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1
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Han NN, Yang JH, Wu GG, Yang JH, Jin JA, Fan NS, Jin RC. Differential size-dependent response patterns and antibiotic resistance development mechanism in anammox consortia. JOURNAL OF HAZARDOUS MATERIALS 2025; 491:137886. [PMID: 40086246 DOI: 10.1016/j.jhazmat.2025.137886] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/05/2025] [Revised: 02/26/2025] [Accepted: 03/06/2025] [Indexed: 03/16/2025]
Abstract
Antibiotic resistance is a global threat to human and animal health. Anaerobic ammonia oxidation (anammox) is an efficient and innovative wastewater treatment technology, which can be served as a promising approach to teat antibiotic wastewater. This study systematically investigated effects of sulfamethazine on the performance, microbial community dynamics and the resistome in anammox systems inoculated with different-sized granular sludge. The activity and performance of small (< 0.5 mm) anammox granules were more susceptible to sulfamethazine stress than those of medium (0.5-1.0 mm) and large (1.0-2.0 mm) granules. Sulfamethazine addition greatly increased the diversity and abundance of mobile genetic elements (MGEs) and antibiotic resistance genes (ARGs). Based on the metagenomic analysis, the horizontal transfer of ARGs in the anammox system was upregulated through bacterial oxidative stress, pili synthesis and type IV secretion system. In addition, two strains of sulfamethazine-resistant bacteria (Pseudomonas asiatica sp. nov. and Pseudomonas shirazica sp. nov.) were isolated from the anammox system. Their whole genome sequencing results showed that the most abundant plasmid was pkF7158B, which mediated the horizontal transfer of two main multidrug resistance genes (cpxR and mexB). This work provides a holistic insight into microbial heterogeneity of different-sized anammox granular sludge and their evolution and resistance development mechanism.
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Affiliation(s)
- Na-Na Han
- School of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China
| | - Jun-Hui Yang
- School of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China
| | - Ge-Ge Wu
- School of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China
| | - Jia-Hui Yang
- School of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 311121, China
| | - Jing-Ao Jin
- School of Engineering, Hangzhou Normal University, Hangzhou 311121, China
| | - Nian-Si Fan
- School of Engineering, Hangzhou Normal University, Hangzhou 311121, China.
| | - Ren-Cun Jin
- School of Engineering, Hangzhou Normal University, Hangzhou 311121, China
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2
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Li Z, Li Z, Peng Y, Zhang M, Wen Y, Lu X, Kan B. Genomic diversity of mcr-carrying plasmids and the role of type IV secretion systems in IncI2 plasmids conjugation. Commun Biol 2025; 8:342. [PMID: 40025288 PMCID: PMC11873049 DOI: 10.1038/s42003-025-07748-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: 05/09/2024] [Accepted: 02/14/2025] [Indexed: 03/04/2025] Open
Abstract
The rapid dissemination of colistin resistance via mcr-carrying plasmids (pMCRs) poses a significant public health challenge. This study examined the genomic diversity and conjugation mechanisms of pMCRs, with a particular focus on the role of type IV secretion systems (T4SS) in IncI2 plasmids. The 868 complete plasmid sequences revealed various replicon types of pMCRs, with IncI2 as the primary epidemic type, and the co-transfer risk of multidrug resistance genes associated with IncHI2. T4SS was identified in 89.9% of pMCRs, with the T4SS sequence exclusively carried by IncI2 being conserved and typical of the VirB/D4 type, consisting of 12 subunits. Conjugation assays confirmed the essential role of the pilus subunit VirB2 and the significant impact of VirB5P3 on conjugation. This was further validated in the in vivo intra-species competitive conjugation of Escherichia coli. Structural predictions show that a hypervariable region at the C-terminus of the pentameric VirB5 co-evolves in sequence with VirB6, and the conserved N-terminal may act as a potential drug target to inhibit the plasmid transfer channel. This study will deepen the understanding of the pMCR epidemic patterns and provide additional insights for controlling the spread of resistant plasmids.
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Affiliation(s)
- Zhe Li
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Zhenpeng Li
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Yao Peng
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Mengke Zhang
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
- School of Light Industry Science and Engineering, Beijing Technology and Business University, Beijing, China
| | - Yuanxi Wen
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
- School of Public Health, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China
| | - Xin Lu
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
| | - Biao Kan
- National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.
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3
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Breidenstein A, Svedberg D, Ter Beek J, Berntsson RPA. Advances in Protein Structure Prediction Highlight Unexpected Commonalities Between Gram-positive and Gram-negative Conjugative T4SSs. J Mol Biol 2025; 437:168924. [PMID: 39746464 DOI: 10.1016/j.jmb.2024.168924] [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: 09/19/2024] [Revised: 12/20/2024] [Accepted: 12/20/2024] [Indexed: 01/04/2025]
Abstract
Despite recent advances in our understanding of the structure and function of conjugative Type 4 Secretion Systems (T4SSs), there is still only very scarce data available for the ones from Gram-positive (G+) bacteria. This is a problem, as conjugative T4SSs are main drivers for the spread of antibiotic resistance genes and virulence factors. Here, we aim to increase our understanding of G+ systems, by using bioinformatic approaches to identify proteins that are conserved in all conjugative T4SS machineries and reviewing the current knowledge available for these components. We then combine this information with the most recent advances in structure prediction technologies to propose a structural model for a G+ T4SS from the model system encoded on pCF10. By doing so, we show that conjugative G+ T4SSs likely have more in common with their Gram-negative counterparts than previously expected, and we highlight the potential of predicted structural models to serve as a starting point for experimental design.
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Affiliation(s)
- Annika Breidenstein
- Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden; Wallenberg Centre for Molecular Medicine & Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden
| | - Dennis Svedberg
- Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden; Wallenberg Centre for Molecular Medicine & Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden
| | - Josy Ter Beek
- Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden; Wallenberg Centre for Molecular Medicine & Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden.
| | - Ronnie P-A Berntsson
- Department of Medical Biochemistry and Biophysics, Umeå University, SE-90187 Umeå, Sweden; Wallenberg Centre for Molecular Medicine & Umeå Centre for Microbial Research, Umeå University, Umeå, Sweden.
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4
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Mok CY, Chu HY, Lam WWL, Au SWN. Structural insights into the assembly pathway of the Helicobacter pylori CagT4SS outer membrane core complex. Structure 2024; 32:1725-1736.e4. [PMID: 39032488 DOI: 10.1016/j.str.2024.06.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2024] [Revised: 05/16/2024] [Accepted: 06/25/2024] [Indexed: 07/23/2024]
Abstract
Cag type IV secretion system (CagT4SS) translocates oncoprotein cytotoxin-associated gene A (CagA) into host cells and plays a key role in the pathogenesis of Helicobacter pylori. The structure of the outer membrane core complex (OMCC) in CagT4SS consists of CagX, CagY, CagM, CagT, and Cag3 in a stoichiometric ratio of 1:1:2:2:5 with 14-fold symmetry. However, the assembly pathway of OMCC remains elusive. Here, we report the crystal structures of CagT and Cag3-CagT complex, and the structural dynamics of Cag3 and CagT using hydrogen deuterium exchange-mass spectrometry (HDX-MS). The interwoven interaction of Cag3 and CagT involves conformational changes of CagT and β strand swapping. In conjunction with biochemical and biophysical assays, we further demonstrate the different oligomerization states of Cag3 and Cag3-CagT complex. Additionally, the association with CagM requires the pre-formation of Cag3-CagT complex. These results demonstrate the generation of different intermediate sub-assemblies and their structural flexibility, potentially representing different building blocks for OMCC assembly.
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Affiliation(s)
- Chin Yu Mok
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Hoi Yee Chu
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Wendy Wai Ling Lam
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China
| | - Shannon Wing Ngor Au
- Center for Protein Science and Crystallography, School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong, China.
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5
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Roberts JR, Tran SC, Frick-Cheng AE, Bryant KN, Okoye CD, McDonald WH, Cover TL, Ohi MD. Subdomains of the Helicobacter pylori Cag T4SS outer membrane core complex exhibit structural independence. Life Sci Alliance 2024; 7:e202302560. [PMID: 38631913 PMCID: PMC11024343 DOI: 10.26508/lsa.202302560] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2023] [Revised: 03/28/2024] [Accepted: 03/28/2024] [Indexed: 04/19/2024] Open
Abstract
The Helicobacter pylori Cag type IV secretion system (Cag T4SS) has an important role in the pathogenesis of gastric cancer. The Cag T4SS outer membrane core complex (OMCC) is organized into three regions: a 14-fold symmetric outer membrane cap (OMC) composed of CagY, CagX, CagT, CagM, and Cag3; a 17-fold symmetric periplasmic ring (PR) composed of CagY and CagX; and a stalk with unknown composition. We investigated how CagT, CagM, and a conserved antenna projection (AP) region of CagY contribute to the structural organization of the OMCC. Single-particle cryo-EM analyses showed that complexes purified from ΔcagT or ΔcagM mutants no longer had organized OMCs, but the PRs remained structured. OMCCs purified from a CagY antenna projection mutant (CagY∆AP) were structurally similar to WT OMCCs, except for the absence of the α-helical antenna projection. These results indicate that CagY and CagX are sufficient for maintaining a stable PR, but the organization of the OMC requires CagY, CagX, CagM, and CagT. Our results highlight an unexpected structural independence of two major subdomains of the Cag T4SS OMCC.
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Affiliation(s)
- Jacquelyn R Roberts
- Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
- Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA
| | - Sirena C Tran
- Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | | | - Kaeli N Bryant
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Chiamaka D Okoye
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - W Hayes McDonald
- Proteomics Laboratory, Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN, USA
- Department of Biochemistry, Vanderbilt University, Nashville, TN USA
| | - Timothy L Cover
- Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN, USA
- Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, USA
- Veterans Affairs Tennessee Valley Healthcare System, Nashville, TN, USA
| | - Melanie D Ohi
- Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI, USA
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6
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Costa TRD, Patkowski JB, Macé K, Christie PJ, Waksman G. Structural and functional diversity of type IV secretion systems. Nat Rev Microbiol 2024; 22:170-185. [PMID: 37814112 PMCID: PMC11290344 DOI: 10.1038/s41579-023-00974-3] [Citation(s) in RCA: 25] [Impact Index Per Article: 25.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/13/2023] [Indexed: 10/11/2023]
Abstract
Considerable progress has been made in recent years in the structural and molecular biology of type IV secretion systems in Gram-negative bacteria. The latest advances have substantially improved our understanding of the mechanisms underlying the recruitment and delivery of DNA and protein substrates to the extracellular environment or target cells. In this Review, we aim to summarize these exciting structural and molecular biology findings and to discuss their functional implications for substrate recognition, recruitment and translocation, as well as the biogenesis of extracellular pili. We also describe adaptations necessary for deploying a breadth of processes, such as bacterial survival, host-pathogen interactions and biotic and abiotic adhesion. We highlight the functional and structural diversity that allows this extremely versatile secretion superfamily to function under different environmental conditions and in different bacterial species. Additionally, we emphasize the importance of further understanding the mechanism of type IV secretion, which will support us in combating antimicrobial resistance and treating type IV secretion system-related infections.
