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Yang C, Zheng YX, Gu HY, Chen H, Li W, Li F, Bi YW, Chen J, Wang FK, Sun QQ, Meng HB, Wu ZH, Yu S, Gu J, Cheng Y. Genomic characteristics, virulence potential, antimicrobial resistance profiles, and phylogenetic insights into Nocardia cyriacigeorgica. Ann Clin Microbiol Antimicrob 2025; 24:22. [PMID: 40188140 PMCID: PMC11972502 DOI: 10.1186/s12941-025-00791-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2025] [Accepted: 03/27/2025] [Indexed: 04/07/2025] Open
Abstract
BACKGROUND Nocardia cyriacigeorgica, an opportunistic pathogen, is increasingly implicated in human infections. This pathogen predominantly causes pulmonary infections, leading to acute, subacute, or chronic necrotizing suppurative lesions, in severe cases, may progress to disseminated infections. Effective clinical diagnosis, prevention, and treatment strategies require a thorough understanding of its biological characteristics and pathogenic mechanisms. However, despite the rising incidence of nocardial diseases, research on the pathogenicity of N. cyriacigeorgica remains limited, primarily focusing on case reports and epidemiological studies. This study aimed to provide a comprehensive analysis of the genomic features, phylogenetic relationships, antimicrobial resistance profiles, and candidate virulence factors of N. cyriacigeorgica strains to inform future investigations into its pathogenesis. METHODS Whole-genome sequencing was conducted on five N. cyriacigeorgica strains isolated from patients with pulmonary infection at our hospital. This analysis utilized a combination of second-generation Illumina HiSeq and third-generation PacBio sequencing technologies. Additionally, publicly available genomic data from 58 strains in the National Center Biotechnology Information database were integrated, resulting in a dataset of 63 genomes. These genomes were subjected to comparative genomic analyses, including phylogenetic reconstruction, pan-genome evaluation, and gene distribution assessments. RESULTS Phylogenetic analysis identified five major clades within N. cyriacigeorgica. ANI analysis further subdivided clade B into five distinct subgroups. Pan-genome analysis revealed clade-specific orthogroups in the distribution of genes assigned to Clusters of Orthologous Groups, with clade A containing the highest number of clade-specific gene families. Comparative genomic analysis uncovered several potential pathogenic genes implicated in host cell invasion, phagosomal maturation arrest, and intracellular survival within macrophages, which were conserved across all analyzed strains. Notable differences in the distribution of enterobactin-encoding genes were observed among the clades. The mce3C gene also displayed variable distributions across clades; however, no correlation was established between its presence and strain source. Among the 63 strains, 27 were found to harbor both mce3C and mce4F genes, which were categorized into five distinct patterns. Furthermore, antibiotic resistance genes, including VanSO, VanRO, erm(O)-Irm, srmB, ermH, bcl, bla1, and cmIR, demonstrated clade-specific distribution patterns. Notably, the genes erm(O)-Irm, srmB, and ermH were associated with the isolation origin of the strains. CONCLUSIONS This study provides a comprehensive evaluation of the genomic characteristics, potential virulence factors, antimicrobial resistance genes, and phylogenetic relationships of N. cyriacigeorgica. The findings offer valuable insights into the mechanisms underlying intracellular survival, replication within macrophages, and pathogen-host interactions in N. cyriacigeorgica infections. These results establish a foundation for future research into the pathogenesis and clinical management of N. cyriacigeorgica.
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Affiliation(s)
- Chen Yang
- National Engineering Research Center of Immunological ProductsDepartment of Microbiology and Biochemical PharmacyCollege of Pharmacy, Army Medical University, Chongqing, 400038, China
| | - Yue-Xin Zheng
- Department of General Surgery, The Sixth Medical Center of PLA General Hospital, Beijing, 100048, China
| | - Hong-Yi Gu
- Department of Public Affairs Management, Tianjin Medical University, Tianjin, 300203, China
| | - Hong Chen
- Department of Clinical Pharmacy, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Wei Li
- Department of Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Fang Li
- Department of Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Yu-Wang Bi
- Department of Information, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Jing Chen
- Department of Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Fu-Kun Wang
- Department of Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Qing-Qing Sun
- Department of Basic Medical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Han-Bing Meng
- Department of Basic Medical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Zuo-Hao Wu
- Department of Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China
| | - Shu Yu
- Department of Laboratory Medicine, People's Hospital of Chongqing Hechuan, Chongqing, 401520, China.
| | - Jiang Gu
- National Engineering Research Center of Immunological ProductsDepartment of Microbiology and Biochemical PharmacyCollege of Pharmacy, Army Medical University, Chongqing, 400038, China.
| | - Yan Cheng
- Department of Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China.
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Parkin LA, Maceren JP, Palande A, Previti ML, Seeliger JC. Metabolic tagging reveals surface-associated lipoproteins in mycobacteria. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.07.631728. [PMID: 39829771 PMCID: PMC11741404 DOI: 10.1101/2025.01.07.631728] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/22/2025]
Abstract
Mycobacteria such as the causative agent of tuberculosis, Mycobacterium tuberculosis, encode over 100 bioinformatically predicted lipoproteins. Despite the importance of these post-translationally modified proteins for mycobacterial survival, many remain experimentally unconfirmed. Here we characterized metabolic incorporation of diverse fatty acid analogues as a facile method of adding chemical groups that enable downstream applications such as detection, crosslinking and enrichment, of not only lipid-modified proteins, but also their protein interactors. Having shown that incorporation is an active process dependent on the lipoprotein biosynthesis pathway, we discovered that lipid-modified proteins are also located at the mycobacterial cell surface. These data counter the commonly held assumption that mycobacteria do not move lipoproteins across the cell envelope and thus have implications for uncovering a novel transport pathway and the roles of lipoproteins at the interface with the host environment. Our findings and the tools we developed will enable the further study of pathways related to lipoprotein function and metabolism in mycobacteria and other bacteria in which lipoproteins remain poorly understood.
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Affiliation(s)
- Lia A. Parkin
- Department of Microbiology and Immunology, Stony Brook, NY 11794, U.S.A
| | | | - Aseem Palande
- Department of Pharmacological Sciences Stony Brook University, Stony Brook, NY 11794, U.S.A
| | - Mary L. Previti
- Department of Pharmacological Sciences Stony Brook University, Stony Brook, NY 11794, U.S.A
| | - Jessica C. Seeliger
- Department of Pharmacological Sciences Stony Brook University, Stony Brook, NY 11794, U.S.A
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Chen Y, Hagopian B, Tan S. Cholesterol metabolism and intrabacterial potassium homeostasis are intrinsically related in Mycobacterium tuberculosis. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.11.10.622811. [PMID: 39605342 PMCID: PMC11601456 DOI: 10.1101/2024.11.10.622811] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/29/2024]
Abstract
Potassium (K+) is the most abundant intracellular cation, but much remains unknown regarding how K+ homeostasis is integrated with other key bacterial biology aspects. Here, we show that K+ homeostasis disruption (CeoBC K+ uptake system deletion) impedes Mycobacterium tuberculosis (Mtb) response to, and growth in, cholesterol, a critical carbon source during infection, with K+ augmenting activity of the Mtb ATPase MceG that is vital for bacterial cholesterol import. Reciprocally, cholesterol directly binds to CeoB, modulating its function, with a residue critical for this interaction identified. Finally, cholesterol binding-deficient CeoB mutant Mtb are attenuated for growth in lipid-rich foamy macrophages and in vivo colonization. Our findings raise the concept of a role for cholesterol as a key co-factor, beyond its role as a carbon source, and illuminate how changes in bacterial intrabacterial K+ levels can act as part of the metabolic adaptation critical for bacterial survival and growth in the host.
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Affiliation(s)
- Yue Chen
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA
| | - Berge Hagopian
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA
| | - Shumin Tan
- Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA
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Jana B, Liu X, Dénéréaz J, Park H, Leshchiner D, Liu B, Gallay C, Zhu J, Veening JW, van Opijnen T. CRISPRi-TnSeq maps genome-wide interactions between essential and non-essential genes in bacteria. Nat Microbiol 2024; 9:2395-2409. [PMID: 39030344 PMCID: PMC11371651 DOI: 10.1038/s41564-024-01759-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2023] [Accepted: 06/12/2024] [Indexed: 07/21/2024]
Abstract
Genetic interactions identify functional connections between genes and pathways, establishing gene functions or druggable targets. Here we use CRISPRi-TnSeq, CRISPRi-mediated knockdown of essential genes alongside TnSeq-mediated knockout of non-essential genes, to map genome-wide interactions between essential and non-essential genes in Streptococcus pneumoniae. Transposon-mutant libraries constructed in 13 CRISPRi strains enabled screening of ~24,000 gene pairs. This identified 1,334 genetic interactions, including 754 negative and 580 positive interactions. Network analyses show that 17 non-essential genes pleiotropically interact with more than half the essential genes tested. Validation experiments confirmed that a 7-gene subset protects against perturbations. Furthermore, we reveal hidden redundancies that compensate for essential gene loss, relationships between cell wall synthesis, integrity and cell division, and show that CRISPRi-TnSeq identifies synthetic and suppressor-type relationships between both functionally linked and disparate genes and pathways. Importantly, in species where CRISPRi and Tn-Seq are established, CRISPRi-TnSeq should be straightforward to implement.
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Affiliation(s)
- Bimal Jana
- Department of Biology, Boston College, Chestnut Hill, MA, USA
- Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA
| | - Xue Liu
- Department of Pathogen Biology, Base for International Science and Technology Cooperation: Carson Cancer Stem Cell Vaccines R&D Center, International Cancer Center, Shenzhen University Health Science Center, Shenzhen, China
- Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland
| | - Julien Dénéréaz
- Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland
| | - Hongshik Park
- Department of Biology, Boston College, Chestnut Hill, MA, USA
| | | | - Bruce Liu
- Department of Biology, Boston College, Chestnut Hill, MA, USA
| | - Clément Gallay
- Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland
| | - Junhao Zhu
- CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
| | - Jan-Willem Veening
- Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.
| | - Tim van Opijnen
- Broad Institute of MIT and Harvard, Cambridge, MA, USA.
- Boston Children's Hospital, Division of Infectious Diseases, Harvard Medical School, Boston, MA, USA.
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Singh L, Karthikeyan S, Thakur KG. Biochemical and structural characterization reveals Rv3400 codes for β-phosphoglucomutase in Mycobacterium tuberculosis. Protein Sci 2024; 33:e4943. [PMID: 38501428 PMCID: PMC10949319 DOI: 10.1002/pro.4943] [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: 08/25/2023] [Revised: 01/22/2024] [Accepted: 02/11/2024] [Indexed: 03/20/2024]
Abstract
Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or β-phosphoglucomutase (β-PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs β-PGM activity and hence, Rv3400 encodes for β-PGM in Mtb. Our data also confirm that Mtb β-PGM is a metal dependent enzyme having broad specificity for divalent metal ions. β-PGM converts β-D-glucose-1-phosphate to β-D-glucose-6-phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of β-PGM in the physiology of Mtb.
