Affiliation(s)
- Ting Wang
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
| | - Yanjun Li
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
- Guangdong Provincial Key Laboratory of Fermentation and Enzyme EngineeringGuangzhou510006China
- Key Laboratory of Industrial Fermentation MicrobiologyMinistry of EducationTianjin University of Science and TechnologyTianjin300457China
| | - Juan Li
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
| | - Dezhi Zhang
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
| | - Ningyun Cai
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
| | - Guihong Zhao
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
| | - Hongkun Ma
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
| | - Can Shang
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
| | - Qian Ma
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
- Key Laboratory of Industrial Fermentation MicrobiologyMinistry of EducationTianjin University of Science and TechnologyTianjin300457China
| | - Qingyang Xu
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
- Key Laboratory of Industrial Fermentation MicrobiologyMinistry of EducationTianjin University of Science and TechnologyTianjin300457China
| | - Ning Chen
- College of BiotechnologyTianjin University of Science and TechnologyTianjin300457China
- Key Laboratory of Industrial Fermentation MicrobiologyMinistry of EducationTianjin University of Science and TechnologyTianjin300457China
| |
Collapse
|
39
|
Zuo F, Zeng Z, Hammarström L, Marcotte H. Inducible Plasmid Self-Destruction (IPSD) Assisted Genome Engineering in Lactobacilli and Bifidobacteria. ACS Synth Biol 2019; 8:1723-1729. [PMID: 31277549 DOI: 10.1021/acssynbio.9b00114] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
Genome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria, but still have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called inducible plasmid self-destruction (IPSD) was successfully used to perform gene knockout and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.
Collapse
Affiliation(s)
- Fanglei Zuo
- Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China
- Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden
| | - Zhu Zeng
- Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, PR China
- Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden
| | - Lennart Hammarström
- Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden
| | - Harold Marcotte
- Department of Laboratory Medicine, Division of Clinical Immunology and Transfusion Medicine, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm SE-141 86, Sweden
| |
Collapse
|
40
|
Hidalgo-Cantabrana C, Goh YJ, Pan M, Sanozky-Dawes R, Barrangou R. Genome editing using the endogenous type I CRISPR-Cas system in Lactobacillus crispatus. Proc Natl Acad Sci U S A 2019; 116:15774-15783. [PMID: 31341082 PMCID: PMC6690032 DOI: 10.1073/pnas.1905421116] [Citation(s) in RCA: 113] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
CRISPR-Cas systems are now widely used for genome editing and transcriptional regulation in diverse organisms. The compact and portable nature of class 2 single effector nucleases, such as Cas9 or Cas12, has facilitated directed genome modifications in plants, animals, and microbes. However, most CRISPR-Cas systems belong to the more prevalent class 1 category, which hinges on multiprotein effector complexes. In the present study, we detail how the native type I-E CRISPR-Cas system, with a 5'-AAA-3' protospacer adjacent motif (PAM) and a 61-nucleotide guide CRISPR RNA (crRNA) can be repurposed for efficient chromosomal targeting and genome editing in Lactobacillus crispatus, an important commensal and beneficial microbe in the vaginal and intestinal tracts. Specifically, we generated diverse mutations encompassing a 643-base pair (bp) deletion (100% efficiency), a stop codon insertion (36%), and a single nucleotide substitution (19%) in the exopolysaccharide priming-glycosyl transferase (p-gtf). Additional genetic targets included a 308-bp deletion (20%) in the prophage DNA packaging Nu1 and a 730-bp insertion of the green fluorescent protein gene downstream of enolase (23%). This approach enables flexible alteration of the formerly genetically recalcitrant species L. crispatus, with potential for probiotic enhancement, biotherapeutic engineering, and mucosal vaccine delivery. These results also provide a framework for repurposing endogenous CRISPR-Cas systems for flexible genome targeting and editing, while expanding the toolbox to include one of the most abundant and diverse systems found in nature.