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Affiliation(s)
- Tiago R D Costa
- Centre for Bacterial Resistance Biology, Department of Life Sciences, Imperial College, London, UK.
| | - Jonasz B Patkowski
- Centre for Bacterial Resistance Biology, Department of Life Sciences, Imperial College, London, UK
| | - Kévin Macé
- Institute of Structural and Molecular Biology, Birkbeck and UCL, London, UK
- Institut de Génétique et Développement de Rennes (IGDR), Université de Rennes and CNRS, Rennes, France
| | - Peter J Christie
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, TX, USA.
| | - Gabriel Waksman
- Institute of Structural and Molecular Biology, Birkbeck and UCL, London, UK.
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7
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Kishida K, Li YG, Ogawa-Kishida N, Khara P, Al Mamun AAM, Bosserman RE, Christie PJ. Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines. PLoS Genet 2024; 20:e1011088. [PMID: 38437248 PMCID: PMC10939261 DOI: 10.1371/journal.pgen.1011088] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2023] [Revised: 03/14/2024] [Accepted: 02/20/2024] [Indexed: 03/06/2024] Open
Abstract
Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate-TraD and TraD-T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.
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Affiliation(s)
- Kouhei Kishida
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, Texas, United States of America
| | - Yang Grace Li
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, Texas, United States of America
| | - Natsumi Ogawa-Kishida
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, Texas, United States of America
| | - Pratick Khara
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, Texas, United States of America
| | - Abu Amar M. Al Mamun
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, Texas, United States of America
| | - Rachel E. Bosserman
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, Texas, United States of America
| | - Peter J. Christie
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, Texas, United States of America
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8
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Ignatiou A, Macé K, Redzej A, Costa TRD, Waksman G, Orlova EV. Structural Analysis of Protein Complexes by Cryo-Electron Microscopy. Methods Mol Biol 2024; 2715:431-470. [PMID: 37930544 DOI: 10.1007/978-1-0716-3445-5_27] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2023]
Abstract
Structural studies of bio-complexes using single particle cryo-Electron Microscopy (cryo-EM) is nowadays a well-established technique in structural biology and has become competitive with X-ray crystallography. Development of digital registration systems for electron microscopy images and algorithms for the fast and efficient processing of the recorded images and their following analysis has facilitated the determination of structures at near-atomic resolution. The latest advances in EM have enabled the determination of protein complex structures at 1.4-3 Å resolution for an extremely broad range of sizes (from ~100 kDa up to hundreds of MDa (Bartesaghi et al., Science 348(6239):1147-1151, 2015; Herzik et al., Nat Commun 10:1032, 2019; Wu et al., J Struct Biol X 4:100020, 2020; Zhang et al., Nat Commun 10:5511, 2019; Zhang et al., Cell Res 30(12):1136-1139, 2020; Yip et al., Nature 587(7832):157-161, 2020; https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year )). In 2022, nearly 1200 structures deposited to the EMDB database were at a resolution of better than 3 Å ( https://www.ebi.ac.uk/emdb/statistics/emdb_resolution_year ).To date, the highest resolutions have been achieved for apoferritin, which comprises a homo-oligomer of high point group symmetry (O432) and has rigid organization together with high stability (Zhang et al., Cell Res 30(12):1136-1139, 2020; Yip et al., Nature 587(7832):157-161, 2020). It has been used as a test object for the assessments of modern cryo-microscopes and processing methods during the last 5 years. In contrast to apoferritin bacterial secretion systems are typical examples of multi protein complexes exhibiting high flexibility owing to their functions relating to the transportation of small molecules, proteins, and DNA into the extracellular space or target cells. This makes their structural characterization extremely challenging (Barlow, Methods Mol Biol 532:397-411, 2009; Costa et al., Nat Rev Microbiol 13:343-359, 2015). The most feasible approach to reveal their spatial organization and functional modification is cryo-electron microscopy (EM). During the last decade, structural cryo-EM has become broadly used for the analysis of the bio-complexes that comprise multiple components and are not amenable to crystallization (Lyumkis, J Biol Chem 294:5181-5197, 2019; Orlova and Saibil, Methods Enzymol 482:321-341, 2010; Orlova and Saibil, Chem Rev 111(12):7710-7748, 2011).In this review, we will describe the basics of sample preparation for cryo-EM, the principles of digital data collection, and the logistics of image analysis focusing on the common steps required for reconstructions of both small and large biological complexes together with refinement of their structures to nearly atomic resolution. The workflow of processing will be illustrated by examples of EM analysis of Type IV Secretion System.
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Affiliation(s)
- Athanasios Ignatiou
- Institute for Structural and Molecular Biology, School of Biological Sciences, Birkbeck College, London, UK
| | - Kévin Macé
- Institute for Structural and Molecular Biology, School of Biological Sciences, Birkbeck College, London, UK
| | - Adam Redzej
- Institute for Structural and Molecular Biology, School of Biological Sciences, Birkbeck College, London, UK
| | - Tiago R D Costa
- Centre for Bacterial Resistance Biology, Department of Life Sciences, Imperial College, London, UK
| | - Gabriel Waksman
- Institute for Structural and Molecular Biology, School of Biological Sciences, Birkbeck College, London, UK
| | - Elena V Orlova
- Institute for Structural and Molecular Biology, School of Biological Sciences, Birkbeck College, London, UK.
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9
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Diepold A. Defining Assembly Pathways by Fluorescence Microscopy. Methods Mol Biol 2024; 2715:383-394. [PMID: 37930541 DOI: 10.1007/978-1-0716-3445-5_24] [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] [Indexed: 11/07/2023]
Abstract
Bacterial secretion systems are among the largest protein complexes in prokaryotes and display remarkably complex architectures. Their assembly often follows clearly defined pathways. Deciphering these pathways not only reveals how bacteria accomplish to build these large functional complexes but can provide crucial information on the interactions and subcomplexes within secretion systems, their distribution within the bacterium, and even functional insights. Fluorescence microscopy provides a powerful tool for biological imaging, which presents an interesting method to accurately define the biogenesis of macromolecular complexes using fluorescently labeled components. Here, I describe the use of this method to decipher the assembly pathway of bacterial secretion systems.
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Affiliation(s)
- Andreas Diepold
- Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
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10
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Zehra M, Heo J, Chung JM, Durie CL. Comparative Analysis of T4SS Molecular Architectures. J Microbiol Biotechnol 2023; 33:1543-1551. [PMID: 37528551 PMCID: PMC10772558 DOI: 10.4014/jmb.2307.07006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2023] [Revised: 07/21/2023] [Accepted: 07/24/2023] [Indexed: 08/03/2023]
Abstract
The recently published high-resolution R388 T4SS structure provides exciting new details about the complete complex of T4SS, including the components making up the stalk and arches, numerous symmetry mismatches between regions of the complex, and an intriguing interpretation of the closed stalk and radial symmetry of the inner membrane complex, which is related to pilus biogenesis assembly. However, there are a few unidentified densities in the electron microscopy map and portions of the identified component sequences for which the structure is not yet known. It is also unclear how well this minimized DNA-transporting T4SS predicts the structure of other T4SSs, such as expanded systems and those that transport proteins rather than DNA. In this review, we evaluate what can be inferred from the recent high-resolution structure of the R388 T4SS with respect to the Cag and Dot/Icm systems. These systems were selected because, given what is currently known about these systems, we expect them to present most structural differences compared to the R388 T4SS structure. Furthermore, we discuss bacterial physiology and diversity, the T4SS structures and their variations between different bacterial species. These insights may prove beneficial for researchers who elucidate the structure and functions of T4SS in different bacterial species.
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Affiliation(s)
- Mishghan Zehra
- Department of Biochemistry, University of Missouri, Columbia, Missouri, USA
| | - Jiwon Heo
- Department of Biotechnology, The Catholic University of Korea, Bucheon-si 14662, Gyeonggi, Republic of Korea
| | - Jeong Min Chung
- Department of Biotechnology, The Catholic University of Korea, Bucheon-si 14662, Gyeonggi, Republic of Korea
| | - Clarissa L Durie
- Department of Biochemistry, University of Missouri, Columbia, Missouri, USA
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11
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Kishida K, Li YG, Ogawa-Kishida N, Khara P, Al Mamun AAM, Bosserman RE, Christie PJ. Chimeric systems composed of swapped Tra subunits between distantly-related F plasmids reveal striking plasticity among type IV secretion machines. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.12.05.570194. [PMID: 38106057 PMCID: PMC10723329 DOI: 10.1101/2023.12.05.570194] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/19/2023]
Abstract
Bacterial type IV secretion systems (T4SSs) are a versatile family of macromolecular translocators, collectively able to recruit diverse DNA and protein substrates and deliver them to a wide range of cell types. Presently, there is little understanding of how T4SSs recognize substrate repertoires and form productive contacts with specific target cells. Although T4SSs are composed of a number of conserved subunits and adopt certain conserved structural features, they also display considerable compositional and structural diversity. Here, we explored the structural bases underlying the functional versatility of T4SSs through systematic deletion and subunit swapping between two conjugation systems encoded by the distantly-related IncF plasmids, pED208 and F. We identified several regions of intrinsic flexibility among the encoded T4SSs, as evidenced by partial or complete functionality of chimeric machines. Swapping of VirD4-like TraD type IV coupling proteins (T4CPs) yielded functional chimeras, indicative of relaxed specificity at the substrate - TraD and TraD - T4SS interfaces. Through mutational analyses, we further delineated domains of the TraD T4CPs contributing to recruitment of cognate vs heterologous DNA substrates. Remarkably, swaps of components comprising the outer membrane core complexes, a few F-specific subunits, or the TraA pilins supported DNA transfer in the absence of detectable pilus production. Among sequenced enterobacterial species in the NCBI database, we identified many strains that harbor two or more F-like plasmids and many F plasmids lacking one or more T4SS components required for self-transfer. We confirmed that host cells carrying co-resident, non-selftransmissible variants of pED208 and F elaborate chimeric T4SSs, as evidenced by transmission of both plasmids. We propose that T4SS plasticity enables the facile assembly of functional chimeras, and this intrinsic flexibility at the structural level can account for functional diversification of this superfamily over evolutionary time and, on a more immediate time-scale, to proliferation of transfer-defective MGEs in nature.