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Affiliation(s)
- Latika Singh
- Division of Protein Science and EngineeringCouncil of Scientific and Industrial Research—Institute of Microbial Technology (CSIR‐IMTECH)ChandigarhIndia
| | - Subramanian Karthikeyan
- Division of Protein Science and EngineeringCouncil of Scientific and Industrial Research—Institute of Microbial Technology (CSIR‐IMTECH)ChandigarhIndia
| | - Krishan Gopal Thakur
- Division of Protein Science and EngineeringCouncil of Scientific and Industrial Research—Institute of Microbial Technology (CSIR‐IMTECH)ChandigarhIndia
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6
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Guo X, Zhang Z, Chen Q, Wang L, Xu X, Wei Z, Zhang Y, Chen K, Wang Z, Lu X, Liang Q. Whole Genome Sequencing Highlights the Pathogenic Profile in Nocardia Keratitis. Invest Ophthalmol Vis Sci 2024; 65:26. [PMID: 38502137 PMCID: PMC10959193 DOI: 10.1167/iovs.65.3.26] [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: 08/26/2023] [Accepted: 02/26/2024] [Indexed: 03/20/2024] Open
Abstract
Purpose Nocardia keratitis is a serious and sight-threatening condition. This study aims to reveal the virulence and antimicrobial resistance gene profile of Nocardia strains using whole genome sequencing. Methods Whole-genome sequencing was performed on 23 cornea-derived Nocardia strains. Together with genomic data from the respiratory tract and the environment, 141 genomes were then utilized for phylogenetic and pan-genome analyses, followed by virulence and antibiotic resistance analysis. The correlations between virulence genes and pathogenicity were experimentally validated, including the characteristics of Nocardia colonies and clinical and histopathological evaluations of Nocardia keratitis mice models. Results Whole-genome sequencing of 141 Nocardia strains revealed a mean of 220 virulence genes contributed to bacterial pathogenesis. The mce gene family analysis led to the categorization of strains from the cornea into groups A, B, and C. The colonies of group C had the largest diameter, height, and fastest growth rate. The size of corneal ulcers and the clinical scores showed a significant increase in mouse models induced by group C. The relative expression levels of pro-inflammatory cytokines (CD4, IFN-γ, IL-6Rα, and TNF-α) in the lesion area exhibited an increasing trend from group A to group C. Antibiotic resistance genes (ARGs) spanned nine distinct drug classes, four resistance mechanisms, and seven primary antimicrobial resistance gene families. Conclusions Whole genome sequencing highlights the pathogenic role of mce gene family in Nocardia keratitis. Its distribution pattern may contribute to the distinct characteristics of the growth of Nocardia colonies and the clinical severity of the mice models.
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Affiliation(s)
- Xiaoyan Guo
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Zijun Zhang
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Qiankun Chen
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Leying Wang
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Xizhan Xu
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Zhenyu Wei
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Yang Zhang
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Kexin Chen
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Zhiqun Wang
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Xinxin Lu
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
| | - Qingfeng Liang
- Beijing Institute of Ophthalmology, Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing, China
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7
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Sturm A, Sun P, Avila-Pacheco J, Clatworthy AE, Bloom-Ackermann Z, Wuo MG, Gomez JE, Jin S, Clish CB, Kiessling LL, Hung DT. Genetic factors affecting storage and utilization of lipids during dormancy in Mycobacterium tuberculosis. mBio 2024; 15:e0320823. [PMID: 38236034 PMCID: PMC10865790 DOI: 10.1128/mbio.03208-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Accepted: 12/11/2023] [Indexed: 01/19/2024] Open
Abstract
Mycobacterium tuberculosis (Mtb) can adopt a non-growing dormant state during infection that may be critical to both active and latent tuberculosis. During dormancy, Mtb is widely tolerant toward antibiotics, a significant obstacle in current anti-tubercular drug regimens, and retains the ability to persist in its environment. We aimed to identify novel mechanisms that permit Mtb to survive dormancy in an in vitro carbon starvation model using transposon insertion sequencing and gene expression analysis. We identified a previously uncharacterized component of the lipid transport machinery, omamC, which was upregulated and required for survival during carbon starvation. We show that OmamC plays a role both in increasing fatty acid stores during growth in rich media and enhancing fatty acid utilization during starvation. Besides its involvement in lipid metabolism, OmamC levels affected the expression of the anti-anti-sigma factor rv0516c and other genes to improve Mtb survival during carbon starvation and increase its tolerance toward rifampicin, a first-line drug effective against non-growing Mtb. Importantly, we show that Mtb can be eradicated during carbon starvation, in an OmamC-dependent manner, by inhibiting lipid metabolism with the lipase inhibitor tetrahydrolipstatin. This work casts new light into the survival processes of non-replicating, drug-tolerant Mtb by identifying new proteins involved in lipid metabolism required for the survival of dormant bacteria and exposing a potential vulnerability that could be exploited for antibiotic discovery.IMPORTANCETuberculosis is a global threat, with ~10 million yearly active cases. Many more people, however, live with "latent" infection, where Mycobacterium tuberculosis survives in a non-replicative form. When latent bacteria activate and regrow, they elicit immune responses and result in significant host damage. Replicating and non-growing bacilli can co-exist; however, non-growing bacteria are considerably less sensitive to antibiotics, thus complicating treatment by necessitating long treatment durations. Here, we sought to identify genes important for bacterial survival in this non-growing state using a carbon starvation model. We found that a previously uncharacterized gene, omamC, is involved in storing and utilizing fatty acids as bacteria transition between these two states. Importantly, inhibiting lipid metabolism using a lipase inhibitor eradicates non-growing bacteria. Thus, targeting lipid metabolism may be a viable strategy for treating the non-growing population in strategies to shorten treatment durations of tuberculosis.
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Affiliation(s)
- Alexander Sturm
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA
- Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
| | - Penny Sun
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA
| | | | - Anne E. Clatworthy
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA
- Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
| | - Zohar Bloom-Ackermann
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA
- Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
| | - Michael G. Wuo
- Department of Chemistry, MIT, Cambridge, Massachusetts, USA
| | - James E. Gomez
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA
- Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
| | - Soomin Jin
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA
| | - Clary B. Clish
- Metabolomics Platform, Broad Institute, Cambridge, Massachusetts, USA
| | | | - Deborah T. Hung
- Infectious Disease and Microbiome Program, Broad Institute, Cambridge, Massachusetts, USA
- Department of Molecular Biology and Center for Computational and Integrative Biology, Massachusetts General Hospital, Boston, Massachusetts, USA
- Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA
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8
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Cooper BF, Ratkevičiūtė G, Clifton LA, Johnston H, Holyfield R, Hardy DJ, Caulton SG, Chatterton W, Sridhar P, Wotherspoon P, Hughes GW, Hall SC, Lovering AL, Knowles TJ. An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system. EMBO Rep 2024; 25:82-101. [PMID: 38228789 PMCID: PMC10897342 DOI: 10.1038/s44319-023-00014-4] [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: 08/02/2023] [Revised: 10/31/2023] [Accepted: 11/10/2023] [Indexed: 01/18/2024] Open
Abstract
The E. coli Paraquat Inducible (Pqi) Pathway is a putative Gram-negative phospholipid transport system. The pathway comprises three components: an integral inner membrane protein (PqiA), a periplasmic spanning MCE family protein (PqiB) and an outer membrane lipoprotein (PqiC). Interactions between all complex components, including stoichiometry, remain uncharacterised; nevertheless, once assembled into their quaternary complex, the trio of Pqi proteins are anticipated to provide a continuous channel between the inner and outer membranes of diderms. Here, we present X-ray structures of both the native and a truncated, soluble construct of the PqiC lipoprotein, providing insight into its biological assembly, and utilise neutron reflectometry to characterise the nature of the PqiB-PqiC-membrane interaction. Finally, we employ phenotypic complementation assays to probe specific PqiC residues, which imply the interaction between PqiB and PqiC is less intimate than previously anticipated.
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Affiliation(s)
- Benjamin F Cooper
- Sir William Dunn School of Pathology, University of Oxford, OX1 3RE, Oxford, UK
| | | | - Luke A Clifton
- ISIS Pulsed Neutron & Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory Harwell Oxford Campus, OX11 OQX, Didcot, UK
| | - Hannah Johnston
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - Rachel Holyfield
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - David J Hardy
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - Simon G Caulton
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - William Chatterton
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - Pooja Sridhar
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - Peter Wotherspoon
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - Gareth W Hughes
- Institute of Cancer and Genomic Sciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - Stephen Cl Hall
- ISIS Pulsed Neutron & Muon Source, Science and Technology Facilities Council, Rutherford Appleton Laboratory Harwell Oxford Campus, OX11 OQX, Didcot, UK
| | - Andrew L Lovering
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK
| | - Timothy J Knowles
- School of Biosciences, University of Birmingham, B15 2TT, Birmingham, UK.
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Rahlwes KC, Dias BR, Campos PC, Alvarez-Arguedas S, Shiloh MU. Pathogenicity and virulence of Mycobacterium tuberculosis. Virulence 2023; 14:2150449. [PMID: 36419223 PMCID: PMC9817126 DOI: 10.1080/21505594.2022.2150449] [Citation(s) in RCA: 61] [Impact Index Per Article: 30.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Accepted: 11/17/2022] [Indexed: 11/27/2022] Open
Abstract
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, an infectious disease with one of the highest morbidity and mortality rates worldwide. Leveraging its highly evolved repertoire of non-protein and protein virulence factors, Mtb invades through the airway, subverts host immunity, establishes its survival niche, and ultimately escapes in the setting of active disease to initiate another round of infection in a naive host. In this review, we will provide a concise synopsis of the infectious life cycle of Mtb and its clinical and epidemiologic significance. We will also take stock of its virulence factors and pathogenic mechanisms that modulate host immunity and facilitate its spread. Developing a greater understanding of the interface between Mtb virulence factors and host defences will enable progress toward improved vaccines and therapeutics to prevent and treat tuberculosis.
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Affiliation(s)
- Kathryn C. Rahlwes
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Beatriz R.S. Dias
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Priscila C. Campos
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Samuel Alvarez-Arguedas
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Michael U. Shiloh
- Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA
- Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX, USA
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10
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Adhyapak P, Liang K, Duan M, Kapoor S. Effect of Host Cholesterol on the Membrane Dynamics of Outer Membrane Lipids of Mycobacteria. Chem Asian J 2023; 18:e202300697. [PMID: 37846643 PMCID: PMC7616960 DOI: 10.1002/asia.202300697] [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: 08/09/2023] [Revised: 10/16/2023] [Accepted: 10/17/2023] [Indexed: 10/18/2023]
Abstract
The ability of Mycobacterium tuberculosis to remain dormant after primary infection represents the prime cause of new TB cases throughout the world. Hence, diagnosis and treatment of individuals hosting dormant mycobacterium is one of the crucial strategies to be adopted for the prevention of Tuberculosis. Among many strategies unleashed by the latent bacterium, one of them is scavenging host cholesterol for carbon source. Cholesterol modifies lipid membranes over many scales and here, its effect on mycobacterial membrane biophysics and the subsequent effect on partitioning of antibiotics into cholesterol- enriched mycobacterial membranes was investigated. Our research showed that cholesterol alters the phase state behavior of mycobacterial outer membrane lipids by enhancing the overall membrane order at the headgroup and acyl chain region and is integrated into both ordered and disordered domains/phases, with a preference for the latter. Exogenous cholesterol further alters the drug partitioning behavior of structurally different drugs, pointing to a larger clinical potential of using more hydrophobic medications to target dormant bacteria.
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Affiliation(s)
- Pranav Adhyapak
- Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076 (India)
| | - Kuan Liang
- National Centre for Magnetic Resonance in Wuhan, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071 Hubei (China)
| | - Mojie Duan
- National Centre for Magnetic Resonance in Wuhan, State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Innovation Academy for Precision Measurement Science and Technology, Chinese Academy of Sciences, Wuhan 430071 Hubei (China)
| | - Shobhna Kapoor
- Department of Chemistry, Indian Institute of Technology Bombay, Mumbai 400076 (India)
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11
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Chen Y, Wang Y, Chng SS. A conserved membrane protein negatively regulates Mce1 complexes in mycobacteria. Nat Commun 2023; 14:5897. [PMID: 37736771 PMCID: PMC10517005 DOI: 10.1038/s41467-023-41578-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2023] [Accepted: 09/10/2023] [Indexed: 09/23/2023] Open
Abstract
Tuberculosis continues to pose a serious threat to global health. Mycobacterium tuberculosis, the causative agent of tuberculosis, is an intracellular pathogen that relies on various mechanisms to survive and persist within the host. Among their many virulence factors, mycobacteria encode Mce systems. Some of these systems are implicated in lipid uptake, but the molecular basis for Mce function(s) is poorly understood. To gain insights into the composition and architecture of Mce systems, we characterized the putative Mce1 complex involved in fatty acid transport. We show that the Mce1 system in Mycobacterium smegmatis comprises a canonical ATP-binding cassette transporter associated with distinct heterohexameric assemblies of substrate-binding proteins. Furthermore, we establish that the conserved membrane protein Mce1N negatively regulates Mce1 function via a unique mechanism involving blocking transporter assembly. Our work offers a molecular understanding of Mce complexes, sheds light on mycobacterial lipid metabolism and its regulation, and informs future anti-mycobacterial strategies.
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Affiliation(s)
- Yushu Chen
- Department of Chemistry, National University of Singapore, Singapore, 117543, Singapore
| | - Yuchun Wang
- Department of Chemistry, National University of Singapore, Singapore, 117543, Singapore
| | - Shu-Sin Chng
- Department of Chemistry, National University of Singapore, Singapore, 117543, Singapore.