Collapse
Affiliation(s)
- Claudio Hidalgo-Cantabrana
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695
| | - Yong Jun Goh
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695
| | - Meichen Pan
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695
| | - Rosemary Sanozky-Dawes
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695
| | - Rodolphe Barrangou
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695
| |
Collapse
|
41
|
Abstract
The Klaenhammer group at North Carolina State University pioneered genomic applications in food microbiology and beneficial lactic acid bacteria used as starter cultures and probiotics. Dr. Todd Klaenhammer was honored to be the first food scientist elected to the National Academy of Sciences (2001). The program was recognized with the highest research awards presented by the American Dairy Science Association (Borden Award 1996), the Institute of Food Technologists (Nicholas Appert Medal, 2007), and the International Dairy Federation (Eli Metchnikoff Award in Biotechnology, 2010) as well as with the Outstanding Achievement Award from the University of Minnesota (2001) and the Oliver Max Gardner Award (2009) for outstanding research across the 16-campus University of North Carolina system. Dr. Klaenhammer is a fellow of the American Association for the Advancement of Science, the American Dairy Science Association, and the Institute of Food Technology. Over his career, six of his PhD graduate students were awarded the annual Kenneth Keller award for the outstanding PhD dissertation that year in the College of Agriculture and Life Sciences. He championed the use of basic microbiology and genomic approaches to set a platform for translational applications of beneficial microbes in foods and their use in food preservation and probiotics and as oral delivery vehicles for vaccines and biotherapeutics. Dr. Klaenhammer was also a founding and co-chief editor of the Annual Review of Food Science and Technology.
Collapse
Affiliation(s)
- Todd Robert Klaenhammer
- Department of Food, Bioprocessing & Nutrition Sciences, North Carolina State University, Raleigh, North Carolina 27695, USA;
| |
Collapse
|
42
|
Zhou C, Liu H, Yuan F, Chai H, Wang H, Liu F, Li Y, Zhang H, Lu F. Development and application of a CRISPR/Cas9 system for Bacillus licheniformis genome editing. Int J Biol Macromol 2019; 122:329-337. [PMID: 30401651 DOI: 10.1016/j.ijbiomac.2018.10.170] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2018] [Revised: 10/24/2018] [Accepted: 10/25/2018] [Indexed: 10/28/2022]
Abstract
A highly efficient genome editing system for Bacillus licheniformis was developed based on single-plasmid CRISPR/Cas9. For highly efficient genome editing the shuttle vector pWH1520 was selected to construct the knockout plasmids. A construct harboring a pS promoter driving cas9 endonuclease expression, a strong pLY-2 promoter driving the transcription of a single guide RNA was demonstrated as being the most effective. To verify the feasibility of the method the uprT gene coding uracil phosphoribosyltransferase was selected as the reporter gene. The efficiency of introducing nucleotide point mutations and single gene deletion reached an editing efficiency of up to 99.2% and 97.3%, respectively. After a upp-deficient strain was engineered, the system and strain were applied to introduce genomic deletions of another two genes, amyL and chiA (encoding amylase and chitinase, respectively) with about 90% deletion efficiency. As two native extracellular proteins with relatively high secretion in the host, amylase and chitinase can hamper the secretion and expression of alkaline protease. It was demonstrated that the mutant with deletions of the two genes effectively improved the alkaline protease yield by 24.8%. The results illustrated that the establishment of a CRISPR/Cas9 system for Bacillus licheniformis is of significance, and confirmed the system's high efficiency. The system provides support for effective molecular modification and metabolic regulation of Bacillus licheniformis, and offers promise for applications in genetic modification of other industrially relevant Bacillus species.