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Affiliation(s)
- Kouhei Kishida
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, 6431 Fannin St, Houston, Texas 77030, United States of America
| | - Yang Grace Li
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, 6431 Fannin St, Houston, Texas 77030, United States of America
| | - Natsumi Ogawa-Kishida
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, 6431 Fannin St, Houston, Texas 77030, United States of America
| | - Pratick Khara
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, 6431 Fannin St, Houston, Texas 77030, United States of America
| | - Abu Amar M Al Mamun
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, 6431 Fannin St, Houston, Texas 77030, United States of America
| | - Rachel E. Bosserman
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, 6431 Fannin St, Houston, Texas 77030, United States of America
| | - Peter J. Christie
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, 6431 Fannin St, Houston, Texas 77030, United States of America
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12
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Valenzuela-Gómez F, Arechaga I, Cabezón E. Nanopore sensing reveals a preferential pathway for the co-translocational unfolding of a conjugative relaxase-DNA complex. Nucleic Acids Res 2023; 51:6857-6869. [PMID: 37264907 PMCID: PMC10359608 DOI: 10.1093/nar/gkad492] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2023] [Revised: 05/05/2023] [Accepted: 05/26/2023] [Indexed: 06/03/2023] Open
Abstract
Bacterial conjugation is the main mechanism for the dissemination of antibiotic resistance genes. A single DNA strand of the conjugative plasmid is transferred across bacterial membranes covalently bound to a large multi-domain protein, named relaxase, which must be unfolded to traverse the secretion channel. Two tyrosine residues of the relaxase (Y18 and Y26 in relaxase TrwC) play an important role in the processing of conjugative DNA. We have used nanopore technology to uncover the unfolding states that take place during translocation of the relaxase-DNA complex. We observed that the relaxase unfolding pathway depends on the tyrosine residue involved in conjugative DNA binding. Transfer of the nucleoprotein complex is faster when DNA is bound to residue Y18. This is the first time in which a protein-DNA complex that is naturally translocated through bacterial membranes has been analyzed by nanopore sensing, opening new horizons to apply this technology to study protein secretion.
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Affiliation(s)
- Fernando Valenzuela-Gómez
- Departamento de Biología Molecular and Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria- CSIC, 39011 Santander, Spain
| | - Ignacio Arechaga
- Departamento de Biología Molecular and Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria- CSIC, 39011 Santander, Spain
| | - Elena Cabezón
- Departamento de Biología Molecular and Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Universidad de Cantabria- CSIC, 39011 Santander, Spain
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13
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Abstract
The versatile type IV secretion system (T4SS) nanomachine plays a pivotal role in bacterial pathogenesis and the propagation of antibiotic resistance determinants throughout microbial populations. In addition to paradigmatic DNA conjugation machineries, diverse T4SSs enable the delivery of multifarious effector proteins to target prokaryotic and eukaryotic cells, mediate DNA export and uptake from the extracellular milieu, and in rare examples, facilitate transkingdom DNA translocation. Recent advances have identified new mechanisms underlying unilateral nucleic acid transport through the T4SS apparatus, highlighting both functional plasticity and evolutionary adaptations that enable novel capabilities. In this review, we describe the molecular mechanisms underscoring DNA translocation through diverse T4SS machineries, emphasizing the architectural features that implement DNA exchange across the bacterial membrane and license transverse DNA release across kingdom boundaries. We further detail how recent studies have addressed outstanding questions surrounding the mechanisms by which nanomachine architectures and substrate recruitment strategies contribute to T4SS functional diversity.
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Affiliation(s)
- Mackenzie E. Ryan
- Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA
| | - Prashant P. Damke
- Department of Veterinary Sciences, University of Kentucky College of Agriculture, Lexington, Kentucky, USA
| | - Carrie L. Shaffer
- Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky, USA
- Department of Veterinary Sciences, University of Kentucky College of Agriculture, Lexington, Kentucky, USA
- Department of Pharmaceutical Sciences, University of Kentucky College of Pharmacy, Lexington, Kentucky, USA
- Markey Cancer Center, University of Kentucky College of Medicine, Lexington, Kentucky, USA
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14
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Cryo-EM structure of the Agrobacteriumtumefaciens T-pilus reveals the importance of positive charges in the lumen. Structure 2022; 31:375-384.e4. [PMID: 36513067 DOI: 10.1016/j.str.2022.11.007] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2022] [Revised: 09/19/2022] [Accepted: 11/14/2022] [Indexed: 12/14/2022]
Abstract
Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance.
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15
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Tarrahimofrad H, Zamani J, Hamblin MR, Darvish M, Mirzaei H. A designed peptide-based vaccine to combat Brucella melitensis, B. suis and B. abortus: Harnessing an epitope mapping and immunoinformatics approach. Biomed Pharmacother 2022; 155:113557. [PMID: 36115112 DOI: 10.1016/j.biopha.2022.113557] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Revised: 08/08/2022] [Accepted: 08/14/2022] [Indexed: 11/19/2022] Open
Abstract
Vaccines against Brucella abortus, B. melitensis and B. suis have been based on weakened or killed bacteria, however there is no recombinant vaccine for disease prevention or therapy. This study attempted to predict IFN-γ epitopes, T cell cytotoxicity, and T lymphocytes in order to produce a multiepitope vaccine based on BtpA, Omp16, Omp28, virB10, Omp25, and Omp31 antigens against B. melitensis, B. abortus, and B. suis. AAY, GPGPG, and EAAAK peptides were used as epitope linkers, while the PADRE sequence was used as a Toll-like receptor 2 (TLR2) and TLR4 agonist. The final construct included 389 amino acids, and was a soluble protein with a molecular weight of 41.3 kDa, and nonallergenic and antigenic properties. Based on molecular docking studies, molecular dynamics simulations such as Gyration, RMSF, and RMSD, as well as tertiary structure validation methods, the modeled protein had a stable structure capable of interacting with TLR2/4. As a result, this novel vaccine may stimulate immune responses in B and T cells, and could prevent infection by B. suis, B. abortus, and B. melitensis.
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Affiliation(s)
- Hossein Tarrahimofrad
- Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
| | - Javad Zamani
- Department of Animal Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
| | - Michael R Hamblin
- Laser Research Centre, Faculty of Health Science, University of Johannesburg, Doornfontein 2028, South Africa
| | - Maryam Darvish
- Department of Medical Biotechnology, School of Medicine, Arak University of Medical Sciences, Arak, Iran.
| | - Hamed Mirzaei
- Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran.
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16
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Abstract
Bacterial type IV secretion systems (T4SSs) are a versatile group of nanomachines that can horizontally transfer DNA through conjugation and deliver effector proteins into a wide range of target cells. The components of T4SSs in gram-negative bacteria are organized into several large subassemblies: an inner membrane complex, an outer membrane core complex, and, in some species, an extracellular pilus. Cryo-electron tomography has been used to define the structures of T4SSs in intact bacteria, and high-resolution structural models are now available for isolated core complexes from conjugation systems, the Xanthomonas citri T4SS, the Helicobacter pylori Cag T4SS, and the Legionella pneumophila Dot/Icm T4SS. In this review, we compare the molecular architectures of these T4SSs, focusing especially on the structures of core complexes. We discuss structural features that are shared by multiple T4SSs as well as evolutionary strategies used for T4SS diversification. Finally, we discuss how structural variations among T4SSs may confer specialized functional properties.
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Affiliation(s)
- Michael J. Sheedlo
- Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota, United States of America
| | - Melanie D. Ohi
- Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan, United States of America
- Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, United States of America
| | - D. Borden Lacy
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
- Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, United States of America
| | - Timothy L. Cover
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, United States of America
- Veterans Affairs Tennessee Valley Healthcare System, Nashville, Tennessee, United States of America
- Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
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17
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Abstract
Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus—the conjugative type IV secretion system (T4SS)—produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament—the conjugative pilus—that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein–protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili. Cryo-electron microscopy structures of a 2.8 megadalton bacterial type IV secretion system encoded by the plasmid R388 and comprising 92 polypeptides provide insights into the stepwise mechanism of pilus assembly.
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18
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Montelongo Hernandez C, Putonti C, Wolfe AJ. Profiling the plasmid conjugation potential of urinary Escherichia coli. Microb Genom 2022; 8:mgen000814. [PMID: 35536743 PMCID: PMC9465074 DOI: 10.1099/mgen.0.000814] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2021] [Accepted: 03/16/2022] [Indexed: 11/18/2022] Open
Abstract
Escherichia coli is often associated with urinary tract infection (UTI). Antibiotic resistance in E. coli is an ongoing challenge in managing UTI. Extrachromosomal elements - plasmids - are vectors for clinically relevant traits, such as antibiotic resistance, with conjugation being one of the main methods for horizontal propagation of plasmids in bacterial populations. Targeting of conjugation components has been proposed as a strategy to curb the spread of plasmid-borne antibiotic resistance. Understanding the types of conjugative systems present in urinary E. coli isolates is fundamental to assessing the viability of this strategy. In this study, we profile two well-studied conjugation systems (F-type and P-type) in the draft genomes of 65 urinary isolates of E. coli obtained from the bladder urine of adult women with and without UTI-like symptoms. Most of these isolates contained plasmids and we found that conjugation genes were abundant/ubiquitous, diverse and often associated with IncF plasmids. To validate conjugation of these urinary plasmids, the plasmids from two urinary isolates, UMB1223 (predicted to have F-type genes) and UMB1284 (predicted to have P-type genes), were transferred by conjugation into the K-12 E. coli strain MG1655. Overall, the findings of this study support the notion that care should be taken in targeting any individual component of a urinary E. coli isolate's conjugation system, given the inherent mechanistic redundancy, gene diversity and different types of conjugation systems in this population.