- Singapore Center for Environmental Life Sciences Engineering, National University of Singapore (SCELSE-NUS), Singapore, 117456, Singapore.
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12
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Bloom BR. A half-century of research on tuberculosis: Successes and challenges. J Exp Med 2023; 220:e20230859. [PMID: 37552470 PMCID: PMC10407785 DOI: 10.1084/jem.20230859] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2023] [Revised: 07/20/2023] [Accepted: 07/21/2023] [Indexed: 08/09/2023] Open
Abstract
Great progress has been made over the past half-century, but TB remains a formidable global health problem, particularly in low- and middle-income countries. Understanding the mechanisms of pathogenesis and necessary and sufficient conditions for protection are critical. The need for inexpensive and sensitive point-of-care diagnostic tests for earlier detection of infection and disease, shorter and less-toxic drug regimens for drug-sensitive and -resistant TB, and a more effective vaccine than BCG is immense. New and better tools, greater support for international research, collaborations, and training will be required to dramatically reduce the burden of this devastating disease which still kills 1.6 million people annually.
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Affiliation(s)
- Barry R. Bloom
- Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, MA, USA
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13
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Winkler KR, Mizrahi V, Warner DF, De Wet TJ. High-throughput functional genomics: A (myco)bacterial perspective. Mol Microbiol 2023; 120:141-158. [PMID: 37278255 PMCID: PMC10953053 DOI: 10.1111/mmi.15103] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2020] [Revised: 04/06/2023] [Accepted: 05/21/2023] [Indexed: 06/07/2023]
Abstract
Advances in sequencing technologies have enabled unprecedented insights into bacterial genome composition and dynamics. However, the disconnect between the rapid acquisition of genomic data and the (much slower) confirmation of inferred genetic function threatens to widen unless techniques for fast, high-throughput functional validation can be applied at scale. This applies equally to Mycobacterium tuberculosis, the leading infectious cause of death globally and a pathogen whose genome, despite being among the first to be sequenced two decades ago, still contains many genes of unknown function. Here, we summarize the evolution of bacterial high-throughput functional genomics, focusing primarily on transposon (Tn)-based mutagenesis and the construction of arrayed mutant libraries in diverse bacterial systems. We also consider the contributions of CRISPR interference as a transformative technique for probing bacterial gene function at scale. Throughout, we situate our analysis within the context of functional genomics of mycobacteria, focusing specifically on the potential to yield insights into M. tuberculosis pathogenicity and vulnerabilities for new drug and regimen development. Finally, we offer suggestions for future approaches that might be usefully applied in elucidating the complex cellular biology of this major human pathogen.
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Affiliation(s)
- Kristy R. Winkler
- Molecular Mycobacteriology Research Unit and DSI/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology and Institute of Infectious Disease and Molecular MedicineUniversity of Cape TownRondeboschSouth Africa
| | - Valerie Mizrahi
- Molecular Mycobacteriology Research Unit and DSI/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology and Institute of Infectious Disease and Molecular MedicineUniversity of Cape TownRondeboschSouth Africa
- Wellcome Centre for Infectious Diseases Research in AfricaUniversity of Cape TownRondeboschSouth Africa
| | - Digby F. Warner
- Molecular Mycobacteriology Research Unit and DSI/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology and Institute of Infectious Disease and Molecular MedicineUniversity of Cape TownRondeboschSouth Africa
- Wellcome Centre for Infectious Diseases Research in AfricaUniversity of Cape TownRondeboschSouth Africa
| | - Timothy J. De Wet
- Molecular Mycobacteriology Research Unit and DSI/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology and Institute of Infectious Disease and Molecular MedicineUniversity of Cape TownRondeboschSouth Africa
- Wellcome Centre for Infectious Diseases Research in AfricaUniversity of Cape TownRondeboschSouth Africa
- Department of Integrative Biomedical SciencesUniversity of Cape TownRondeboschSouth Africa
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14
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Chen J, Fruhauf A, Fan C, Ponce J, Ueberheide B, Bhabha G, Ekiert DC. Structure of an endogenous mycobacterial MCE lipid transporter. Nature 2023; 620:445-452. [PMID: 37495693 DOI: 10.1038/s41586-023-06366-0] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2022] [Accepted: 06/22/2023] [Indexed: 07/28/2023]
Abstract
To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.
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Affiliation(s)
- James Chen
- Department of Cell Biology, New York University School of Medicine, New York, NY, USA
| | - Alice Fruhauf
- Department of Cell Biology, New York University School of Medicine, New York, NY, USA
| | - Catherine Fan
- Department of Cell Biology, New York University School of Medicine, New York, NY, USA
| | - Jackeline Ponce
- Proteomics Laboratory, Division of Advanced Research Technologies, New York University School of Medicine, New York, NY, USA
| | - Beatrix Ueberheide
- Proteomics Laboratory, Division of Advanced Research Technologies, New York University School of Medicine, New York, NY, USA
- Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY, USA
- Department of Neurology, New York University School of Medicine, New York, NY, USA
| | - Gira Bhabha
- Department of Cell Biology, New York University School of Medicine, New York, NY, USA.
| | - Damian C Ekiert
- Department of Cell Biology, New York University School of Medicine, New York, NY, USA.
- Department of Microbiology, New York University School of Medicine, New York, NY, USA.
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15
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Adefisayo OO, Curtis ER, Smith CM. Mycobacterial Genetic Technologies for Probing the Host-Pathogen Microenvironment. Infect Immun 2023; 91:e0043022. [PMID: 37249448 PMCID: PMC10269127 DOI: 10.1128/iai.00430-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/31/2023] Open
Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is one of the oldest and most successful pathogens in the world. Diverse selective pressures encountered within host cells have directed the evolution of unique phenotypic traits, resulting in the remarkable evolutionary success of this largely obligate pathogen. Despite centuries of study, the genetic repertoire utilized by Mtb to drive virulence and host immune evasion remains to be fully understood. Various genetic approaches have been and continue to be developed to tackle the challenges of functional gene annotation and validation in an intractable organism such as Mtb. In vitro and ex vivo systems remain the primary approaches to generate and confirm hypotheses that drive a general understanding of mycobacteria biology. However, it remains of great importance to characterize genetic requirements for successful infection within a host system as in vitro and ex vivo studies fail to fully replicate the complex microenvironment experienced by Mtb. In this review, we evaluate the employment of the mycobacterial genetic toolkit to probe the host-pathogen interface by surveying the current state of mycobacterial genetic studies within host systems, with a major focus on the murine model. Specifically, we discuss the different ways that these tools have been utilized to examine various aspects of infection, including bacterial survival/virulence, bacterial evasion of host immunity, and development of novel antibacterial/vaccine strategies.
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Affiliation(s)
| | - Erin R. Curtis
- Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA
| | - Clare M. Smith
- Molecular Genetics and Microbiology, Duke University, Durham, North Carolina, USA
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16
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Jana B, Liu X, Dénéréaz J, Park H, Leshchiner D, Liu B, Gallay C, Veening JW, van Opijnen T. CRISPRi-TnSeq: A genome-wide high-throughput tool for bacterial essential-nonessential genetic interaction mapping. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.05.31.543074. [PMID: 37398100 PMCID: PMC10312587 DOI: 10.1101/2023.05.31.543074] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/04/2023]
Abstract
Genetic interaction networks can help identify functional connections between genes and pathways, which can be leveraged to establish (new) gene function, drug targets, and fill pathway gaps. Since there is no optimal tool that can map genetic interactions across many different bacterial strains and species, we develop CRISPRi-TnSeq, a genome-wide tool that maps genetic interactions between essential genes and nonessential genes through the knockdown of a targeted essential gene (CRISPRi) and the simultaneous knockout of individual nonessential genes (Tn-Seq). CRISPRi-TnSeq thereby identifies, on a genome-wide scale, synthetic and suppressor-type relationships between essential and nonessential genes, enabling the construction of essential-nonessential genetic interaction networks. To develop and optimize CRISPRi-TnSeq, CRISPRi strains were obtained for 13 essential genes in Streptococcus pneumoniae, involved in different biological processes including metabolism, DNA replication, transcription, cell division and cell envelope synthesis. Transposon-mutant libraries were constructed in each strain enabling screening of ∼24,000 gene-gene pairs, which led to the identification of 1,334 genetic interactions, including 754 negative and 580 positive genetic interactions. Through extensive network analyses and validation experiments we identify a set of 17 pleiotropic genes, of which a subset tentatively functions as genetic capacitors, dampening phenotypic outcomes and protecting against perturbations. Furthermore, we focus on the relationships between cell wall synthesis, integrity and cell division and highlight: 1) how essential gene knockdown can be compensated by rerouting flux through nonessential genes in a pathway; 2) the existence of a delicate balance between Z-ring formation and localization, and septal and peripheral peptidoglycan (PG) synthesis to successfully accomplish cell division; 3) the control of c-di-AMP over intracellular K + and turgor, and thereby modulation of the cell wall synthesis machinery; 4) the dynamic nature of cell wall protein CozEb and its effect on PG synthesis, cell shape morphology and envelope integrity; 5) functional dependency between chromosome decatenation and segregation, and the critical link with cell division, and cell wall synthesis. Overall, we show that CRISPRi-TnSeq uncovers genetic interactions between closely functionally linked genes and pathways, as well as disparate genes and pathways, highlighting pathway dependencies and valuable leads for gene function. Importantly, since both CRISPRi and Tn-Seq are widely used tools, CRISPRi-TnSeq should be relatively easy to implement to construct genetic interaction networks across many different microbial strains and species.
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17
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Wei W, Zhao Y, Zhang C, Yu M, Wu Z, Xu L, Peng K, Wu Z, Li Y, Wang X. Whole-genome sequencing and transcriptome-characterized in vitro evolution of aminoglycoside resistance in Mycobacterium tuberculosis. Microb Genom 2023; 9. [PMID: 37224060 DOI: 10.1099/mgen.0.001022] [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: 05/26/2023] Open
Abstract
Antibiotic resistance of Mycobacterium tuberculosis (Mtb) is a major public health concern worldwide. Therefore, it is of great significance to characterize the mutational pathways by which susceptible Mtb evolves into drug resistance. In this study, we used laboratory evolution to explore the mutational pathways of aminoglycoside resistance. The level of resistance in amikacin inducing Mtb was also associated with changes in susceptibility to other anti-tuberculosis drugs such as isoniazid, levofloxacin and capreomycin. Whole-genome sequencing (WGS) revealed that the induced resistant Mtb strains had accumulated diverse mutations. We found that rrs A1401G was the predominant mutation in aminoglycoside-resistant clinical Mtb isolates from Guangdong. In addition, this study provided global insight into the characteristics of the transcriptome in four representative induced strains and revealed that rrs mutated and unmutated aminoglycoside-resistant Mtb strains have different transcriptional profiles. WGS analysis and transcriptional profiling of Mtb strains during evolution revealed that Mtb strains harbouring rrs A1401G have an evolutionary advantage over other drug-resistant strains under the pressure of aminoglycosides because of their ultra-high resistance level and low physiological impact on the strain. The results of this study should advance our understanding of aminoglycoside resistance mechanisms.
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Affiliation(s)
- Wenjing Wei
- Center for Tuberculosis Control of Guangdong Province, Key Laboratory of Translational Medicine of Guangdong, Guangzhou 510630, PR China
| | - Yuchuan Zhao
- Center for Tuberculosis Control of Guangdong Province, Key Laboratory of Translational Medicine of Guangdong, Guangzhou 510630, PR China
| | - Chenchen Zhang
- Center for Tuberculosis Control of Guangdong Province, Key Laboratory of Translational Medicine of Guangdong, Guangzhou 510630, PR China
| | - Meiling Yu
- Center for Tuberculosis Control of Guangdong Province, Key Laboratory of Translational Medicine of Guangdong, Guangzhou 510630, PR China
| | - Zhuhua Wu
- Center for Tuberculosis Control of Guangdong Province, Key Laboratory of Translational Medicine of Guangdong, Guangzhou 510630, PR China
| | - Liuyue Xu
- Center for Tuberculosis Control of Guangdong Province, Key Laboratory of Translational Medicine of Guangdong, Guangzhou 510630, PR China
| | - Kehao Peng
- Center for Tuberculosis Control of Guangdong Province, Key Laboratory of Translational Medicine of Guangdong, Guangzhou 510630, PR China
| | - Zhilong Wu
- Foshan Fourth People's Hospital, Foshan 528000, PR China
| | - Yanxia Li
- Foshan Fourth People's Hospital, Foshan 528000, PR China
| | - Xuezhi Wang
- Foshan Fourth People's Hospital, Foshan 528000, PR China
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18
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Fieweger RA, Wilburn KM, Montague CR, Roszkowski EK, Kelly CM, Southard TL, Sondermann H, Nazarova EV, VanderVen BC. MceG stabilizes the Mce1 and Mce4 transporters in Mycobacterium tuberculosis. J Biol Chem 2023; 299:102910. [PMID: 36642182 PMCID: PMC9947336 DOI: 10.1016/j.jbc.2023.102910] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2022] [Revised: 12/28/2022] [Accepted: 12/29/2022] [Indexed: 01/15/2023] Open
Abstract
Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously, it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.