Collapse
Affiliation(s)
- Cuixia Zhou
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China
| | - Huan Liu
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China
| | - Feiyan Yuan
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China
| | - Haonan Chai
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China
| | - Haikuan Wang
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China
| | - Fufeng Liu
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China
| | - Yu Li
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China
| | - Huitu Zhang
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China.
| | - Fuping Lu
- State Key Laboratory of Food Nutrition and Safety, College of Biotechnology, Tianjin University of Science &Technology, Tianjin 022, PR China.
| |
Collapse
|
43
|
Klotz C, Barrangou R. Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications. Front Microbiol 2018; 9:2264. [PMID: 30333802 PMCID: PMC6176008 DOI: 10.3389/fmicb.2018.02264] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2018] [Accepted: 09/05/2018] [Indexed: 02/06/2023] Open
Abstract
Lactic acid bacteria (LAB) are frequently harnessed for the delivery of biomolecules to mucosal tissues. Several species of Lactobacillus are commonly employed for this task, of which a subset are known to possess surface-layers (S-layers). S-layers are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost coating of many prokaryotic cell envelopes. Their periodicity and abundance have made them a target for numerous biotechnological applications. In the following review, we examine the multi-faceted S-layer protein (Slp), and its use in both heterologous protein expression systems and mucosal vaccine delivery frameworks, through its diverse genetic components: the strong native promoter, capable of synthesizing as many as 500 Slp subunits per second; the signal peptide that stimulates robust secretion of recombinant proteins; and the structural domains, which can be harnessed for both cell surface display of foreign peptides or adhesion enhancement of a host bacterium. Although numerous studies have established vaccine platforms based on one or more components of the Lactobacillus S-layer, this area of research still remains largely in its infancy, thus this review is meant to not only highlight past works, but also advocate for the future usage of Slps in biotherapeutic research.
Collapse
Affiliation(s)
- Courtney Klotz
- Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC, United States
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States
| | - Rodolphe Barrangou
- Genomic Sciences Graduate Program, North Carolina State University, Raleigh, NC, United States
- Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC, United States
| |
Collapse
|
44
|
Hatti-Kaul R, Chen L, Dishisha T, Enshasy HE. Lactic acid bacteria: from starter cultures to producers of chemicals. FEMS Microbiol Lett 2018; 365:5087731. [DOI: 10.1093/femsle/fny213] [Citation(s) in RCA: 87] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2018] [Accepted: 08/29/2018] [Indexed: 12/26/2022] Open
Affiliation(s)
- Rajni Hatti-Kaul
- Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden
| | - Lu Chen
- Biotechnology, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden
| | - Tarek Dishisha
- Department of Microbiology and Immunology, Faculty of Pharmacy, Beni-Suef University, 62511 Beni-Suef, Egypt
| | - Hesham El Enshasy
- Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM), 81 310 Skudai, Johor, Malaysia
- City of Scientific Research and Technology Applications, New Burg Al Arab, Alexandria, Egypt
| |
Collapse
|
45
|
Guo Y, Li X, Yang Y, Wu Z, Zeng X, Nadari F, Pan D. Molecular cloning, expression and adhesion analysis of silent slpB of Lactobacillus acidophilus NCFM. AMB Express 2018; 8:103. [PMID: 29936673 PMCID: PMC6015585 DOI: 10.1186/s13568-018-0631-2] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2018] [Accepted: 06/14/2018] [Indexed: 11/10/2022] Open
Abstract
The slpB gene of Lactobacillus acidophilus NCFM, which differs from the slpA gene and is silent under normal conditions, was successfully amplified and ligated to the corresponding available sites on a recombinant pET-28a vector. Then the pET-28a-slpB vector was transformed into Escherichia coli DH (DE3) and the fusion His-slpB protein was expressed by induction with 1 mM IPTG for 14 h at 37 °C. The resulting His-slpB protein (SB) had a relative molecular weight of 48 kDa. It was purified using a Ni-NTA column and was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot contrastive analysis. The slpA protein (SA) from L. acidophilus NCFM was extracted and purified. It had a relative molecular weight of 46 kDa. Circular dichroism measurements suggested that the two S-layer proteins had a high β-sheet content and a low α-helix structure content. In an adhesion experiment, SA displayed higher adhesive capability towards Caco-2 cells than did SB. The results suggest that these two S-layer proteins could have biotechnological applications.