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Affiliation(s)
- Cesar Montelongo Hernandez
- Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
| | - Catherine Putonti
- Bioinformatics Program, Loyola University Chicago, Chicago, IL 60660, USA
- Department of Biology, Loyola University Chicago, Chicago, IL 60660, USA
| | - Alan J. Wolfe
- Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
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19
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Cheng E, Dorjsuren D, Lehman S, Larson CL, Titus SA, Sun H, Zakharov A, Rai G, Heinzen RA, Simeonov A, Machner MP. A Comprehensive Phenotypic Screening Strategy to Identify Modulators of Cargo Translocation by the Bacterial Type IVB Secretion System. mBio 2022; 13:e0024022. [PMID: 35258332 PMCID: PMC9040768 DOI: 10.1128/mbio.00240-22] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2022] [Accepted: 02/08/2022] [Indexed: 02/03/2023] Open
Abstract
Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.7 mouse macrophages. Using a fluorescence resonance energy transfer (FRET)-based detection assay in a bacteria/macrophage coculture format, we screened a library of over 18,000 compounds and, upon vetting compound candidates in a variety of in vitro and cell-based secondary screens, isolated several hits that efficiently interfered with biological processes that depend on a functional T4SS, such as intracellular bacterial proliferation or lysosomal avoidance, but had no detectable effect on L. pneumophila growth in culture medium, conditions under which the T4SS is dispensable. Notably, the same hit compounds also attenuated, to varying degrees, effector delivery by the closely related T4SS from Coxiella burnetii, notably without impacting growth of this organism within synthetic media. Together, these results support the idea that interference with T4SS function is a possible therapeutic intervention strategy, and the emerging compounds provide tools to interrogate at a molecular level the regulation and dynamics of these virulence-critical translocation machines. IMPORTANCE Multi-drug-resistant pathogens are an emerging threat to human health. Because conventional antibiotics target not only the pathogen but also eradicate the beneficial microbiota, they often cause additional clinical complications. Thus, there is an urgent need for the development of "smarter" therapeutics that selectively target pathogens without affecting beneficial commensals. The bacterial type IV secretion system (T4SS) is essential for the virulence of a variety of pathogens but dispensable for bacterial viability in general and can, thus, be considered a pathogen's Achilles heel. By identifying small molecules that interfere with cargo delivery by the T4SS from two important human pathogens, Legionella pneumophila and Coxiella burnetii, our study represents the first step in our pursuit toward precision medicine by developing pathogen-selective therapeutics capable of treating the infections without causing harm to commensal bacteria.
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Affiliation(s)
- Eric Cheng
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA
| | - Dorjbal Dorjsuren
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA
| | - Stephanie Lehman
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA
| | - Charles L. Larson
- Laboratory of Bacteriology, Coxiella Pathogenesis Section, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana, USA
| | - Steven A. Titus
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA
| | - Hongmao Sun
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA
| | - Alexey Zakharov
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA
| | - Ganesha Rai
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA
| | - Robert A. Heinzen
- Laboratory of Bacteriology, Coxiella Pathogenesis Section, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Montana, USA
| | - Anton Simeonov
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, USA
| | - Matthias P. Machner
- Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA
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20
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Liu X, Khara P, Baker ML, Christie PJ, Hu B. Structure of a type IV secretion system core complex encoded by multi-drug resistance F plasmids. Nat Commun 2022; 13:379. [PMID: 35046412 PMCID: PMC8770708 DOI: 10.1038/s41467-022-28058-5] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2021] [Accepted: 01/04/2022] [Indexed: 11/09/2022] Open
Abstract
Bacterial type IV secretion systems (T4SSs) are largely responsible for the proliferation of multi-drug resistance. We solved the structure of the outer-membrane core complex (OMCCF) of a T4SS encoded by a conjugative F plasmid at <3.0 Å resolution by cryoelectron microscopy. The OMCCF consists of a 13-fold symmetrical outer ring complex (ORC) built from 26 copies of TraK and TraV C-terminal domains, and a 17-fold symmetrical central cone (CC) composed of 17 copies of TraB β-barrels. Domains of TraV and TraB also bind the CC and ORC substructures, establishing that these proteins undergo an intraprotein symmetry alteration to accommodate the C13:C17 symmetry mismatch. We present evidence that other pED208-encoded factors stabilize the C13:C17 architecture and define the importance of TraK, TraV and TraB domains to T4SSF function. This work identifies OMCCF structural motifs of proposed importance for structural transitions associated with F plasmid dissemination and F pilus biogenesis.
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Affiliation(s)
- Xiangan Liu
- Department of Microbiology and Molecular Genetics, McGovern Medical School, 6431 Fannin St, Houston, TX, 77030, USA
| | - Pratick Khara
- Department of Microbiology and Molecular Genetics, McGovern Medical School, 6431 Fannin St, Houston, TX, 77030, USA
| | - Matthew L Baker
- Department of Biochemistry and Molecular Biology, McGovern Medical School, 6431 Fannin St, Houston, TX, 77030, USA
| | - Peter J Christie
- Department of Microbiology and Molecular Genetics, McGovern Medical School, 6431 Fannin St, Houston, TX, 77030, USA.
| | - Bo Hu
- Department of Microbiology and Molecular Genetics, McGovern Medical School, 6431 Fannin St, Houston, TX, 77030, USA.
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21
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Xiong X, Li B, Zhou Z, Gu G, Li M, Liu J, Jiao H. The VirB System Plays a Crucial Role in Brucella Intracellular Infection. Int J Mol Sci 2021; 22:ijms222413637. [PMID: 34948430 PMCID: PMC8707931 DOI: 10.3390/ijms222413637] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2021] [Revised: 12/09/2021] [Accepted: 12/15/2021] [Indexed: 01/18/2023] Open
Abstract
Brucellosis is a highly prevalent zoonotic disease caused by Brucella. Brucella spp. are gram-negative facultative intracellular parasitic bacteria. Its intracellular survival and replication depend on a functional virB system, an operon encoded by VirB1–VirB12. Type IV secretion system (T4SS) encoded by the virB operon is an important virulence factor of Brucella. It can subvert cellular pathway and induce host immune response by secreting effectors, which promotes Brucella replication in host cells and induce persistent infection. Therefore, this paper summarizes the function and significance of the VirB system, focusing on the structure of the VirB system where VirB T4SS mediates biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV), the effectors of T4SS and the cellular pathways it subverts, which will help better understand the pathogenic mechanism of Brucella and provide new ideas for clinical vaccine research and development.
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Affiliation(s)
- Xue Xiong
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (X.X.); (B.L.); (Z.Z.); (G.G.); (M.L.)
| | - Bowen Li
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (X.X.); (B.L.); (Z.Z.); (G.G.); (M.L.)
| | - Zhixiong Zhou
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (X.X.); (B.L.); (Z.Z.); (G.G.); (M.L.)
| | - Guojing Gu
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (X.X.); (B.L.); (Z.Z.); (G.G.); (M.L.)
| | - Mengjuan Li
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (X.X.); (B.L.); (Z.Z.); (G.G.); (M.L.)
| | - Jun Liu
- Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Yujinxiang Street 573, Changchun 130122, China
- Correspondence: (J.L.); (H.J.)
| | - Hanwei Jiao
- College of Veterinary Medicine, Southwest University, Chongqing 402460, China; (X.X.); (B.L.); (Z.Z.); (G.G.); (M.L.)
- National Center of Technology Innovation for Pigs, Chongqing 402460, China
- Veterinary Scientific Engineering Research Center, Chongqing 402460, China
- Immunology Research Center, Medical Research Institute, Southwest University, Chongqing 402460, China
- Correspondence: (J.L.); (H.J.)
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22
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Ge T, Jiang H, Tan EH, Johnson SB, Larkin RP, Charkowski AO, Secor G, Hao J. Pangenomic Analysis of Dickeya dianthicola Strains Related to the Outbreak of Blackleg and Soft Rot of Potato in the United States. PLANT DISEASE 2021; 105:3946-3955. [PMID: 34213964 DOI: 10.1094/pdis-03-21-0587-re] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Dickeya dianthicola has caused an outbreak of blackleg and soft rot of potato in the eastern half of the United States since 2015. To investigate genetic diversity of the pathogen, a comparative analysis was conducted on genomes of D. dianthicola strains. Whole genomes of 16 strains from the United States outbreak were assembled and compared with 16 previously sequenced genomes of D. dianthicola isolated from potato or carnation. Among the 32 strains, eight distinct clades were distinguished based on phylogenomic analysis. The outbreak strains were grouped into three clades, with the majority of the strains in clade I. Clade I strains were unique and homogeneous, suggesting a recent incursion of this strain into potato production from alternative hosts or environmental sources. The pangenome of the 32 strains contained 6,693 genes, 3,377 of which were core genes. By screening primary protein subunits associated with virulence from all U.S. strains, we found that many virulence-related gene clusters, such as plant cell wall degrading enzyme genes, flagellar and chemotaxis related genes, two-component regulatory genes, and type I/II/III secretion system genes, were highly conserved but that type IV and type VI secretion system genes varied. The clade I strains encoded two clusters of type IV secretion systems, whereas the clade II and III strains encoded only one cluster. Clade I and II strains encoded one more VgrG/PAAR spike protein than did clade III. Thus, we predicted that the presence of additional virulence-related genes may have enabled the unique clade I strain to become predominant in the U.S. outbreak.
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Affiliation(s)
- Tongling Ge
- School of Food and Agriculture, University of Maine, Orono, ME 04469
| | - He Jiang
- School of Food and Agriculture, University of Maine, Orono, ME 04469
| | - Ek Han Tan
- School of Biology and Ecology, University of Maine, Orono, ME 04469
| | | | - Robert P Larkin
- USDA-ARS, New England Plant, Soil, and Water Laboratory, University of Maine, Orono, ME 04469
| | - Amy O Charkowski
- Department of Agricultural Biology, Colorado State University, Fort Collins, CO 80523
| | - Gary Secor
- Department of Plant Pathology, North Dakota State University, Fargo, ND58108
| | - Jianjun Hao
- School of Food and Agriculture, University of Maine, Orono, ME 04469
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23
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Amin H, Ilangovan A, Costa TRD. Architecture of the outer-membrane core complex from a conjugative type IV secretion system. Nat Commun 2021; 12:6834. [PMID: 34824240 PMCID: PMC8617172 DOI: 10.1038/s41467-021-27178-8] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2021] [Accepted: 11/03/2021] [Indexed: 11/30/2022] Open
Abstract
Conjugation is one of the most important processes that bacteria utilize to spread antibiotic resistance genes among bacterial populations. Interbacterial DNA transfer requires a large double membrane-spanning nanomachine called the type 4 secretion system (T4SS) made up of the inner-membrane complex (IMC), the outer-membrane core complex (OMCC) and the conjugative pilus. The iconic F plasmid-encoded T4SS has been central in understanding conjugation for several decades, however atomic details of its structure are not known. Here, we report the structure of a complete conjugative OMCC encoded by the pED208 plasmid from E. coli, solved by cryo-electron microscopy at 3.3 Å resolution. This 2.1 MDa complex has a unique arrangement with two radial concentric rings, each having a different symmetry eventually contributing to remarkable differences in protein stoichiometry and flexibility in comparison to other OMCCs. Our structure suggests that F-OMCC is a highly dynamic complex, with implications for pilus extension and retraction during conjugation.