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Affiliation(s)
- Rachael A Fieweger
- Microbiology & Immunology, College of Veterinary Medicine, Cornell University, Ithaca New York, USA
| | - Kaley M Wilburn
- Microbiology & Immunology, College of Veterinary Medicine, Cornell University, Ithaca New York, USA
| | - Christine R Montague
- Microbiology & Immunology, College of Veterinary Medicine, Cornell University, Ithaca New York, USA
| | - Emma K Roszkowski
- Microbiology & Immunology, College of Veterinary Medicine, Cornell University, Ithaca New York, USA
| | - Carolyn M Kelly
- Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca New York, USA
| | - Teresa L Southard
- Biomedical Sciences; College of Veterinary Medicine, Cornell University, Ithaca New York, USA
| | - Holger Sondermann
- Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca New York, USA
| | - Evgeniya V Nazarova
- Microbiology & Immunology, College of Veterinary Medicine, Cornell University, Ithaca New York, USA
| | - Brian C VanderVen
- Microbiology & Immunology, College of Veterinary Medicine, Cornell University, Ithaca New York, USA.
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19
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Chen J, Fruhauf A, Fan C, Ponce J, Ueberheide B, Bhabha G, Ekiert D. Structure of an endogenous mycobacterial MCE lipid transporter. RESEARCH SQUARE 2023:rs.3.rs-2412186. [PMID: 36711512 PMCID: PMC9882608 DOI: 10.21203/rs.3.rs-2412186/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 05/17/2023]
Abstract
To replicate inside human macrophages and cause the disease tuberculosis, Mycobacterium tuberculosis ( Mtb ) must scavenge a variety of nutrients from the host 1,2 . The Mammalian Cell Entry (MCE) proteins are important virulence factors in Mtb 1,3 , where they are encoded in large gene clusters and have been implicated in the transport of fatty acids 4â€"7 and cholesterol 1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and how the ~10 proteins encoded in a mycobacterial mce gene cluster might assemble to transport cargo across the cell envelope remains unknown. Here we report the cryo-EM structure of the endogenous Mce1 fatty acid import machine from Mycobacterium smegmatis , a non-pathogenic relative of Mtb . The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex, long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Unexpectedly, our structural data revealed the presence of a previously unknown subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated fatty acid import across the mycobacterial cell envelope.
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20
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Hogan AM, Cardona ST. Gradients in gene essentiality reshape antibacterial research. FEMS Microbiol Rev 2022; 46:fuac005. [PMID: 35104846 PMCID: PMC9075587 DOI: 10.1093/femsre/fuac005] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2021] [Revised: 01/14/2022] [Accepted: 01/24/2022] [Indexed: 02/03/2023] Open
Abstract
Essential genes encode the processes that are necessary for life. Until recently, commonly applied binary classifications left no space between essential and non-essential genes. In this review, we frame bacterial gene essentiality in the context of genetic networks. We explore how the quantitative properties of gene essentiality are influenced by the nature of the encoded process, environmental conditions and genetic background, including a strain's distinct evolutionary history. The covered topics have important consequences for antibacterials, which inhibit essential processes. We argue that the quantitative properties of essentiality can thus be used to prioritize antibacterial cellular targets and desired spectrum of activity in specific infection settings. We summarize our points with a case study on the core essential genome of the cystic fibrosis pathobiome and highlight avenues for targeted antibacterial development.
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Affiliation(s)
- Andrew M Hogan
- Department of Microbiology, University of Manitoba, 45 Chancellor's Circle, Winnipeg, Manitoba R3T 2N2, Canada
| | - Silvia T Cardona
- Department of Microbiology, University of Manitoba, 45 Chancellor's Circle, Winnipeg, Manitoba R3T 2N2, Canada
- Department of Medical Microbiology and Infectious Diseases, Max Rady College of Medicine, University of Manitoba, Room 543 - 745 Bannatyne Avenue, Winnipeg, Manitoba, R3E 0J9, Canada
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21
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Grønberg C, Hu Q, Mahato DR, Longhin E, Salustros N, Duelli A, Lyu P, Bågenholm V, Eriksson J, Rao KU, Henderson DI, Meloni G, Andersson M, Croll T, Godaly G, Wang K, Gourdon P. Structure and ion-release mechanism of P IB-4-type ATPases. eLife 2021; 10:73124. [PMID: 34951590 PMCID: PMC8880997 DOI: 10.7554/elife.73124] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Accepted: 12/17/2021] [Indexed: 11/13/2022] Open
Abstract
Transition metals, such as zinc, are essential micronutrients in all organisms, but also highly toxic in excessive amounts. Heavy-metal transporting P-type (PIB) ATPases are crucial for homeostasis, conferring cellular detoxification and redistribution through transport of these ions across cellular membranes. No structural information is available for the PIB-4-ATPases, the subclass with the broadest cargo scope, and hence even their topology remains elusive. Here we present structures and complementary functional analyses of an archetypal PIB‑4‑ATPase, sCoaT from Sulfitobacter sp. NAS14-1. The data disclose the architecture, devoid of classical so-called heavy metal binding domains, and provides fundamentally new insights into the mechanism and diversity of heavy-metal transporters. We reveal several novel P-type ATPase features, including a dual role in heavy-metal release and as an internal counter ion of an invariant histidine. We also establish that the turn-over of PIB‑ATPases is potassium independent, contrasting to many other P-type ATPases. Combined with new inhibitory compounds, our results open up for efforts in e.g. drug discovery, since PIB-4-ATPases function as virulence factors in many pathogens.
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Affiliation(s)
- Christina Grønberg
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen N, Denmark
| | - Qiaoxia Hu
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
| | | | - Elena Longhin
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark
| | - Nina Salustros
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen N, Denmark
| | - Annette Duelli
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen N, Denmark
| | - Pin Lyu
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen N, Denmark
| | - Viktoria Bågenholm
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen N, Denmark
| | | | | | | | - Gabriele Meloni
- Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, United States
| | | | - Tristan Croll
- Department of Haematology, University of Cambridge, Cambridge, United Kingdom
| | - Gabriela Godaly
- Department of Laboratory Medicine, Umeå University, Umeå, Sweden
| | - Kaituo Wang
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen N, Denmark
| | - Pontus Gourdon
- Department of Biomedical Sciences, University of Copenhagen, Copenhagen N, Denmark
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22
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Asthana P, Singh D, Pedersen JS, Hynönen MJ, Sulu R, Murthy AV, Laitaoja M, Jänis J, Riley LW, Venkatesan R. Structural insights into the substrate-binding proteins Mce1A and Mce4A from Mycobacterium tuberculosis. IUCRJ 2021; 8:757-774. [PMID: 34584737 PMCID: PMC8420772 DOI: 10.1107/s2052252521006199] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/07/2021] [Accepted: 06/15/2021] [Indexed: 05/28/2023]
Abstract
Mycobacterium tuberculosis (Mtb), which is responsible for more than a million deaths annually, uses lipids as the source of carbon and energy for its survival in the latent phase of infection. Mtb cannot synthesize all of the lipid molecules required for its growth and pathogenicity. Therefore, it relies on transporters such as the mammalian cell entry (Mce) complexes to import lipids from the host across the cell wall. Despite their importance for the survival and pathogenicity of Mtb, information on the structural properties of these proteins is not yet available. Each of the four Mce complexes in Mtb (Mce1-4) comprises six substrate-binding proteins (SBPs; MceA-F), each of which contains four conserved domains (N-terminal transmembrane, MCE, helical and C-terminal unstructured tail domains). Here, the properties of the various domains of Mtb Mce1A and Mce4A, which are involved in the import of mycolic/fatty acids and cholesterol, respectively, are reported. In the crystal structure of the MCE domain of Mce4A (MtMce4A39-140) a domain-swapped conformation is observed, whereas solution studies, including small-angle X-ray scattering (SAXS), indicate that all Mce1A and Mce4A domains are predominantly monomeric. Further, structural comparisons show interesting differences from the bacterial homologs MlaD, PqiB and LetB, which form homohexamers when assembled as functional transporter complexes. These data, and the fact that there are six SBPs in each Mtb mce operon, suggest that the MceA-F SBPs from Mce1-4 may form heterohexamers. Also, interestingly, the purification and SAXS analysis showed that the helical domains interact with the detergent micelle, suggesting that when assembled the helical domains of MceA-F may form a hydrophobic pore for lipid transport, as observed in EcPqiB. Overall, these data highlight the unique structural properties of the Mtb Mce SBPs.
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Affiliation(s)
- Pooja Asthana
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
| | - Dhirendra Singh
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
| | - Jan Skov Pedersen
- Department of Chemistry and Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus, Denmark
| | - Mikko J. Hynönen
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
| | - Ramita Sulu
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
| | - Abhinandan V. Murthy
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
| | - Mikko Laitaoja
- Department of Chemistry, University of Eastern Finland, Joensuu, Finland
| | - Janne Jänis
- Department of Chemistry, University of Eastern Finland, Joensuu, Finland
| | - Lee W. Riley
- School of Public Health, University of California, Berkeley, California, USA
| | - Rajaram Venkatesan
- Faculty of Biochemistry and Molecular Medicine, University of Oulu, Oulu, Finland
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23
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Effect of the deletion of lprG and p55 genes in the K10 strain of Mycobacterium avium subspecies paratuberculosis. Res Vet Sci 2021; 138:1-10. [PMID: 34087563 DOI: 10.1016/j.rvsc.2021.05.019] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Revised: 04/23/2021] [Accepted: 05/25/2021] [Indexed: 11/21/2022]
Abstract
The lprG-p55 operon of Mycobacterium tuberculosis, M. bovis and M. avium strain D4ER has been identified as a virulence factor involved in the transport of toxic compounds. LprG is a lipoprotein that modulates the host immune response against mycobacteria, whereas P55 is an efflux pump that provides resistance to several drugs. In the present study we search for, and characterize, lprg and p55, putative virulence genes in Mycobacterium avium subsp. paratuberculosis (MAP) to generate a live-attenuated strain of MAP that may be useful in the future as live-attenuated vaccine. For this purpose, we generated and evaluated two mutants of MAP strain K10: one mutant lacking the lprG gene (ΔlprG) and the other lacking both genes lprG and p55 (ΔlprG-p55). None of the mutant strains showed altered susceptibility to first-line and second-line antituberculosis drugs or ethidium bromide, only the double mutant had two-fold increase in clarithromycin susceptibility compared with the wild-type strain. The deletion of lprG and of lprG-p55 reduced the replication of MAP in bovine macrophages; however, only the mutant in lprG-p55 grew faster in liquid media and showed reduced viability in macrophages and in a mouse model. Considering that the deletion of both genes lprG-p55, but not that of lprG alone, showed a reduced replication in vivo, we can speculate that p55 contributes to the survival of MAP in this animal model.