Collapse
|
46
|
d-Alanyl-d-Alanine Ligase as a Broad-Host-Range Counterselection Marker in Vancomycin-Resistant Lactic Acid Bacteria. J Bacteriol 2018; 200:JB.00607-17. [PMID: 29686137 PMCID: PMC5996685 DOI: 10.1128/jb.00607-17] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2017] [Accepted: 04/16/2018] [Indexed: 12/03/2022] Open
Abstract
The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Lactobacillus. Finally, our system represents the first “plug and play” counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium. IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)—an essential enzyme involved in peptidoglycan synthesis—increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in Lactobacillus; 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.
Collapse
|
47
|
Wang W, He J, Pan D, Wu Z, Guo Y, Zeng X, Lian L. Metabolomics analysis of Lactobacillus plantarum ATCC 14917 adhesion activity under initial acid and alkali stress. PLoS One 2018; 13:e0196231. [PMID: 29795550 PMCID: PMC5967736 DOI: 10.1371/journal.pone.0196231] [Citation(s) in RCA: 43] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2017] [Accepted: 04/09/2018] [Indexed: 12/25/2022] Open
Abstract
The adhesion ability of Lactobacillus plantarum affects retention time in the human gastro-intestinal tract, as well as influencing the interaction with their host. In this study, the relationship between the adhesion activity of, and metabolic changes in, L. plantarum ATCC 14917 under initial acid and alkali stress was evaluated by analyzing auto-aggregation, protein adhesion and cell adhesion in vitro. Based on scanning electron microscope (SEM) and transmission electron microscope (TEM) analysis, the morphology of the bacteria became thickset and the thickness of their cell walls decreased under initial alkali stress. The fold changes of auto-aggregation, adhere to mucin and HT-29 cell lines of L. plantarum ATCC 14917 in the acid group were increased by 1.141, 1.125 and 1.156, respectively. But decreased significantly in the alkali group (fold changes with 0.842, 0.728 and 0.667). Adhesion-related protein increased in the acid group but declined in the alkali group at the mRNA expression level according to real time polymerase chain reaction (RT-PCR) analysis. The changes in the metabolite profiles of L. plantarum ATCC 14917 were characterized using Ultra-Performance Liquid Chromatography-Electrospray ionization-Quadrupole-Time of Flight-mass spectrometry (UPLS-ESI-Q-TOF-MS). In the alkali group, the content of a lot of substances involved in the energy and amino acid metabolism decreased, but the content of some substances involved in the energy metabolism was slightly increased in the acid group. These findings demonstrate that energy metabolism is positively correlated with the adhesion ability of L. plantarum ATCC 14917. The amino-acids metabolism, especially the amino acids related to pH-homeostasis mechanisms (lysine, aspartic acid, arginine, proline and glutamic acid), showed an obvious effect on the adhesion ability of L. plantarum ATCC 14917. This investigation provides a better understanding of L. plantarum's adhesion mechanisms under initial pH stress.