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Affiliation(s)
- Himani Amin
- grid.7445.20000 0001 2113 8111MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, SW7 2AZ UK
| | - Aravindan Ilangovan
- School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK.
| | - Tiago R. D. Costa
- grid.7445.20000 0001 2113 8111MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College, London, SW7 2AZ UK
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24
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Kitao T, Kubori T, Nagai H. Recent advances in structural studies of the Legionella pneumophila Dot/Icm type IV secretion system. Microbiol Immunol 2021; 66:67-74. [PMID: 34807482 PMCID: PMC9302130 DOI: 10.1111/1348-0421.12951] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2021] [Accepted: 11/15/2021] [Indexed: 11/29/2022]
Abstract
The intracellular bacterial pathogen Legionella pneumophila utilizes the Dot/Icm type IV secretion system to translocate approximately 300 effector proteins to establish a replicative niche known as the Legionella‐containing vacuole. The Dot/Icm system is classified as a type IVB secretion system, which is evolutionarily closely related to the I‐type conjugation systems and is distinct from type IVA secretion systems, such as the Agrobacterium VirB/D4 system. Although both type IVA and IVB systems directly transport nucleic acids or proteins into the cytosol of recipient cells, the components and architecture of type IVB systems are much more complex than those of type IVA systems. Taking full advantage of rapidly developing cryo‐electron microscopy techniques, the structural details of the transport apparatus and coupling complexes in the Dot/Icm system have been clarified in the past few years. In this review, we summarize recent progress in the structural studies of the L. pneumophila type IVB secretion system and the insights gained into the mechanisms of substrate recognition and transport.
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Affiliation(s)
- Tomoe Kitao
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Gifu, 501-1194, Japan
| | - Tomoko Kubori
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Gifu, 501-1194, Japan.,G-CHAIN, Gifu University, Gifu, Gifu, 501-1194, Japan
| | - Hiroki Nagai
- Department of Microbiology, Graduate School of Medicine, Gifu University, Gifu, Gifu, 501-1194, Japan.,G-CHAIN, Gifu University, Gifu, Gifu, 501-1194, Japan
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25
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In Situ Visualization of the pKM101-Encoded Type IV Secretion System Reveals a Highly Symmetric ATPase Energy Center. mBio 2021; 12:e0246521. [PMID: 34634937 PMCID: PMC8510550 DOI: 10.1128/mbio.02465-21] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Bacterial conjugation systems are members of the type IV secretion system (T4SS) superfamily. T4SSs can be classified as “minimized” or “expanded” based on whether they are composed of a core set of signature subunits or additional system-specific components. Prototypical minimized systems mediating Agrobacterium tumefaciens transfer DNA (T-DNA) and pKM101 and R388 plasmid transfer are built from subunits generically named VirB1 to VirB11 and VirD4. We visualized the pKM101-encoded T4SS in its native cellular context by in situ cryo-electron tomography (CryoET). The T4SSpKM101 is composed of an outer membrane core complex (OMCC) connected by a thin stalk to an inner membrane complex (IMC). The OMCC exhibits 14-fold symmetry and resembles that of the T4SSR388 analyzed previously by single-particle electron microscopy. The IMC is highly symmetrical and exhibits 6-fold symmetry. It is dominated by a hexameric collar in the periplasm and a cytoplasmic complex composed of a hexamer of dimers of the VirB4-like TraB ATPase. The IMC closely resembles equivalent regions of three expanded T4SSs previously visualized by in situ CryoET but differs strikingly from the IMC of the purified T4SSR388, whose cytoplasmic complex instead presents as two side-by-side VirB4 hexamers. Analyses of mutant machines lacking each of the three ATPases required for T4SSpKM101 function supplied evidence that TraBB4 as well as VirB11-like TraG contribute to distinct stages of machine assembly. We propose that the VirB4-like ATPases, configured as hexamers of dimers at the T4SS entrance, orchestrate IMC assembly and recruitment of the spatially dynamic VirB11 and VirD4 ATPases to activate the T4SS for substrate transfer.
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26
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Sheedlo MJ, Durie CL, Chung JM, Chang L, Roberts J, Swanson M, Lacy DB, Ohi MD. Cryo-EM reveals new species-specific proteins and symmetry elements in the Legionella pneumophila Dot/Icm T4SS. eLife 2021; 10:e70427. [PMID: 34519271 PMCID: PMC8486379 DOI: 10.7554/elife.70427] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2021] [Accepted: 09/14/2021] [Indexed: 01/21/2023] Open
Abstract
Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly, the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.
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Affiliation(s)
- Michael J Sheedlo
- Department of Pharmacology, University of MinnesotaMinneapolisUnited States
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical CenterNashvilleUnited States
| | - Clarissa L Durie
- Life Sciences Institute, University of MichiganAnn ArborUnited States
| | - Jeong Min Chung
- Life Sciences Institute, University of MichiganAnn ArborUnited States
- Department of Biotechnology, The Catholic University of KoreaGyeonggiRepublic of Korea
| | - Louise Chang
- Life Sciences Institute, University of MichiganAnn ArborUnited States
| | - Jacquelyn Roberts
- Life Sciences Institute, University of MichiganAnn ArborUnited States
| | - Michele Swanson
- Department of Microbiology and Immunology, University Of MichiganAnn ArborUnited States
| | - Dana Borden Lacy
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical CenterNashvilleUnited States
- The Veterans Affairs Tennessee Valley Healthcare SystemNasvhilleUnited States
| | - Melanie D Ohi
- Life Sciences Institute, University of MichiganAnn ArborUnited States
- Department of Cell and Developmental Biology, University of MichiganAnn ArborUnited States
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27
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Burns DL. Secretion of Pertussis Toxin from Bordetella pertussis. Toxins (Basel) 2021; 13:toxins13080574. [PMID: 34437445 PMCID: PMC8402538 DOI: 10.3390/toxins13080574] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Revised: 08/12/2021] [Accepted: 08/16/2021] [Indexed: 11/24/2022] Open
Abstract
Production and secretion of pertussis toxin (PT) is essential for the virulence of Bordetella pertussis. Due to the large oligomeric structure of PT, transport of the toxin across bacterial membrane barriers represents a significant hurdle that the bacteria must overcome in order to maintain pathogenicity. During the secretion process, PT undergoes a two-step transport process. The first step involves transport of the individual polypeptide chains of PT across the inner membrane utilizing a generalized secretion pathway, most likely the bacterial Sec system. The second step involves the use of a specialized apparatus to transport the toxin across the outer membrane of the bacterial cell. This apparatus, which has been termed the Ptl transporter and which is unique to the PT secretion pathway, is a member of the type IV family of bacterial transporters. Here, the current understanding of the PT secretion process is reviewed including a description of the Ptl proteins that assemble to form the transporter, the general structure of type IV transporters, the known similarities and differences between canonical type IV substrate transport and Ptl-mediated transport of PT, as well as the known sequence of events in the assembly and secretion of PT.
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Affiliation(s)
- Drusilla L Burns
- Center for Biologics Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, MD 20993, USA
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28
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Mathioudaki E, Arvaniti K, Muenke C, Drakonaki A, Vranakis I, Koutantou M, Psaroulaki A, Xie H, Tsiotis G. Expression, purification and characterization of the IcmG and IcmK proteins of the type IVB secretion system from Coxiella burnetii. Protein Expr Purif 2021; 186:105905. [PMID: 33989770 DOI: 10.1016/j.pep.2021.105905] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2020] [Revised: 04/15/2021] [Accepted: 05/06/2021] [Indexed: 10/21/2022]
Abstract
Coxiella burnetii, the causative agent of Q fever, is an intracellular bacterial pathogen. Studies on Coxiella have shown that a type IVB secretion system (T4BSS) contributes to the establishment of the infection by transferring protein molecules. In this report, we focus on two core proteins of the Coxiella T4BSS, namely the IcmG/DotF protein (CBU_1626) and the IcmK/DotH protein (CBU_1628). Here we present a method for the recombinant expression of IcmG and IcmK in E. coli. IcmG was purified by Strep-Tactin affinity chromatography and size exclusion chromatography, while for the purification of IcmK an additional anion exchange chromatography step was introduced. The yields of the purified IcmG and IcmK proteins were 1.2 mg/L and 3 mg/L, respectively. The purified proteins showed predominant band on SDS-PAGE gel of 37 kDa for the IcmG and 40 kDa for the IcmK. Protein folding is confirmed by circular dichroism spectroscopy. The dynamic light scattering experiment indicated that IcmG and IcmK existed in a homogenous form. Further Blue native PAGE indicates the presences of a monomeric form for the IcmK and IcmG. Our work lays the basis for functional exploration and structural determination of IcmG and IcmK proteins of Coxiella's secretion system.
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Affiliation(s)
- Eirini Mathioudaki
- Division of Biochemistry, Department of Chemistry, University of Crete, GR-71003, Voutes, Greece
| | - Katerina Arvaniti
- Division of Biochemistry, Department of Chemistry, University of Crete, GR-71003, Voutes, Greece
| | - Cornelia Muenke
- Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, D-60438, Frankfurt am Main, Germany
| | - Athina Drakonaki
- Division of Biochemistry, Department of Chemistry, University of Crete, GR-71003, Voutes, Greece
| | - Iosif Vranakis
- Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete, GR-71110, Heraklion, Greece
| | - Myrto Koutantou
- Division of Biochemistry, Department of Chemistry, University of Crete, GR-71003, Voutes, Greece
| | - Anna Psaroulaki
- Department of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, Medical School, University of Crete, GR-71110, Heraklion, Greece
| | - Hao Xie
- Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, D-60438, Frankfurt am Main, Germany.
| | - Georgios Tsiotis
- Division of Biochemistry, Department of Chemistry, University of Crete, GR-71003, Voutes, Greece.