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24
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Halder V, McDonnell B, Uthayakumar D, Usher J, Shapiro RS. Genetic interaction analysis in microbial pathogens: unravelling networks of pathogenesis, antimicrobial susceptibility and host interactions. FEMS Microbiol Rev 2021; 45:fuaa055. [PMID: 33145589 DOI: 10.1093/femsre/fuaa055] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Accepted: 10/16/2020] [Indexed: 12/13/2022] Open
Abstract
Genetic interaction (GI) analysis is a powerful genetic strategy that analyzes the fitness and phenotypes of single- and double-gene mutant cells in order to dissect the epistatic interactions between genes, categorize genes into biological pathways, and characterize genes of unknown function. GI analysis has been extensively employed in model organisms for foundational, systems-level assessment of the epistatic interactions between genes. More recently, GI analysis has been applied to microbial pathogens and has been instrumental for the study of clinically important infectious organisms. Here, we review recent advances in systems-level GI analysis of diverse microbial pathogens, including bacterial and fungal species. We focus on important applications of GI analysis across pathogens, including GI analysis as a means to decipher complex genetic networks regulating microbial virulence, antimicrobial drug resistance and host-pathogen dynamics, and GI analysis as an approach to uncover novel targets for combination antimicrobial therapeutics. Together, this review bridges our understanding of GI analysis and complex genetic networks, with applications to diverse microbial pathogens, to further our understanding of virulence, the use of antimicrobial therapeutics and host-pathogen interactions. .
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Affiliation(s)
- Viola Halder
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada
| | - Brianna McDonnell
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada
| | - Deeva Uthayakumar
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada
| | - Jane Usher
- Medical Research Council Centre for Medical Mycology, University of Exeter, Geoffrey Pope Building, Stocker Road, Exeter EX4 4QD, UK
| | - Rebecca S Shapiro
- Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada
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25
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Evidence for the Mycobacterial Mce4 Transporter Being a Multiprotein Complex. J Bacteriol 2021; 203:JB.00685-20. [PMID: 33649150 DOI: 10.1128/jb.00685-20] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Accepted: 02/24/2021] [Indexed: 01/01/2023] Open
Abstract
Mycobacteria possess Mce transporters that import lipids and are thought to function analogously to ATP-binding cassette (ABC) transporters. However, whereas ABC transporters import substrates using a single solute-binding protein (SBP) to deliver a substrate to permease proteins in the membrane, mycobacterial Mce transporters have a potential for six SBPs (MceA to MceF) working with a pair of permeases (YrbEA and YrbEB), a cytoplasmic ATPase (MceG), and multiple Mce-associated membrane (Mam) and orphaned Mam (Omam) proteins to transport lipids. In this study, we used the model mycobacterium Mycobacterium smegmatis to study the requirement for individual Mce, Mam, and Omam proteins in Mce4 transport of cholesterol. All of the Mce4 and Mam4 proteins we investigated were required for cholesterol uptake. However, not all Omam proteins, which are encoded by genes outside mce loci, proved to contribute to cholesterol import. OmamA and OmamB were required for cholesterol import, while OmamC, OmamD, OmamE, and OmamF were not. In the absence of any single Mce4, Mam4, or Omam protein that we tested, the abundance of Mce4A and Mce4E declined. This relationship between the levels of Mce4A and Mce4E and these additional proteins suggests a network of interactions that assemble and/or stabilize a multiprotein Mce4 transporter complex. Further support for Mce transporters being multiprotein complexes was obtained by immunoprecipitation-mass spectrometry, in which we identified every single Mce, YrbE, MceG, Mam, and Omam protein with a role in cholesterol transport as associating with Mce4A. This study represents the first time any of these Mce4 transporter proteins has been shown to associate.IMPORTANCE How lipids travel between membranes of diderm bacteria is a challenging mechanistic question because lipids, which are hydrophobic molecules, must traverse a hydrophilic periplasm. This question is even more complex for mycobacteria, which have a unique cell envelope that is highly impermeable to molecules. A growing body of knowledge identifies Mce transporters as lipid importers for mycobacteria. Here, using protein stability experiments and immunoprecipitation-mass spectrometry, we provide evidence for mycobacterial Mce transporters existing as multiprotein complexes.
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26
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Rosiana S, Zhang L, Kim GH, Revtovich AV, Uthayakumar D, Sukumaran A, Geddes-McAlister J, Kirienko NV, Shapiro RS. Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans. Genetics 2021; 217:iyab003. [PMID: 33724419 PMCID: PMC8045720 DOI: 10.1093/genetics/iyab003] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2020] [Accepted: 12/31/2020] [Indexed: 12/14/2022] Open
Abstract
Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans' ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.
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Affiliation(s)
- Sierra Rosiana
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON NIG 2W1, Canada
| | - Liyang Zhang
- Department of BioSciences, Rice University, Houston, TX 77005, USA
| | - Grace H Kim
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON NIG 2W1, Canada
| | | | - Deeva Uthayakumar
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON NIG 2W1, Canada
| | - Arjun Sukumaran
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON NIG 2W1, Canada
| | | | | | - Rebecca S Shapiro
- Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON NIG 2W1, Canada
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27
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Chang DPS, Guan XL. Metabolic Versatility of Mycobacterium tuberculosis during Infection and Dormancy. Metabolites 2021; 11:88. [PMID: 33540752 PMCID: PMC7913082 DOI: 10.3390/metabo11020088] [Citation(s) in RCA: 30] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2020] [Revised: 01/16/2021] [Accepted: 01/29/2021] [Indexed: 12/18/2022] Open
Abstract
Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a highly successful intracellular pathogen with the ability to withstand harsh conditions and reside long-term within its host. In the dormant and persistent states, the bacterium tunes its metabolism and is able to resist the actions of antibiotics. One of the main strategies Mtb adopts is through its metabolic versatility-it is able to cometabolize a variety of essential nutrients and direct these nutrients simultaneously to multiple metabolic pathways to facilitate the infection of the host. Mtb further undergo extensive remodeling of its metabolic pathways in response to stress and dormancy. In recent years, advancement in systems biology and its applications have contributed substantially to a more coherent view on the intricate metabolic networks of Mtb. With a more refined appreciation of the roles of metabolism in mycobacterial infection and drug resistance, and the success of drugs targeting metabolism, there is growing interest in further development of anti-TB therapies that target metabolism, including lipid metabolism and oxidative phosphorylation. Here, we will review current knowledge revolving around the versatility of Mtb in remodeling its metabolism during infection and dormancy, with a focus on central carbon metabolism and lipid metabolism.
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Affiliation(s)
| | - Xue Li Guan
- Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921, Singapore;
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28
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Harnagel A, Lopez Quezada L, Park SW, Baranowski C, Kieser K, Jiang X, Roberts J, Vaubourgeix J, Yang A, Nelson B, Fay A, Rubin E, Ehrt S, Nathan C, Lupoli TJ. Nonredundant functions of Mycobacterium tuberculosis chaperones promote survival under stress. Mol Microbiol 2020; 115:272-289. [PMID: 32996193 DOI: 10.1111/mmi.14615] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2020] [Revised: 09/21/2020] [Accepted: 09/22/2020] [Indexed: 12/12/2022]
Abstract
Bacterial chaperones ClpB and DnaK, homologs of the respective eukaryotic heat shock proteins Hsp104 and Hsp70, are essential in the reactivation of toxic protein aggregates that occur during translation or periods of stress. In the pathogen Mycobacterium tuberculosis (Mtb), the protective effect of chaperones extends to survival in the presence of host stresses, such as protein-damaging oxidants. However, we lack a full understanding of the interplay of Hsps and other stress response genes in mycobacteria. Here, we employ genome-wide transposon mutagenesis to identify the genes that support clpB function in Mtb. In addition to validating the role of ClpB in Mtb's response to oxidants, we show that HtpG, a homolog of Hsp90, plays a distinct role from ClpB in the proteotoxic stress response. While loss of neither clpB nor htpG is lethal to the cell, loss of both through genetic depletion or small molecule inhibition impairs recovery after exposure to host-like stresses, especially reactive nitrogen species. Moreover, defects in cells lacking clpB can be complemented by overexpression of other chaperones, demonstrating that Mtb's stress response network depends upon finely tuned chaperone expression levels. These results suggest that inhibition of multiple chaperones could work in concert with host immunity to disable Mtb.
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Affiliation(s)
- Alexa Harnagel
- Department of Chemistry, New York University, New York, NY, USA
| | - Landys Lopez Quezada
- Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA
| | - Sae Woong Park
- Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA
| | - Catherine Baranowski
- Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Karen Kieser
- Department of Immunology and Infectious Disease, Harvard T.H. Chan School of Public Health, Boston, MA, USA
| | - Xiuju Jiang
- Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA
| | - Julia Roberts
- Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA
| | - Julien Vaubourgeix
- Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA
| | - Amy Yang
- Department of Chemistry, New York University, New York, NY, USA
| | - Brock Nelson
- Department of Chemistry, New York University, New York, NY, USA
| | - Allison Fay
- Immunology Program, Sloan Kettering Institute, New York, NY, USA
| | - Eric Rubin
- Immunology Program, Sloan Kettering Institute, New York, NY, USA
| | - Sabine Ehrt
- Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA
| | - Carl Nathan
- Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA
| | - Tania J Lupoli
- Department of Chemistry, New York University, New York, NY, USA.,Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA
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29
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Baena A, Vasco E, Pastrana M, Alzate JF, Barrera LF, Martínez A. New Conjugated Compound T5 Epidioxy-Sterol-ANB Inhibits the Growth of Mycobacterium tuberculosis Affecting the Cholesterol and Folate Pathways. Front Microbiol 2020; 11:537935. [PMID: 33072006 PMCID: PMC7533559 DOI: 10.3389/fmicb.2020.537935] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2020] [Accepted: 08/13/2020] [Indexed: 11/13/2022] Open
Abstract
The upsurge and persistence of drug resistant strains of Mycobacterium tuberculosis (Mtb) is an important limitant to the battery of drugs available for the elimination of tuberculosis (TB). To avoid future scarcity of antibiotics against Mtb, it is important to discover new effective anti-mycobacterial agents. In this study, we present data from a series of experiments to determine in vitro and in vivo anti-mycobacterial activity of a library of epidioxy-sterol analogs. We test 15 compounds for their ability to reduce the viability of Mtb. We found that one compound called T5 epidioxy-sterol-ANB display significant potency against Mtb in vitro specifically inside macrophages but without effectivity in axenic cultures. A viability assay confirms that this T5 compound is less toxic for macrophages in vitro as compared to the current Mtb drug Rifampicin at higher concentrations. We use a transcriptomic analysis of Mtb inside macrophages after T5 epidioxy-sterol-ANB treatment, and we found a significant down-regulation of enzymes involved in the cholesterol and folic acid pathways. In vivo, significant differences were found in the lungs and spleen CFUs of Mtb infected mice treated with the T5 epidioxy-sterol-ANB as compared with the untreated control group, which provides additional evidence of the effectivity of the T5 compound. Altogether these results confirm the potential of this T5 epidioxy-sterol-ANB compound against Mtb.
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Affiliation(s)
- Andres Baena
- Grupo de Inmunología Celular e Inmunogenética, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
| | - Emanuel Vasco
- Grupo de Inmunología Celular e Inmunogenética, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
| | - Manuel Pastrana
- Grupo de Productos Naturales Marinos, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia, Medellín, Colombia
| | - Juan F Alzate
- Grupo de Parasitología, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia.,Centro Nacional de Secuenciación Genómica, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
| | - Luis F Barrera
- Grupo de Inmunología Celular e Inmunogenética, Facultad de Medicina, Universidad de Antioquia, Medellín, Colombia
| | - Alejandro Martínez
- Grupo de Productos Naturales Marinos, Facultad de Ciencias Farmacéuticas y Alimentarias, Universidad de Antioquia, Medellín, Colombia
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30
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Smith AA, Villarreal-Ramos B, Mendum TA, Williams KJ, Jones GJ, Wu H, McFadden J, Vordermeier HM, Stewart GR. Genetic screening for the protective antigenic targets of BCG vaccination. Tuberculosis (Edinb) 2020; 124:101979. [PMID: 32814303 DOI: 10.1016/j.tube.2020.101979] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 07/10/2020] [Accepted: 07/13/2020] [Indexed: 11/18/2022]
Abstract
Bovine tuberculosis is an important animal health problem and the predominant cause of zoonotic tuberculosis worldwide. It results in serious economic burden due to losses in productivity and the cost of control programmes. Control could be greatly improved by the introduction of an efficacious cattle vaccine but the most likely candidate, BCG, has several limitations including variable efficacy. Augmentation of BCG with a subunit vaccine booster has been shown to increase protection but the selection of antigens has hitherto been left largely to serendipity. In the present study, we take a rational approach to identify the protective antigens of BCG, selecting a BCG transposon mutant library in naïve and BCG-vaccinated cattle. Ten mutants had increased relative survival in vaccinated compared to naïve cattle, consistent with loss of protective antigen targets making the mutants less visible to the BCG immune response. The immunogenicity of three putative protective antigens, BCG_0116, BCG_0205 (YrbE1B) and BCG_1448 (PPE20) was investigated using peptide pools and PBMCs from BCG vaccinated cattle. BCG vaccination induced PBMC to release elevated levels of IP10, IL-17a and IL-10 in response to all three antigens. Taken together, the data supports the further study of these antigens for use in subunit vaccines.