Collapse
Affiliation(s)
- Wenwen Wang
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Marine Science School, Ningbo University, Ningbo, Zhejiang, P. R. China
| | - Jiayi He
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Marine Science School, Ningbo University, Ningbo, Zhejiang, P. R. China
| | - Daodong Pan
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Marine Science School, Ningbo University, Ningbo, Zhejiang, P. R. China
- Department of Food Science and Nutrition, Ginling College, Nanjing Normal University, Nanjing, P. R. China
- * E-mail: (DDP); (ZW)
| | - Zhen Wu
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Marine Science School, Ningbo University, Ningbo, Zhejiang, P. R. China
- * E-mail: (DDP); (ZW)
| | - Yuxing Guo
- Department of Food Science and Nutrition, Ginling College, Nanjing Normal University, Nanjing, P. R. China
| | - Xiaoqun Zeng
- Key Laboratory of Animal Protein Deep Processing Technology of Zhejiang, Marine Science School, Ningbo University, Ningbo, Zhejiang, P. R. China
| | - Liwei Lian
- Ningbo Dairy Group, Ningbo, Zhejiang, China
| |
Collapse
|
48
|
Bumgardner SA, Zhang L, LaVoy AS, Andre B, Frank CB, Kajikawa A, Klaenhammer TR, Dean GA. Nod2 is required for antigen-specific humoral responses against antigens orally delivered using a recombinant Lactobacillus vaccine platform. PLoS One 2018; 13:e0196950. [PMID: 29734365 PMCID: PMC5937747 DOI: 10.1371/journal.pone.0196950] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2018] [Accepted: 04/23/2018] [Indexed: 12/27/2022] Open
Abstract
Safe and efficacious orally-delivered mucosal vaccine platforms are desperately needed to combat the plethora of mucosally transmitted pathogens. Lactobacillus spp. have emerged as attractive candidates to meet this need and are known to activate the host innate immune response in a species- and strain-specific manner. For selected bacterial isolates and mutants, we investigated the role of key innate immune pathways required for induction of innate and subsequent adaptive immune responses. Co-culture of murine macrophages with L. gasseri (strain NCK1785), L. acidophilus (strain NCFM), or NCFM-derived mutants—NCK2025 and NCK2031—elicited an M2b-like phenotype associated with TH2 skewing and immune regulatory function. For NCFM, this M2b phenotype was dependent on expression of lipoteichoic acid and S layer proteins. Through the use of macrophage genetic knockouts, we identified Toll-like receptor 2 (TLR2), the cytosolic nucleotide-binding oligomerization domain containing 2 (NOD2) receptor, and the inflammasome-associated caspase-1 as contributors to macrophage activation, with NOD2 cooperating with caspase-1 to induce inflammasome derived interleukin (IL)-1β in a pyroptosis-independent fashion. Finally, utilizing an NCFM-based mucosal vaccine platform with surface expression of human immunodeficiency virus type 1 (HIV-1) Gag or membrane proximal external region (MPER), we demonstrated that NOD2 signaling is required for antigen-specific mucosal and systemic humoral responses. We show that lactobacilli differentially utilize innate immune pathways and highlight NOD2 as a key mediator of macrophage function and antigen-specific humoral responses to a Lactobacillus acidophilus mucosal vaccine platform.
Collapse
Affiliation(s)
- Sara A. Bumgardner
- Center for Comparative Medicine and Translational Research, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina, United States of America
| | - Lin Zhang
- Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America
| | - Alora S. LaVoy
- Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America
| | - Barbara Andre
- Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America
| | - Chad B. Frank
- Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America
| | - Akinobu Kajikawa
- Department of Applied Biology and Chemistry, Tokyo University of Agriculture, Setagaya, Tokyo, Japan
| | - Todd R. Klaenhammer
- Department of Food, Bioprocessing, & Nutrition Sciences, North Carolina State University, Raleigh, North Carolina, United States of America
| | - Gregg A. Dean
- Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, United States of America
- * E-mail:
| |
Collapse
|
49
|
do Carmo FLR, Rabah H, De Oliveira Carvalho RD, Gaucher F, Cordeiro BF, da Silva SH, Le Loir Y, Azevedo V, Jan G. Extractable Bacterial Surface Proteins in Probiotic-Host Interaction. Front Microbiol 2018; 9:645. [PMID: 29670603 PMCID: PMC5893755 DOI: 10.3389/fmicb.2018.00645] [Citation(s) in RCA: 34] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2017] [Accepted: 03/19/2018] [Indexed: 01/09/2023] Open
Abstract
Some Gram-positive bacteria, including probiotic ones, are covered with an external proteinaceous layer called a surface-layer. Described as a paracrystalline layer and formed by the self-assembly of a surface-layer-protein (Slp), this optional structure is peculiar. The surface layer per se is conserved and encountered in many prokaryotes. However, the sequence of the corresponding Slp protein is highly variable among bacterial species, or even among strains of the same species. Other proteins, including surface layer associated proteins (SLAPs), and other non-covalently surface-bound proteins may also be extracted with this surface structure. They can be involved a various functions. In probiotic Gram-positives, they were shown by different authors and experimental approaches to play a role in key interactions with the host. Depending on the species, and sometime on the strain, they can be involved in stress tolerance, in survival within the host digestive tract, in adhesion to host cells or mucus, or in the modulation of intestinal inflammation. Future trends include the valorization of their properties in the formation of nanoparticles, coating and encapsulation, and in the development of new vaccines.