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29
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pCTX-M3-Structure, Function, and Evolution of a Multi-Resistance Conjugative Plasmid of a Broad Recipient Range. Int J Mol Sci 2021; 22:ijms22094606. [PMID: 33925677 PMCID: PMC8125031 DOI: 10.3390/ijms22094606] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Revised: 04/23/2021] [Accepted: 04/26/2021] [Indexed: 11/17/2022] Open
Abstract
pCTX-M3 is the archetypic member of the IncM incompatibility group of conjugative plasmids (recently referred to as IncM2). It is responsible for the worldwide dissemination of numerous antibiotic resistance genes, including those coding for extended-spectrum β-lactamases and conferring resistance to aminoglycosides. The IncM plasmids acquired during evolution diverse mobile genetic elements found in one or two multiple resistance regions, MRR(s), grouping antibiotic resistance genes as well as mobile genetic elements or their remnants. The IncM plasmids can be found in bacteria inhabiting various environments. The information on the structure and biology of pCTX-M3 is integrated in this review. It focuses on the functional modules of pCTX-M3 responsible for its replication, stable maintenance, and conjugative transfer, indicating that the host range of the pCTX-M3 replicon is limited to representatives of the family Enterobacteriaceae (Enterobacterales ord. nov.), while the range of recipients of its conjugation system is wide, comprising Alpha-, Beta-, and Gammaproteobacteria, and also Firmicutes.
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30
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Patterson LL, Byerly CD, McBride JW. Anaplasmataceae: Dichotomous Autophagic Interplay for Infection. Front Immunol 2021; 12:642771. [PMID: 33912170 PMCID: PMC8075259 DOI: 10.3389/fimmu.2021.642771] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Accepted: 03/15/2021] [Indexed: 12/19/2022] Open
Abstract
Autophagy is a vital conserved degradative process that maintains cellular homeostasis by recycling or eliminating dysfunctional cellular organelles and proteins. More recently, autophagy has become a well-recognized host defense mechanism against intracellular pathogens through a process known as xenophagy. On the host-microbe battlefield many intracellular bacterial pathogens have developed the ability to subvert xenophagy to establish infection. Obligately intracellular bacterial pathogens of the Anaplasmataceae family, including Ehrlichia chaffeensis, Anaplasma phaogocytophilium and Orientia tsutsugamushi have developed a dichotomous strategy to exploit the host autophagic pathway to obtain nutrients while escaping lysosomal destruction for intracellular survival within the host cell. In this review, the recent findings regarding how these master manipulators engage and inhibit autophagy for infection are explored. Future investigation to understand mechanisms used by Anaplasmataceae to exploit autophagy may advance novel antimicrobial therapies and provide new insights into how intracellular microbes exploit autophagy to survive.
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Affiliation(s)
- LaNisha L Patterson
- Department of Pathology, University of Texas Medical Branch, Galveston, TX, United States
| | - Caitlan D Byerly
- Department of Pathology, University of Texas Medical Branch, Galveston, TX, United States
| | - Jere W McBride
- Department of Pathology, University of Texas Medical Branch, Galveston, TX, United States.,Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, United States.,Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, United States.,Sealy Institute for Vaccine Sciences, University of Texas Medical Branch, Galveston, TX, United States.,Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, TX, United States
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31
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Jaboulay C, Godeux AS, Doublet P, Vianney A. Regulatory Networks of the T4SS Control: From Host Cell Sensing to the Biogenesis and the Activity during the Infection. J Mol Biol 2021; 433:166892. [PMID: 33636165 DOI: 10.1016/j.jmb.2021.166892] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2020] [Revised: 02/17/2021] [Accepted: 02/17/2021] [Indexed: 02/03/2023]
Abstract
Delivery of effectors, DNA or proteins, that hijack host cell processes to the benefit of bacteria is a mechanism widely used by bacterial pathogens. It is achieved by complex effector injection devices, the secretion systems, among which Type 4 Secretion Systems (T4SSs) play a key role in bacterial virulence of numerous animal and plant pathogens. Considerable progress has recently been made in the structure-function analyses of T4SSs. Nevertheless, the signals and processes that trigger machine assembly and activity during infection, as well as those involved in substrate recognition and transfer, are complex and still poorly understood. In this review, we aim at summarizing the last updates of the knowledge on signaling pathways that regulate the biogenesis and the activity of T4SSs in important bacterial pathogens.
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Affiliation(s)
- C Jaboulay
- CIRI, Centre International de Recherche en Infectiologie, (Team: Legionella pathogenesis), Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007 Lyon, France.
| | - A S Godeux
- CIRI, Centre International de Recherche en Infectiologie, (Team: Horigene), Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007 Lyon, France
| | - P Doublet
- CIRI, Centre International de Recherche en Infectiologie, (Team: Legionella pathogenesis), Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007 Lyon, France
| | - A Vianney
- CIRI, Centre International de Recherche en Infectiologie, (Team: Legionella pathogenesis), Univ Lyon, Inserm, U1111, Université Claude Bernard Lyon 1, CNRS, UMR5308, ENS de Lyon, F-69007 Lyon, France
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32
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Costa TRD, Harb L, Khara P, Zeng L, Hu B, Christie PJ. Type IV secretion systems: Advances in structure, function, and activation. Mol Microbiol 2021; 115:436-452. [PMID: 33326642 DOI: 10.1111/mmi.14670] [Citation(s) in RCA: 119] [Impact Index Per Article: 29.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2020] [Revised: 12/12/2020] [Accepted: 12/13/2020] [Indexed: 12/14/2022]
Abstract
Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved "core" subunits that respectively elaborate "minimized" systems in Gram-positive or -negative bacteria. However, many "expanded" T4SSs are built from "core" subunits plus numerous others that are system-specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure-function relationships for model Gram-negative bacterial T4SSs, including "minimized" systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and "expanded" systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid-encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long-standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.
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Affiliation(s)
- Tiago R D Costa
- MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK
| | - Laith Harb
- Department of Biochemistry and Biophysics and Center for Phage Technology, Texas A&M University, College Station, TX, USA
| | - Pratick Khara
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, TX, USA
| | - Lanying Zeng
- Department of Biochemistry and Biophysics and Center for Phage Technology, Texas A&M University, College Station, TX, USA
| | - Bo Hu
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, TX, USA
| | - Peter J Christie
- Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, TX, USA
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33
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Umrekar TR, Cohen E, Drobnič T, Gonzalez-Rodriguez N, Beeby M. CryoEM of bacterial secretion systems: A primer for microbiologists. Mol Microbiol 2020; 115:366-382. [PMID: 33140482 DOI: 10.1111/mmi.14637] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2020] [Revised: 10/27/2020] [Accepted: 10/29/2020] [Indexed: 12/11/2022]
Abstract
"CryoEM" has come of age, enabling considerable structural insights into many facets of molecular biology. Here, we present a primer for microbiologists to understand the capabilities and limitations of two complementary cryoEM techniques for studying bacterial secretion systems. The first, single particle analysis, determines the structures of purified protein complexes to resolutions sufficient for molecular modeling, while the second, electron cryotomography and subtomogram averaging, tends to determine more modest resolution structures of protein complexes in intact cells. We illustrate these abilities with examples of insights provided into how secretion systems work by cryoEM, with a focus on type III secretion systems.
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Affiliation(s)
| | - Eli Cohen
- Department of Life Sciences, Imperial College London, London, UK
| | - Tina Drobnič
- Department of Life Sciences, Imperial College London, London, UK
| | | | - Morgan Beeby
- Department of Life Sciences, Imperial College London, London, UK
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34
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Sheedlo MJ, Chung JM, Sawhney N, Durie CL, Cover TL, Ohi MD, Lacy DB. Cryo-EM reveals species-specific components within the Helicobacter pylori Cag type IV secretion system core complex. eLife 2020; 9:e59495. [PMID: 32876048 PMCID: PMC7511236 DOI: 10.7554/elife.59495] [Citation(s) in RCA: 35] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2020] [Accepted: 09/01/2020] [Indexed: 02/06/2023] Open
Abstract
The pathogenesis of Helicobacter pylori-associated gastric cancer is dependent on delivery of CagA into host cells through a type IV secretion system (T4SS). The H. pylori Cag T4SS includes a large membrane-spanning core complex containing five proteins, organized into an outer membrane cap (OMC), a periplasmic ring (PR) and a stalk. Here, we report cryo-EM reconstructions of a core complex lacking Cag3 and an improved map of the wild-type complex. We define the structures of two unique species-specific components (Cag3 and CagM) and show that Cag3 is structurally similar to CagT. Unexpectedly, components of the OMC are organized in a 1:1:2:2:5 molar ratio (CagY:CagX:CagT:CagM:Cag3). CagX and CagY are components of both the OMC and the PR and bridge the symmetry mismatch between these regions. These results reveal that assembly of the H. pylori T4SS core complex is dependent on incorporation of interwoven species-specific components.
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Affiliation(s)
- Michael J Sheedlo
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical CenterNashvilleUnited States
| | - Jeong Min Chung
- Life Sciences Institute, University of MichiganAnn ArborUnited States
| | - Neha Sawhney
- Department of Medicine, Vanderbilt University School of MedicineNashvilleUnited States
| | - Clarissa L Durie
- Life Sciences Institute, University of MichiganAnn ArborUnited States
| | - Timothy L Cover
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical CenterNashvilleUnited States
- Department of Medicine, Vanderbilt University School of MedicineNashvilleUnited States
- Veterans Affairs Tennessee Valley Healthcare SystemNashvilleUnited States
| | - Melanie D Ohi
- Life Sciences Institute, University of MichiganAnn ArborUnited States
- Department of Cell and Developmental Biology, University of MichiganAnn ArborUnited States
| | - D Borden Lacy
- Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical CenterNashvilleUnited States
- Veterans Affairs Tennessee Valley Healthcare SystemNashvilleUnited States
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35
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Koch B, Callaghan MM, Tellechea-Luzardo J, Seeger AY, Dillard JP, Krasnogor N. Protein interactions within and between two F-type type IV secretion systems. Mol Microbiol 2020; 114:823-838. [PMID: 32738086 DOI: 10.1111/mmi.14582] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2020] [Revised: 07/20/2020] [Accepted: 07/20/2020] [Indexed: 01/03/2023]
Abstract
Bacterial type IV secretion systems (T4SSs) can mediate conjugation. The T4SS from Neisseria gonorrhoeae possesses the unique ability to mediate DNA secretion into the extracellular environment. The N. gonorrhoeae T4SS can be grouped with F-type conjugative T4SSs based on homology. We tested 17 proteins important for DNA secretion by N. gonorrhoeae for protein interactions. The BACTH-TM bacterial two-hybrid system was successfully used to study periplasmic interactions. By determining if the same interactions were observed for F-plasmid T4SS proteins and when one interaction partner was replaced by the corresponding protein from the other T4SS, we aimed to identify features associated with the unique function of the N. gonorrhoeae T4SS as well as generic features of F-type T4SSs. For both systems, we observed already described interactions shared by homologs from other T4SSs as well as new and described interactions between F-type T4SS-specific proteins. Furthermore, we demonstrate, for the first-time, interactions between proteins with homology to the conserved T4SS outer membrane core proteins and F-type-specific proteins and we confirmed two of them by co-purification. The F-type-specific protein TraHN was found to localize to the outer membrane and the presence of significant amounts of TraHN in the outer membrane requires TraGN .