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MESH Headings
- Animals
- Antigens, Bacterial/administration & dosage
- Antigens, Bacterial/genetics
- Antigens, Bacterial/immunology
- BCG Vaccine/administration & dosage
- BCG Vaccine/immunology
- Cattle
- Cytokines/immunology
- Cytokines/metabolism
- DNA Transposable Elements
- Immunogenicity, Vaccine
- Leukocytes, Mononuclear/immunology
- Leukocytes, Mononuclear/metabolism
- Leukocytes, Mononuclear/microbiology
- Mutation
- Mycobacterium tuberculosis/genetics
- Mycobacterium tuberculosis/immunology
- Tuberculosis, Bovine/immunology
- Tuberculosis, Bovine/metabolism
- Tuberculosis, Bovine/microbiology
- Tuberculosis, Bovine/prevention & control
- Vaccination/veterinary
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Affiliation(s)
- Alex A Smith
- Department of Microbial Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK
| | - Bernardo Villarreal-Ramos
- Department of Bacteriology, Animal and Plant Health Agency, Weybridge, KT15 3NB, UK; Centre of Excellence for Bovine Tuberculosis, Institute for Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, Wales, SY23 3DA, UK.
| | - Tom A Mendum
- Department of Microbial Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK
| | - Kerstin J Williams
- Department of Microbial Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK
| | - Gareth J Jones
- Department of Bacteriology, Animal and Plant Health Agency, Weybridge, KT15 3NB, UK
| | - Huihai Wu
- Department of Microbial Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK
| | - Johnjoe McFadden
- Department of Microbial Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK
| | - H Martin Vordermeier
- Department of Bacteriology, Animal and Plant Health Agency, Weybridge, KT15 3NB, UK; Centre of Excellence for Bovine Tuberculosis, Institute for Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, Wales, SY23 3DA, UK.
| | - Graham R Stewart
- Department of Microbial Sciences, School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK.
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31
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McClean CM, Tobin DM. Early cell-autonomous accumulation of neutral lipids during infection promotes mycobacterial growth. PLoS One 2020; 15:e0232251. [PMID: 32407412 PMCID: PMC7224534 DOI: 10.1371/journal.pone.0232251] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2019] [Accepted: 04/12/2020] [Indexed: 11/19/2022] Open
Abstract
Lipids represent an important source of nutrition for infecting mycobacteria, accumulating within the necrotic core of granulomas and present in foamy macrophages associated with mycobacterial infection. In order to better understand the timing, process and importance of lipid accumulation, we developed methods for direct in vivo visualization and quantification of this process using the zebrafish-M. marinum larval model of infection. We find that neutral lipids accumulate cell-autonomously in mycobacterium-infected macrophages in vivo during early infection, with detectable levels of accumulation by two days post-infection. Treatment with ezetimibe, an FDA-approved drug, resulted in decreased levels of free cholesterol and neutral lipids, and a reduction of bacterial growth in vivo. The effect of ezetimibe in reducing bacterial growth was dependent on the mce4 operon, a key bacterial determinant of lipid utilization. Thus, in vivo, lipid accumulation can occur cell-autonomously at early timepoints of mycobacterial infection, and limitation of this process results in decreased bacterial burden.
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Affiliation(s)
- Colleen M. McClean
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America
- Department of Immunology, Duke University School of Medicine, Durham, North Carolina, United States of America
- Medical Scientist Training Program, Duke University School of Medicine, Durham, North Carolina, United States of America
| | - David M. Tobin
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America
- Department of Immunology, Duke University School of Medicine, Durham, North Carolina, United States of America
- * E-mail:
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32
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Mycobacterium tuberculosis YrbE3A Promotes Host Innate Immune Response by Targeting NF-κB/JNK Signaling. Microorganisms 2020; 8:microorganisms8040584. [PMID: 32316659 PMCID: PMC7232258 DOI: 10.3390/microorganisms8040584] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2020] [Revised: 04/14/2020] [Accepted: 04/15/2020] [Indexed: 11/24/2022] Open
Abstract
Mycobacterium tuberculosis is considered a successful pathogen with multiple strategies to undermine host immunity. The YrbE3A is encoded by Rv1964 within the RD15 region present in the genome of Mtb, but missing in M. bovis, M. bovis BCG (Pasteur) strain, and M. smegmatis (Ms). However, little is known about its function. In this study, the YrbE3A gene was cloned into pMV261 and expressed in Ms and BCG, while the strains with the vector served as the controls. The YrbE3A was expressed on the mycobacterial membrane, and the purified protein could stimulate RAW264.7 cells to produce IL-6. Furthermore, the effect of the recombinant strains on cytokine secretion by RAW264.7 was confirmed, which varied with the host strains. Ms_YrbE3A increased significantly higher levels of TNF-α and IL-6 than did Ms_vec, while BCG_YrbE3A enhanced higher TNF-α than BCG_vec. The pathways associated with NF-κB p65 and MAPK p38/JNK, other than Erk1/2, regulated this process. In addition, mice were infected with Ms_YrbE3A and Ms-vec and were kinetically examined. Compared to Ms-vec, Ms_YrbE3A induced more serious inflammatory damage, higher levels of TNF-α and IL-6, higher numbers of lymphocytes, neutrophils, and monocytes in a time-dependent way, but lower lung bacterial load in lung. These findings may contribute to a better understanding of Mtb pathogenesis.
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33
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Alonso MN, Malaga W, Mc Neil M, Jackson M, Romano MI, Guilhot C, Santangelo MP. Efficient method for targeted gene disruption by homologous recombination in Mycobacterium avium subspecie paratuberculosis. Res Microbiol 2020; 171:203-210. [PMID: 32283218 DOI: 10.1016/j.resmic.2020.04.001] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2020] [Revised: 04/02/2020] [Accepted: 04/03/2020] [Indexed: 10/24/2022]
Abstract
Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.
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Affiliation(s)
- Maria Natalia Alonso
- IABIMO Instituto de Agrobiotecnología y Biología Molecular, INTA-CONICET, Los Reseros y Nicolas Repetto 1686, Hurlingham, Buenos Aires, Argentina.
| | - Wladimir Malaga
- Institut de Pharmacologie et de Biologie Structurale, IPBS, University of Toulouse, CNRS, UPS, BP64182 205 Route de Narbonne, 31077 Toulouse Cedex 04, France.
| | - Michael Mc Neil
- Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
| | - Mary Jackson
- Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.
| | - Maria Isabel Romano
- IABIMO Instituto de Agrobiotecnología y Biología Molecular, INTA-CONICET, Los Reseros y Nicolas Repetto 1686, Hurlingham, Buenos Aires, Argentina.
| | - Christophe Guilhot
- Institut de Pharmacologie et de Biologie Structurale, IPBS, University of Toulouse, CNRS, UPS, BP64182 205 Route de Narbonne, 31077 Toulouse Cedex 04, France.
| | - María Paz Santangelo
- IABIMO Instituto de Agrobiotecnología y Biología Molecular, INTA-CONICET, Los Reseros y Nicolas Repetto 1686, Hurlingham, Buenos Aires, Argentina.
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Hemati Z, Derakhshandeh A, Haghkhah M, Chaubey KK, Gupta S, Singh M, Singh SV, Dhama K. Mammalian cell entry operons; novel and major subset candidates for diagnostics with special reference to Mycobacterium avium subspecies paratuberculosis infection. Vet Q 2020; 39:65-75. [PMID: 31282842 PMCID: PMC6830979 DOI: 10.1080/01652176.2019.1641764] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Mammalian cell entry (mce) genes are the components of the mce operon and play a vital role in the entry of Mycobacteria into the mammalian cell and their survival within phagocytes and epithelial cells. Mce operons are present in the DNA of Mycobacteria and translate proteins associated with the invasion and long-term existence of these pathogens in macrophages. The exact mechanism of action of mce genes and their functions are not clear yet. However, with the loss of these genes Mycobacteria lose their pathogenicity. Mycobacterium avium subspecies paratuberculosis (MAP), the etiological agent of Johne’s disease, is the cause of chronic enteritis of animals and significantly affects economic impact on the livestock industry. Since MAP is not inactivated during pasteurization, human population is continuously at the risk of getting exposed to MAP infection through consumption of dairy products. There is need for new candidate genes and/or proteins for developing improved diagnostic assays for the diagnosis of MAP infection and for the control of disease. Increasing evidences showed that expression of mce genes is important for the virulence of MAP. Whole-genome DNA microarray representing MAP revealed that there are 14 large sequence polymorphisms with LSPP12 being the most widely conserved MAP-specific region that included a cluster of six homologs of mce-family involved in lipid metabolism. On the other hand, LSP11 comprising part of mce2 operon was absent in MAP isolates. This review summarizes the advancement of research on mce genes of Mycobacteria with special reference to the MAP infection.
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Affiliation(s)
- Zahra Hemati
- Department of Pathobiology, School of Veterinary Medicine, Shiraz University , Shiraz , Iran
| | - Abdollah Derakhshandeh
- Department of Pathobiology, School of Veterinary Medicine, Shiraz University , Shiraz , Iran
| | - Masoud Haghkhah
- Department of Pathobiology, School of Veterinary Medicine, Shiraz University , Shiraz , Iran
| | - Kundan Kumar Chaubey
- Department of Biotechnology, Institute of Applied Sciences and Humanities, GLA University , Mathura , India
| | - Saurabh Gupta
- Department of Biotechnology, Institute of Applied Sciences and Humanities, GLA University , Mathura , India
| | - Manju Singh
- Department of Biotechnology, Institute of Applied Sciences and Humanities, GLA University , Mathura , India
| | - Shoorvir V Singh
- Department of Biotechnology, Institute of Applied Sciences and Humanities, GLA University , Mathura , India
| | - Kuldeep Dhama
- Department of Pathology, Indian Veterinary Research Institute , Bareilly , India
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35
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Fenn K, Wong CT, Darbari VC. Mycobacterium tuberculosis Uses Mce Proteins to Interfere With Host Cell Signaling. Front Mol Biosci 2020; 6:149. [PMID: 31998747 PMCID: PMC6961568 DOI: 10.3389/fmolb.2019.00149] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2019] [Accepted: 12/05/2019] [Indexed: 12/15/2022] Open
Abstract
Tuberculosis continues to be the main cause for mortality by an infectious agent, making Mycobacterium tuberculosis one of the most successful pathogens to survive for long durations within human cells. In order to survive against host defenses, M. tuberculosis modulates host cell signaling. It employs many proteins to achieve this and the Mce proteins are emerging as one group that play a role in host cell signaling in addition to their primary role as lipid/sterol transporters. Mce proteins belong to the conserved Mce/MlaD superfamily ubiquitous in diderm bacteria and chloroplasts. In mycobacteria, mce operons, encode for six different Mce proteins that assemble with inner membrane permeases into complexes that span across the mycobacterial cell wall. Their involvement in signaling modulation is varied and they have been shown to bind ERK1/2 to alter host cytokine expression; eEF1A1 to promote host cell proliferation and integrins for host cell adherence and entry. Recently, structures of prokaryotic Mce/MlaD proteins have been determined, giving an insight into the conserved domain. In this mini-review, we discuss current evidence for the role of mycobacterial Mce proteins in host cell signaling and structural characteristics of the protein-protein interactions coordinated by the human proteins to modulate the host signaling.