Collapse
Affiliation(s)
- Fillipe L R do Carmo
- Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil.,STLO, Agrocampus Ouest, INRA, Rennes, France
| | - Houem Rabah
- STLO, Agrocampus Ouest, INRA, Rennes, France.,Pôle Agronomique Ouest, Rennes, France
| | | | - Floriane Gaucher
- STLO, Agrocampus Ouest, INRA, Rennes, France.,Bioprox, Levallois-Perret, France
| | - Barbara F Cordeiro
- Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
| | - Sara H da Silva
- Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
| | | | - Vasco Azevedo
- Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil
| | - Gwénaël Jan
- STLO, Agrocampus Ouest, INRA, Rennes, France
| |
Collapse
|
50
|
Lactobacillus acidophilus Metabolizes Dietary Plant Glucosides and Externalizes Their Bioactive Phytochemicals. mBio 2017; 8:mBio.01421-17. [PMID: 29162708 PMCID: PMC5698550 DOI: 10.1128/mbio.01421-17] [Citation(s) in RCA: 77] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Therapeutically active glycosylated phytochemicals are ubiquitous in the human diet. The human gut microbiota (HGM) modulates the bioactivities of these compounds, which consequently affect host physiology and microbiota composition. Despite a significant impact on human health, the key players and the underpinning mechanisms of this interplay remain uncharacterized. Here, we demonstrate the growth of Lactobacillus acidophilus on mono- and diglucosyl dietary plant glycosides (PGs) possessing small aromatic aglycones. Transcriptional analysis revealed the upregulation of host interaction genes and identified two loci that encode phosphotransferase system (PTS) transporters and phospho-β-glucosidases, which mediate the uptake and deglucosylation of these compounds, respectively. Inactivating these transport and hydrolysis genes abolished or severely reduced growth on PG, establishing the specificity of the loci to distinct groups of PGs. Following intracellular deglucosylation, the aglycones of PGs are externalized, rendering them available for absorption by the host or for further modification by other microbiota taxa. The PG utilization loci are conserved in L. acidophilus and closely related lactobacilli, in correlation with versatile growth on these compounds. Growth on the tested PG appeared more common among human gut lactobacilli than among counterparts from other ecologic niches. The PGs that supported the growth of L. acidophilus were utilized poorly or not at all by other common HGM strains, underscoring the metabolic specialization of L. acidophilus. These findings highlight the role of human gut L. acidophilus and select lactobacilli in the bioconversion of glycoconjugated phytochemicals, which is likely to have an important impact on the HGM and human host. Thousands of therapeutically active plant-derived compounds are widely present in berries, fruits, nuts, and beverages like tea and wine. The bioactivity and bioavailability of these compounds, which are typically glycosylated, are altered by microbial bioconversions in the human gut. Remarkably, little is known about the bioconversion of PGs by the gut microbial community, despite the significance of this metabolic facet to human health. Our work provides the first molecular insights into the metabolic routes of diet relevant and therapeutically active PGs by Lactobacillus acidophilus and related human gut lactobacilli. This taxonomic group is adept at metabolizing the glucoside moieties of select PG and externalizes their aglycones. The study highlights an important role of lactobacilli in the bioconversion of dietary PG and presents a framework from which to derive molecular insights into their metabolism by members of the human gut microbiota.
Collapse
|