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Affiliation(s)
- Birgit Koch
- Interdisciplinary Computing and Complex BioSystems (ICOS), School of Computing Science, Newcastle University, Newcastle upon Tyne, UK
| | - Melanie M Callaghan
- Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA
| | - Jonathan Tellechea-Luzardo
- Interdisciplinary Computing and Complex BioSystems (ICOS), School of Computing Science, Newcastle University, Newcastle upon Tyne, UK
| | - Ami Y Seeger
- Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA
| | - Joseph P Dillard
- Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA
| | - Natalio Krasnogor
- Interdisciplinary Computing and Complex BioSystems (ICOS), School of Computing Science, Newcastle University, Newcastle upon Tyne, UK
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36
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Clairfeuille T, Buchholz KR, Li Q, Verschueren E, Liu P, Sangaraju D, Park S, Noland CL, Storek KM, Nickerson NN, Martin L, Dela Vega T, Miu A, Reeder J, Ruiz-Gonzalez M, Swem D, Han G, DePonte DP, Hunter MS, Gati C, Shahidi-Latham S, Xu M, Skelton N, Sellers BD, Skippington E, Sandoval W, Hanan EJ, Payandeh J, Rutherford ST. Structure of the essential inner membrane lipopolysaccharide-PbgA complex. Nature 2020; 584:479-483. [PMID: 32788728 DOI: 10.1038/s41586-020-2597-x] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2018] [Accepted: 07/10/2020] [Indexed: 12/21/2022]
Abstract
Lipopolysaccharide (LPS) resides in the outer membrane of Gram-negative bacteria where it is responsible for barrier function1,2. LPS can cause death as a result of septic shock, and its lipid A core is the target of polymyxin antibiotics3,4. Despite the clinical importance of polymyxins and the emergence of multidrug resistant strains5, our understanding of the bacterial factors that regulate LPS biogenesis is incomplete. Here we characterize the inner membrane protein PbgA and report that its depletion attenuates the virulence of Escherichia coli by reducing levels of LPS and outer membrane integrity. In contrast to previous claims that PbgA functions as a cardiolipin transporter6-9, our structural analyses and physiological studies identify a lipid A-binding motif along the periplasmic leaflet of the inner membrane. Synthetic PbgA-derived peptides selectively bind to LPS in vitro and inhibit the growth of diverse Gram-negative bacteria, including polymyxin-resistant strains. Proteomic, genetic and pharmacological experiments uncover a model in which direct periplasmic sensing of LPS by PbgA coordinates the biosynthesis of lipid A by regulating the stability of LpxC, a key cytoplasmic biosynthetic enzyme10-12. In summary, we find that PbgA has an unexpected but essential role in the regulation of LPS biogenesis, presents a new structural basis for the selective recognition of lipids, and provides opportunities for future antibiotic discovery.
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Affiliation(s)
| | - Kerry R Buchholz
- Infectious Diseases, Genentech Inc., South San Francisco, CA, USA
| | - Qingling Li
- Microchemistry, Proteomics & Lipidomics, Genentech Inc., South San Francisco, CA, USA
| | - Erik Verschueren
- Microchemistry, Proteomics & Lipidomics, Genentech Inc., South San Francisco, CA, USA
| | - Peter Liu
- Microchemistry, Proteomics & Lipidomics, Genentech Inc., South San Francisco, CA, USA
| | - Dewakar Sangaraju
- Drug Metabolism & Pharmacokinetics, Genentech Inc., South San Francisco, CA, USA
| | - Summer Park
- Translational Immunology, Genentech Inc., South San Francisco, CA, USA
| | - Cameron L Noland
- Structural Biology, Genentech Inc., South San Francisco, CA, USA
| | - Kelly M Storek
- Infectious Diseases, Genentech Inc., South San Francisco, CA, USA
| | | | - Lynn Martin
- BioMolecular Resources, Genentech Inc., South San Francisco, CA, USA
| | - Trisha Dela Vega
- BioMolecular Resources, Genentech Inc., South San Francisco, CA, USA
| | - Anh Miu
- Biochemical & Cellular Pharmacology, Genentech Inc., South San Francisco, CA, USA
| | - Janina Reeder
- Bioinformatics & Computational Biology, Genentech Inc., South San Francisco, CA, USA
| | - Maria Ruiz-Gonzalez
- Discovery Chemistry Departments, Genentech Inc., South San Francisco, CA, USA
| | - Danielle Swem
- Infectious Diseases, Genentech Inc., South San Francisco, CA, USA
| | - Guanghui Han
- Microchemistry, Proteomics & Lipidomics, Genentech Inc., South San Francisco, CA, USA
| | - Daniel P DePonte
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA, USA
| | - Mark S Hunter
- Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, CA, USA
| | - Cornelius Gati
- Bioscience Division, SLAC National Accelerator Laboratory, Menlo Park, CA, USA.,Stanford University, Department of Structural Biology, Stanford, CA, USA
| | | | - Min Xu
- Translational Immunology, Genentech Inc., South San Francisco, CA, USA
| | - Nicholas Skelton
- Discovery Chemistry Departments, Genentech Inc., South San Francisco, CA, USA
| | - Benjamin D Sellers
- Discovery Chemistry Departments, Genentech Inc., South San Francisco, CA, USA
| | - Elizabeth Skippington
- Bioinformatics & Computational Biology, Genentech Inc., South San Francisco, CA, USA
| | - Wendy Sandoval
- Microchemistry, Proteomics & Lipidomics, Genentech Inc., South San Francisco, CA, USA
| | - Emily J Hanan
- Discovery Chemistry Departments, Genentech Inc., South San Francisco, CA, USA.
| | - Jian Payandeh
- Structural Biology, Genentech Inc., South San Francisco, CA, USA. .,Infectious Diseases, Genentech Inc., South San Francisco, CA, USA.
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37
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Li YG, Christie PJ. The TraK accessory factor activates substrate transfer through the pKM101 type IV secretion system independently of its role in relaxosome assembly. Mol Microbiol 2020; 114:214-229. [PMID: 32239779 DOI: 10.1111/mmi.14507] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2020] [Revised: 03/24/2020] [Indexed: 12/12/2022]
Abstract
A large subfamily of the type IV secretion systems (T4SSs), termed the conjugation systems, transmit mobile genetic elements (MGEs) among many bacterial species. In the initiating steps of conjugative transfer, DNA transfer and replication (Dtr) proteins assemble at the origin-of-transfer (oriT) sequence as the relaxosome, which nicks the DNA strand destined for transfer and couples the nicked substrate with the VirD4-like substrate receptor. Here, we defined contributions of the Dtr protein TraK, a predicted member of the Ribbon-Helix-Helix (RHH) family of DNA-binding proteins, to transfer of DNA and protein substrates through the pKM101-encoded T4SS. Using a combination of cross-linking/affinity pull-downs and two-hybrid assays, we determined that TraK self-associates as a probable tetramer and also forms heteromeric contacts with pKM101-encoded TraI relaxase, VirD4-like TraJ receptor, and VirB11-like and VirB4-like ATPases, TraG and TraB, respectively. TraK also promotes stable TraJ-TraB complex formation and stimulates binding of TraI with TraB. Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the TrapKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance.
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Affiliation(s)
- Yang Grace Li
- Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, TX, USA
| | - Peter J Christie
- Department of Microbiology and Molecular Genetics, McGovern Medical School, Houston, TX, USA
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38
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Abstract
The translocation of proteins across membranes is a fundamental cellular function. Bacteria have evolved a striking array of pathways for delivering proteins into or across cytoplasmic membranes and, when present, outer membranes. Translocated proteins can form part of the membrane landscape, reside in the periplasmic space situated between the inner and outer membranes of Gram-negative bacteria, deposit on the cell surface, or be released to the extracellular milieu or injected directly into target cells. One protein translocation system, the general secretory pathway, is conserved in all domains of life. A second, the twin-arginine translocation pathway, is also phylogenetically distributed among most bacteria and plant chloroplasts. While all cell types have evolved additional systems dedicated to the translocation of protein cargoes, the number of such systems in bacteria is now known to exceed nine. These dedicated protein translocation systems, which include the types 1 through 9 secretion systems (T1SSs-T9SSs), the chaperone-usher pathway, and type IV pilus system, are the subject of this review. Most of these systems were originally identified and have been extensively characterized in Gram-negative or diderm (two-membrane) species. It is now known that several of these systems also have been adapted to function in Gram-positive or monoderm (single-membrane) species, and at least one pathway is found only in monoderms. This review briefly summarizes the distinctive mechanistic and structural features of each dedicated pathway, as well as the shared properties, that together account for the broad biological diversity of protein translocation in bacteria.
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Affiliation(s)
- Peter J Christie
- Department of Microbiology and Molecular Genetics, McGovern Medical School, 6431 Fannin St., Houston, TX, USA.
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39
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The Helicobacter pylori Cag Type IV Secretion System. Trends Microbiol 2020; 28:682-695. [PMID: 32451226 DOI: 10.1016/j.tim.2020.02.004] [Citation(s) in RCA: 85] [Impact Index Per Article: 17.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2019] [Revised: 01/30/2020] [Accepted: 02/25/2020] [Indexed: 12/24/2022]
Abstract
Colonization of the human stomach with Helicobacter pylori strains containing the cag pathogenicity island is a risk factor for development of gastric cancer. The cag pathogenicity island contains genes encoding a secreted effector protein (CagA) and components of a type IV secretion system (Cag T4SS). The molecular architecture of the H. pylori Cag T4SS is substantially more complex than that of prototype T4SSs in other bacterial species. In this review, we discuss recent discoveries pertaining to the structure and function of the Cag T4SS and its role in gastric cancer pathogenesis.