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Affiliation(s)
- Katherine Fenn
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
| | - Chi Tung Wong
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
| | - Vidya Chandran Darbari
- School of Biological and Chemical Sciences, Queen Mary University of London, London, United Kingdom
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36
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Borgers K, Vandewalle K, Festjens N, Callewaert N. A guide to Mycobacterium mutagenesis. FEBS J 2019; 286:3757-3774. [PMID: 31419030 DOI: 10.1111/febs.15041] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2019] [Revised: 07/05/2019] [Accepted: 08/12/2019] [Indexed: 12/18/2022]
Abstract
The genus Mycobacterium includes several pathogens that cause severe disease in humans, like Mycobacterium tuberculosis (M. tb), the infectious agent causing tuberculosis. Genetic tools to engineer mycobacterial genomes, in a targeted or random fashion, have provided opportunities to investigate M. tb infection and pathogenesis. Furthermore, they have allowed the identification and validation of potential targets for the diagnosis, prevention, and treatment of tuberculosis. This review describes the various methods that are available for the generation of mutants in Mycobacterium species, focusing specifically on tools for altering slow-growing mycobacteria from the M. tb complex. Among others, it incorporates the recent new molecular biological technologies (e.g. ORBIT) to rapidly and/or genome-wide comprehensively obtain targeted mutants in mycobacteria. As such, this review can be used as a guide to select the appropriate genetic tools to generate mycobacterial mutants of interest, which can be used as tools to aid understanding of M. tb infection or to help developing TB intervention strategies.
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Affiliation(s)
- Katlyn Borgers
- VIB-UGhent Center for Medical Biotechnology, Belgium.,Department of Biochemistry and Microbiology, Ghent University, Belgium
| | - Kristof Vandewalle
- VIB-UGhent Center for Medical Biotechnology, Belgium.,Department of Biochemistry and Microbiology, Ghent University, Belgium
| | - Nele Festjens
- VIB-UGhent Center for Medical Biotechnology, Belgium.,Department of Biochemistry and Microbiology, Ghent University, Belgium
| | - Nico Callewaert
- VIB-UGhent Center for Medical Biotechnology, Belgium.,Department of Biochemistry and Microbiology, Ghent University, Belgium
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37
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Olivera ER, Luengo JM. Steroids as Environmental Compounds Recalcitrant to Degradation: Genetic Mechanisms of Bacterial Biodegradation Pathways. Genes (Basel) 2019; 10:E512. [PMID: 31284586 PMCID: PMC6678751 DOI: 10.3390/genes10070512] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2019] [Revised: 07/02/2019] [Accepted: 07/03/2019] [Indexed: 12/29/2022] Open
Abstract
Steroids are perhydro-1,2-cyclopentanophenanthrene derivatives that are almost exclusively synthesised by eukaryotic organisms. Since the start of the Anthropocene, the presence of these molecules, as well as related synthetic compounds (ethinylestradiol, dexamethasone, and others), has increased in different habitats due to farm and municipal effluents and discharge from the pharmaceutical industry. In addition, the highly hydrophobic nature of these molecules, as well as the absence of functional groups, makes them highly resistant to biodegradation. However, some environmental bacteria are able to modify or mineralise these compounds. Although steroid-metabolising bacteria have been isolated since the beginning of the 20th century, the genetics and catabolic pathways used have only been characterised in model organisms in the last few decades. Here, the metabolic alternatives used by different bacteria to metabolise steroids (e.g., cholesterol, bile acids, testosterone, and other steroid hormones), as well as the organisation and conservation of the genes involved, are reviewed.
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Affiliation(s)
- Elías R Olivera
- Departamento Biología Molecular (Área Bioquímica y Biología Molecular), Universidad de León, 24007 León, Spain.
| | - José M Luengo
- Departamento Biología Molecular (Área Bioquímica y Biología Molecular), Universidad de León, 24007 León, Spain
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38
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Hauer A, Michelet L, Cochard T, Branger M, Nunez J, Boschiroli ML, Biet F. Accurate Phylogenetic Relationships Among Mycobacterium bovis Strains Circulating in France Based on Whole Genome Sequencing and Single Nucleotide Polymorphism Analysis. Front Microbiol 2019; 10:955. [PMID: 31130937 PMCID: PMC6509552 DOI: 10.3389/fmicb.2019.00955] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2019] [Accepted: 04/16/2019] [Indexed: 12/13/2022] Open
Abstract
In recent years the diversity of the French Mycobacterium bovis population responsible for bovine tuberculosis (bTB) outbreaks since 1970 has been described in detail. To further understand bTB evolution in France, we used single nucleotide polymorphisms (SNPs) based on whole genome sequence versus classical genotyping methods in order to identify accurate phylogenetic relationships between M. bovis strains. Whole genome sequencing was carried out on a selection of 87 strains which reflect the French M. bovis population’s genetic diversity. Sequences were compared to the M. bovis reference genome AF2122/97. Comparison among the 87 genomes revealed 9,170 sites where at least one strain shows a SNP with respect to the reference genome; 1,172 are intergenic and 7,998 in coding sequences, of which 2,880 are synonymous and 5,118 non-synonymous. SNP-based phylogenetic analysis using these 9,170 SNP is congruent with the cluster defined by spoligotyping and multilocus variable number of tandem repeat analysis typing. In addition, some SNPs were identified as specific to genotypic groups. These findings suggest new SNP targets that can be used for the development of high-resolving methods for genotyping as well as for studying M. bovis evolution and transmission patterns. The detection of non-synonymous SNPs on virulence genes enabled us to distinguish different clusters. Our results seem to indicate that genetically differentiated clusters could also display distinctive phenotypic traits.
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Affiliation(s)
- Amandine Hauer
- University Paris-Est - ANSES, French Reference Laboratory for Tuberculosis, Maisons-Alfort, France.,ISP, INRA, UMR 1282, Université de Tours, Nouzilly, France
| | - Lorraine Michelet
- University Paris-Est - ANSES, French Reference Laboratory for Tuberculosis, Maisons-Alfort, France
| | | | - Maxime Branger
- ISP, INRA, UMR 1282, Université de Tours, Nouzilly, France
| | - Javier Nunez
- Animal and Plant Health Agency, Addlestone, United Kingdom
| | - Maria-Laura Boschiroli
- University Paris-Est - ANSES, French Reference Laboratory for Tuberculosis, Maisons-Alfort, France
| | - Franck Biet
- ISP, INRA, UMR 1282, Université de Tours, Nouzilly, France
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39
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Nazarova EV, Montague CR, Huang L, La T, Russell D, VanderVen BC. The genetic requirements of fatty acid import by Mycobacterium tuberculosis within macrophages. eLife 2019; 8:e43621. [PMID: 30735132 PMCID: PMC6368401 DOI: 10.7554/elife.43621] [Citation(s) in RCA: 47] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2018] [Accepted: 01/10/2019] [Indexed: 12/11/2022] Open
Abstract
Mycobacterium tuberculosis (Mtb) imports and metabolizes fatty acids to maintain infection within human macrophages. Although this is a well-established paradigm, the bacterial factors required for fatty acid import are poorly understood. Previously, we found that LucA and Mce1 are required for fatty acid import in Mtb (Nazarova et al., 2017). Here, we identified additional Mtb mutants that have a reduced ability to import a fluorescent fatty acid substrate during infection within macrophages. This screen identified the novel genes as rv2799 and rv0966c as be necessary for fatty acid import and confirmed the central role for Rv3723/LucA and putative components of the Mce1 fatty acid transporter (Rv0200/OmamB, Rv0172/Mce1D, and Rv0655/MceG) in this process.
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Affiliation(s)
- Evgeniya V Nazarova
- Department of Microbiology and Immunology, College of Veterinary MedicineCornell UniversityIthacaUnited States
| | - Christine R Montague
- Department of Microbiology and Immunology, College of Veterinary MedicineCornell UniversityIthacaUnited States
| | - Lu Huang
- Department of Microbiology and Immunology, College of Veterinary MedicineCornell UniversityIthacaUnited States
| | - Thuy La
- Department of Microbiology and Immunology, College of Veterinary MedicineCornell UniversityIthacaUnited States
| | - David Russell
- Department of Microbiology and Immunology, College of Veterinary MedicineCornell UniversityIthacaUnited States
| | - Brian C VanderVen
- Department of Microbiology and Immunology, College of Veterinary MedicineCornell UniversityIthacaUnited States
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40
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diCenzo GC, Mengoni A, Fondi M. Tn-Core: A Toolbox for Integrating Tn-seq Gene Essentiality Data and Constraint-Based Metabolic Modeling. ACS Synth Biol 2019; 8:158-169. [PMID: 30525460 DOI: 10.1021/acssynbio.8b00432] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The design of synthetic cells requires a detailed understanding of the relevance of genes and gene networks underlying complex cellular phenotypes. Transposon-sequencing (Tn-seq) and constraint-based metabolic modeling can be used to probe the core genetic and metabolic networks underlying a biological process. Integrating these highly complementary experimental and in silico approaches has the potential to yield a highly comprehensive understanding of the core networks of a cell. Specifically, it can facilitate the interpretation of Tn-seq data sets and identify gaps in the data that could hinder the engineering of the cellular system, while also providing refined models for the accurate predictions of cellular metabolism. Here, we present Tn-Core, the first easy-to-use computational pipeline specifically designed for integrating Tn-seq data with metabolic modeling, prepared for use by both experimental and computational biologists. Tn-Core is a MATLAB toolbox that contains several custom functions, and it is built upon existing functions within the COBRA Toolbox and the TIGER Toolbox. Tn-Core takes as input a genome-scale metabolic model, Tn-seq data, and optionally RNA-seq data, and returns: (i) a context-specific core metabolic model; (ii) an evaluation of redundancies within core metabolic pathways, and optionally (iii) a refined genome-scale metabolic model. A simple, user-friendly workflow, requiring limited knowledge of metabolic modeling, is provided that allows users to run the analyses and export the data as easy-to-explore files of value to both experimental and computational biologists. We demonstrate the utility of Tn-Core using Sinorhizobium meliloti, Pseudomonas aeruginosa, and Rhodobacter sphaeroides genome-scale metabolic reconstructions as case studies.
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Affiliation(s)
- George C. diCenzo
- Department of Biology, University of Florence, Sesto Fiorentino, Florence, 50019, Italy
| | - Alessio Mengoni
- Department of Biology, University of Florence, Sesto Fiorentino, Florence, 50019, Italy
| | - Marco Fondi
- Department of Biology, University of Florence, Sesto Fiorentino, Florence, 50019, Italy
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41
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Klobucar K, Brown ED. Use of genetic and chemical synthetic lethality as probes of complexity in bacterial cell systems. FEMS Microbiol Rev 2018; 42:4563584. [PMID: 29069427 DOI: 10.1093/femsre/fux054] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Accepted: 10/23/2017] [Indexed: 12/22/2022] Open
Abstract
Different conditions and genomic contexts are known to have an impact on gene essentiality and interactions. Synthetic lethal interactions occur when a combination of perturbations, either genetic or chemical, result in a more profound fitness defect than expected based on the effect of each perturbation alone. Synthetic lethality in bacterial systems has long been studied; however, during the past decade, the emerging fields of genomics and chemical genomics have led to an increase in the scale and throughput of these studies. Here, we review the concepts of genomics and chemical genomics in the context of synthetic lethality and their revolutionary roles in uncovering novel biology such as the characterization of genes of unknown function and in antibacterial drug discovery. We provide an overview of the methodologies, examples and challenges of both genetic and chemical synthetic lethal screening platforms. Finally, we discuss how to apply genetic and chemical synthetic lethal approaches to rationalize the synergies of drugs, screen for new and improved antibacterial therapies and predict drug mechanism of action.
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Affiliation(s)
- Kristina Klobucar
- Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1280 Main St West, Hamilton, ON L8N 3Z5, Canada
| | - Eric D Brown
- Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1280 Main St West, Hamilton, ON L8N 3Z5, Canada
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42
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Rameshwaram NR, Singh P, Ghosh S, Mukhopadhyay S. Lipid metabolism and intracellular bacterial virulence: key to next-generation therapeutics. Future Microbiol 2018; 13:1301-1328. [DOI: 10.2217/fmb-2018-0013] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
Lipid metabolism is thought to play a key role in the pathogenicity of several intracellular bacteria. Bacterial lipolytic enzymes hydrolyze lipids from the host cell to release free fatty acids which are used as an energy source and building blocks for the synthesis of cell envelope and also to modulate host immune responses. In this review, we discussed the role of lipid metabolism and lipolytic enzymes in the life cycle and virulence of Mycobacterium tuberculosis and other intracellular bacteria. The lipolytic enzymes appear to be potential candidates for developing novel therapeutics by targeting lipid metabolism for controlling M. tuberculosis and other intracellular pathogenic bacteria. [Formula: see text]
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Affiliation(s)
- Nagender Rao Rameshwaram
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting & Diagnostics (CDFD), Inner Ring Road, Uppal, Hyderabad, India. 500 039
| | - Parul Singh
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting & Diagnostics (CDFD), Inner Ring Road, Uppal, Hyderabad, India. 500 039
- Graduate Studies, Manipal University, Manipal, Karnataka, India. 576 104
| | - Sudip Ghosh
- Molecular Biology Division, National Institute of Nutrition (ICMR), Jamai-Osmania PO, Hyderabad, India. 500 007
| | - Sangita Mukhopadhyay
- Laboratory of Molecular Cell Biology, Centre for DNA Fingerprinting & Diagnostics (CDFD), Inner Ring Road, Uppal, Hyderabad, India. 500 039
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43
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Westermann AJ. Regulatory RNAs in Virulence and Host-Microbe Interactions. Microbiol Spectr 2018; 6:10.1128/microbiolspec.rwr-0002-2017. [PMID: 30003867 PMCID: PMC11633609 DOI: 10.1128/microbiolspec.rwr-0002-2017] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2017] [Indexed: 02/06/2023] Open
Abstract
Bacterial regulatory RNAs are key players in adaptation to changing environmental conditions and response to diverse cellular stresses. However, while regulatory RNAs of bacterial pathogens have been intensely studied under defined conditions in vitro, characterization of their role during the infection of eukaryotic host organisms is lagging behind. This review summarizes our current understanding of the contribution of the different classes of regulatory RNAs and RNA-binding proteins to bacterial virulence and illustrates their role in infection by reviewing the mechanisms of some prominent representatives of each class. Emerging technologies are described that bear great potential for global, unbiased studies of virulence-related RNAs in bacterial model and nonmodel pathogens in the future. The review concludes by deducing common principles of RNA-mediated gene expression control of virulence programs in different pathogens, and by defining important open questions for upcoming research in the field.
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Affiliation(s)
- Alexander J Westermann
- Institute of Molecular Infection Biology, University of Würzburg
- Helmholtz Institute for RNA-Based Infection Research, D-97080 Würzburg, Germany
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44
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Wilburn KM, Fieweger RA, VanderVen BC. Cholesterol and fatty acids grease the wheels of Mycobacterium tuberculosis pathogenesis. Pathog Dis 2018; 76:4931720. [PMID: 29718271 PMCID: PMC6251666 DOI: 10.1093/femspd/fty021] [Citation(s) in RCA: 117] [Impact Index Per Article: 16.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2017] [Accepted: 03/06/2018] [Indexed: 01/23/2023] Open
Abstract
Tuberculosis is a distinctive disease in which the causative agent, Mycobacterium tuberculosis, can persist in humans for decades by avoiding clearance from host immunity. During infection, M. tuberculosis maintains viability by extracting and utilizing essential nutrients from the host, and this is a prerequisite for all of the pathogenic activities that are deployed by the bacterium. In particular, M. tuberculosis preferentially acquires and metabolizes host-derived lipids (fatty acids and cholesterol), and the bacterium utilizes these substrates to cause and maintain disease. In this review, we discuss our current understanding of lipid utilization by M. tuberculosis, and we describe how these pathways promote pathogenesis to fuel metabolic processes in the bacillus. Finally, we highlight weaknesses in these pathways that potentially can be targeted for drug discovery.
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Affiliation(s)
- Kaley M Wilburn
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14850, USA
| | - Rachael A Fieweger
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14850, USA
| | - Brian C VanderVen
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14850, USA
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45
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Engineering phytosterol transport system in Mycobacterium sp. strain MS136 enhances production of 9α-hydroxy-4-androstene-3,17-dione. Biotechnol Lett 2018; 40:673-678. [DOI: 10.1007/s10529-018-2520-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2017] [Accepted: 01/24/2018] [Indexed: 01/05/2023]
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46
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Barquist L, Westermann AJ, Vogel J. Molecular phenotyping of infection-associated small non-coding RNAs. Philos Trans R Soc Lond B Biol Sci 2017; 371:rstb.2016.0081. [PMID: 27672158 DOI: 10.1098/rstb.2016.0081] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 05/19/2016] [Indexed: 02/07/2023] Open
Abstract
Infection is a complicated balance, with both pathogen and host struggling to tilt the result in their favour. Bacterial infection biology has relied on forward genetics for many of its advances, defining phenotype in terms of replication in model systems. However, many known virulence factors fail to produce robust phenotypes, particularly in the systems most amenable to genetic manipulation, such as cell-culture models. This has particularly been limiting for the study of the bacterial regulatory small RNAs (sRNAs) in infection. We argue that new sequencing-based technologies can work around this problem by providing a 'molecular phenotype', defined in terms of the specific transcriptional dysregulation in the infection system induced by gene deletion. We illustrate this using the example of our recent study of the PinT sRNA using dual RNA-seq, that is, simultaneous RNA sequencing of host and pathogen during infection. We additionally discuss how other high-throughput technologies, in particular genetic interaction mapping using transposon insertion sequencing, may be used to further dissect molecular phenotypes. We propose a strategy for how high-throughput technologies can be integrated in the study of non-coding regulators as well as bacterial virulence factors, enhancing our ability to rapidly generate hypotheses with regards to their function.This article is part of the themed issue 'The new bacteriology'.
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Affiliation(s)
- Lars Barquist
- RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Straße 2/D15, 97080 Würzburg, Germany
| | - Alexander J Westermann
- RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Straße 2/D15, 97080 Würzburg, Germany
| | - Jörg Vogel
- RNA Biology Group, Institute for Molecular Infection Biology, University of Würzburg, Josef-Schneider-Straße 2/D15, 97080 Würzburg, Germany Research Centre for Infectious Diseases (ZINF), University of Würzburg, 97070 Würzburg, Germany
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47
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Application of Distributive Conjugal DNA Transfer in Mycobacterium smegmatis To Establish a Genome-Wide Synthetic Genetic Array. J Bacteriol 2017; 199:JB.00410-17. [PMID: 28784812 DOI: 10.1128/jb.00410-17] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2017] [Accepted: 07/27/2017] [Indexed: 11/20/2022] Open
Abstract
Genetic redundancy can obscure phenotypic effects of single-gene mutations. Two individual mutations may be viable separately but are lethal when combined, thus synthetically linking the two gene products in an essential process. Synthetic genetic arrays (SGAs), in which defined mutations are combined, provide a powerful approach to identify novel genetic interactions and redundant pathways. A genome-scale SGA can offer an initial assignment of function to hypothetical genes by uncovering interactions with known genes or pathways. Here, we take advantage of the chromosomal conjugation system of Mycobacterium smegmatis to combine individual donor and recipient mutations on a genome-wide scale. We demonstrated the feasibility of a high-throughput mycobacterial SGA (mSGA) screen by using mutants of esx3, fxbA, and recA as query genes, which were combined with an arrayed library of transposon mutants by conjugation. The mSGA identified interacting genes that we had predicted and, most importantly, identified novel interacting genes-encoding both proteins and a noncoding RNA (ncRNA). In combination with other molecular genetic approaches, the mSGA has great potential to both reduce the high number of conserved hypothetical protein annotations in mycobacterial genomes and further define mycobacterial pathways and gene interactions.IMPORTANCE Mycobacterium smegmatis is the model organism of choice for the study of mycobacterial pathogens, because it is a fast-growing nonpathogenic species harboring many genes that are conserved throughout mycobacteria. In this work, we describe a synthetic genetic array (mSGA) approach for M. smegmatis, which combines mutations on a genome-wide scale with high efficiency. Analysis of the double mutant strains enables the identification of interacting genes and pathways that are normally hidden by redundant biological pathways. The mSGA is a powerful genetic tool that enables functions to be assigned to the many conserved hypothetical genes found in all mycobacterial species.
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48
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García-Fernández J, Papavinasasundaram K, Galán B, Sassetti CM, García JL. Molecular and functional analysis of the mce4 operon in Mycobacterium smegmatis. Environ Microbiol 2017; 19:3689-3699. [PMID: 28752922 DOI: 10.1111/1462-2920.13869] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2017] [Revised: 07/11/2017] [Accepted: 07/24/2017] [Indexed: 11/30/2022]
Abstract
Mycobacterium smegmatis contains 6 homologous mce (mammalian cell entry) operons which have been proposed to encode ABC-like import systems. The mce operons encode up to 10 different proteins of unknown function that are not present in conventional ABC transporters. We have analysed the consequences of individually deleting each of the genes of the mce4 operon of M. smegmatis, which mediates the transport of cholesterol. None of the mce4 mutants were able to grow in cholesterol suggesting that all these genes are required for its uptake and that none of them can be replaced by the homologous genes of the other mce operons. This result suggests that different mce operons do not provide redundant capabilities and that M. smegmatis, in contrast with Mycobacterium tuberculosis, is not able to use alternative systems to import cholesterol in the analysed culture conditions. Either deletion of the entire mce4 operon or single point mutations that eliminate the transport function cause a phenotype similar to the one observed in a mutant lacking all 6 mce operons suggesting a pleiotropic role for this system.
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Affiliation(s)
- Julia García-Fernández
- Department of Environmental Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
| | - Kadamba Papavinasasundaram
- Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA
| | - Beatriz Galán
- Department of Environmental Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
| | - Christopher M Sassetti
- Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, MA, USA
| | - José L García
- Department of Environmental Biology, Centro de Investigaciones Biológicas (CIB-CSIC), Madrid, Spain
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Abstract
All bacteria utilize pathways to export proteins from the cytoplasm to the bacterial cell envelope or extracellular space. Many exported proteins function in essential physiological processes or in virulence. Consequently, the responsible protein export pathways are commonly essential and/or are important for pathogenesis. The general Sec protein export pathway is conserved and essential in all bacteria, and it is responsible for most protein export. The energy for Sec export is provided by the SecA ATPase. Mycobacteria and some Gram-positive bacteria have two SecA paralogs: SecA1 and SecA2. SecA1 is essential and works with the canonical Sec pathway to perform the bulk of protein export. The nonessential SecA2 exports a smaller subset of proteins and is required for the virulence of pathogens such as Mycobacterium tuberculosis. In this article, we review our current understanding of the mechanism of the SecA1 and SecA2 export pathways and discuss some of their better-studied exported substrates. We focus on proteins with established functions in M. tuberculosis pathogenesis and proteins that suggest potential roles for SecA1 and SecA2 in M. tuberculosis dormancy.
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50
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Nazarova EV, Montague CR, La T, Wilburn KM, Sukumar N, Lee W, Caldwell S, Russell DG, VanderVen BC. Rv3723/LucA coordinates fatty acid and cholesterol uptake in Mycobacterium tuberculosis. eLife 2017; 6:e26969. [PMID: 28708968 PMCID: PMC5487216 DOI: 10.7554/elife.26969] [Citation(s) in RCA: 119] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2017] [Accepted: 06/07/2017] [Indexed: 01/05/2023] Open
Abstract
Pathogenic bacteria have evolved highly specialized systems to extract essential nutrients from their hosts. Mycobacterium tuberculosis (Mtb) scavenges lipids (cholesterol and fatty acids) to maintain infections in mammals but mechanisms and proteins responsible for the import of fatty acids in Mtb were previously unknown. Here, we identify and determine that the previously uncharacterized protein Rv3723/LucA, functions to integrate cholesterol and fatty acid uptake in Mtb. Rv3723/LucA interacts with subunits of the Mce1 and Mce4 complexes to coordinate the activities of these nutrient transporters by maintaining their stability. We also demonstrate that Mce1 functions as a fatty acid transporter in Mtb and determine that facilitating cholesterol and fatty acid import via Rv3723/LucA is required for full bacterial virulence in vivo. These data establish that fatty acid and cholesterol assimilation are inexorably linked in Mtb and reveals a key function for Rv3723/LucA in in coordinating thetransport of both these substrates.
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Affiliation(s)
- Evgeniya V Nazarova
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
| | - Christine R Montague
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
| | - Thuy La
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
| | - Kaley M Wilburn
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
| | - Neelima Sukumar
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
| | - Wonsik Lee
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
| | - Shannon Caldwell
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
| | - David G Russell
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
| | - Brian C VanderVen
- Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, United States
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