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40
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Darbari VC, Ciccone J, Patel JS, Islam B, Agarwal PK, Haider S. Electrostatic Switching Controls Channel Dynamics of the Sensor Protein VirB10 in A. tumefaciens Type IV Secretion System. ACS OMEGA 2020; 5:3271-3281. [PMID: 32118142 PMCID: PMC7045316 DOI: 10.1021/acsomega.9b03313] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/07/2019] [Accepted: 01/20/2020] [Indexed: 05/19/2023]
Abstract
Type IV secretion systems are large nanomachines assembled across the bacterial cell envelope for effector translocation and conjugation. VirB10 traverses the inner and outer membranes, sensing cellular signals for coordinating the conformational switch for pilus biogenesis and/or secretion. Mutations uncoupling secretion from pilus biogenesis were identified in Agrobacterium tumefaciens VirB10 including a gating defect mutation G272R that made VirB10 unresponsive to intracellular ATP, causing unregulated secretion of VirE2 in a contact-independent manner. Comparative long-timescale molecular dynamics of the wild type and G272R mutant of the A. tumefaciens VirB10CTD tetradecamer reveals how the G272R mutation locks the oligomer in a rigid conformation by swapping the ionic interactions between the loops from the β-barrel close to the inner leaflet of the outer membrane. This electrostatic switching changes the allosteric communication pathway from the extracellular loop to the base of the barrel, suggesting that the local conformational dynamics in the loops can gate information across VirB10.
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Affiliation(s)
- Vidya Chandran Darbari
- School
of Biological and Chemical Sciences, Queen
Mary University of London, Mile End Road, London E1 4NS, United Kingdom
- E-mail: . Tel: +44 (0)
20 7882 6360 (V.C.D.)
| | - Jonah Ciccone
- Department
of Pharmaceutical and Biological Chemistry, University College London School of Pharmacy, WC1N 1AX London, United Kingdom
| | - Jagdish Suresh Patel
- Department
of Biological Sciences, University of Idaho, C/O IRIC 333, 875 Perimeter MS 1122, Moscow, Idaho 83844-1122, United States
| | - Barira Islam
- Centre
for Biomarker Research, School of Applied Sciences, University of Huddersfield, HD1 3DH Huddersfield, United Kingdom
| | - Pratul K Agarwal
- Department
of Biochemistry & Cellular and Molecular Biology Department, University of Tennessee-Knoxville, Knoxville, Tennessee 37996, United States
| | - Shozeb Haider
- Department
of Pharmaceutical and Biological Chemistry, University College London School of Pharmacy, WC1N 1AX London, United Kingdom
- E-mail: . Tel: +44 (0) 20 7753 5883 (S.H.)
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41
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Park D, Chetrit D, Hu B, Roy CR, Liu J. Analysis of Dot/Icm Type IVB Secretion System Subassemblies by Cryoelectron Tomography Reveals Conformational Changes Induced by DotB Binding. mBio 2020; 11:e03328-19. [PMID: 32071271 PMCID: PMC7029142 DOI: 10.1128/mbio.03328-19] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Accepted: 01/03/2020] [Indexed: 12/23/2022] Open
Abstract
Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane.IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.
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Affiliation(s)
- Donghyun Park
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA
- Microbial Sciences Institute, Yale University, West Haven, Connecticut, USA
| | - David Chetrit
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Bo Hu
- Department of Microbiology and Molecular Genetics, McGovern Medical School, The University of Texas Health Science Center at Houston, Houston, Texas, USA
| | - Craig R Roy
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA
| | - Jun Liu
- Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA
- Microbial Sciences Institute, Yale University, West Haven, Connecticut, USA
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42
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Das A. Identification of a Carboxy-Terminal Glutamine-Rich Domain in Agrobacterium tumefaciens Coupling Protein VirD4 Required for Recognition of T-Strand DNA and Not VirE2 as a Substrate for Transfer to Plant Cells. MOLECULAR PLANT-MICROBE INTERACTIONS : MPMI 2020; 33:166-172. [PMID: 31855496 DOI: 10.1094/mpmi-04-19-0099-r] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
Agrobacterium tumefaciens transfers DNA and proteins to a plant cell inciting crown gall tumor disease on most plants. VirD4 targets the DNA and protein substrates to a type IV secretion (T4S) apparatus for translocation into the plant cell. Several bacteria with VirD4 homologs use T4S for intercellular export of microbial macromolecules to eukaryotic and prokaryotic hosts. How the VirD4 proteins recognize the diverse substrates is not well understood. To identify functional domains of A. tumefaciens pTiA6 VirD4, we introduced random 19-codon and targeted 10-codon insertions throughout the coding region. Analysis of 21 mutants showed that only the carboxy-terminal end of VirD4 is tolerant of an insertion. Sequence comparison of VirD4 proteins of Agrobacterium spp. and their close relative, Rhizobium etli, showed that these proteins contain a highly conserved C-terminal end, but the immediate upstream regions share no discernible sequence similarity. The conserved region sequence is rich in the amino acid glutamine (6/13 Q). Using site-specific and deletion mutagenesis, we demonstrated that the conserved Q-rich region is required for VirD4 function and for the specific recognition of VirD2-linked T-strand DNA as a substrate for translocation to plants. The Q-rich region is not required for the transfer of a second A. tumefaciens substrate, VirE2, to plants or a promiscuous Escherichia coli IncQ plasmid to another A. tumefaciens strain. We identified Q-rich sequences at or near the C terminus of several VirD4 homologs, including the E. coli F plasmid TraD. In F TraD, the Q-rich sequence maps to a region required specifically for the conjugative transfer of the F plasmid.
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Affiliation(s)
- Anath Das
- Department of Biochemistry, Molecular Biology and Biophysics, and Microbial and Plant Genomics Institute, University of Minnesota, Minneapolis, MN 55455, U.S.A
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43
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Chang YW, Shaffer CL, Rettberg LA, Ghosal D, Jensen GJ. In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System. Cell Rep 2019; 23:673-681. [PMID: 29669273 PMCID: PMC5931392 DOI: 10.1016/j.celrep.2018.03.085] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2017] [Revised: 01/16/2018] [Accepted: 03/19/2018] [Indexed: 02/06/2023] Open
Abstract
The type IV secretion system (T4SS) is a versatile nanomachine that translocates diverse effector molecules between microbes and into eukaryotic cells. Here, using electron cryotomography, we reveal the molecular architecture of the Helicobacter pylori cag T4SS. Although most components are unique to H. pylori, the cag T4SS exhibits remarkable architectural similarity to other T4SSs. Our images revealed that, when H. pylori encounters host cells, the bacterium elaborates membranous tubes perforated by lateral ports. Sub-tomogram averaging of the cag T4SS machinery revealed periplasmic densities associated with the outer membrane, a central stalk, and peripheral wing-like densities. Additionally, we resolved pilus-like rod structures extending from the cag T4SS into the inner membrane, as well as densities within the cytoplasmic apparatus corresponding to a short central barrel surrounded by four longer barrels. Collectively, these studies reveal the structure of a dynamic molecular machine that evolved to function in the human gastric niche.
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Affiliation(s)
- Yi-Wei Chang
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Carrie L Shaffer
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Lee A Rettberg
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; Howard Hughes Medical Institute, Pasadena, CA 91125, USA
| | - Debnath Ghosal
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA
| | - Grant J Jensen
- Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; Howard Hughes Medical Institute, Pasadena, CA 91125, USA.
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44
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Bayer-Santos E, Cenens W, Matsuyama BY, Oka GU, Di Sessa G, Mininel IDV, Alves TL, Farah CS. The opportunistic pathogen Stenotrophomonas maltophilia utilizes a type IV secretion system for interbacterial killing. PLoS Pathog 2019; 15:e1007651. [PMID: 31513674 PMCID: PMC6759196 DOI: 10.1371/journal.ppat.1007651] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2019] [Revised: 09/24/2019] [Accepted: 08/09/2019] [Indexed: 12/11/2022] Open
Abstract
Bacterial type IV secretion systems (T4SS) are a highly diversified but evolutionarily related family of macromolecule transporters that can secrete proteins and DNA into the extracellular medium or into target cells. It was recently shown that a subtype of T4SS harboured by the plant pathogen Xanthomonas citri transfers toxins into target cells. Here, we show that a similar T4SS from the multi-drug-resistant opportunistic pathogen Stenotrophomonas maltophilia is proficient in killing competitor bacterial species. T4SS-dependent duelling between S. maltophilia and X. citri was observed by time-lapse fluorescence microscopy. A bioinformatic search of the S. maltophilia K279a genome for proteins containing a C-terminal domain conserved in X. citri T4SS effectors (XVIPCD) identified twelve putative effectors and their cognate immunity proteins. We selected a putative S. maltophilia effector with unknown function (Smlt3024) for further characterization and confirmed that it is indeed secreted in a T4SS-dependent manner. Expression of Smlt3024 in the periplasm of E. coli or its contact-dependent delivery via T4SS into E. coli by X. citri resulted in reduced growth rates, which could be counteracted by expression of its cognate inhibitor Smlt3025 in the target cell. Furthermore, expression of the VirD4 coupling protein of X. citri can restore the function of S. maltophilia ΔvirD4, demonstrating that effectors from one species can be recognized for transfer by T4SSs from another species. Interestingly, Smlt3024 is homologous to the N-terminal domain of large Ca2+-binding RTX proteins and the crystal structure of Smlt3025 revealed a topology similar to the iron-regulated protein FrpD from Neisseria meningitidis which has been shown to interact with the RTX protein FrpC. This work expands our current knowledge about the function of bacteria-killing T4SSs and increases the panel of effectors known to be involved in T4SS-mediated interbacterial competition, which possibly contribute to the establishment of S. maltophilia in clinical and environmental settings.
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Affiliation(s)
- Ethel Bayer-Santos
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil
- Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, São Paulo, Brazil
| | - William Cenens
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil
| | - Bruno Yasui Matsuyama
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil
| | - Gabriel Umaji Oka
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil
| | - Giancarlo Di Sessa
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil
| | - Izabel Del Valle Mininel
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil
| | - Tiago Lubiana Alves
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil
| | - Chuck Shaker Farah
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, São Paulo, Brazil
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45
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Makes caterpillars floppy-like effector-containing MARTX toxins require host ADP-ribosylation factor (ARF) proteins for systemic pathogenicity. Proc Natl Acad Sci U S A 2019; 116:18031-18040. [PMID: 31427506 PMCID: PMC6731672 DOI: 10.1073/pnas.1905095116] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
MARTX toxins present across multiple bacterial genera are primary virulence factors that facilitate initial colonization, dissemination, and lethality in a wide range of hosts, including humans. Upon entry into host cells, the toxins undergo a processing event to release their disease-related modularly structured effector domains. However, the mechanisms underlying processing and activation of diverse effector domains within the toxins remain unclear. Here, we use biochemical and structural biological approaches, in combination with cellular microbiological experiments, to demonstrate how Makes caterpillars floppy-like effector (MCF) or its homolog-containing MARTX toxins process effector modules and fully activate effectors. MCF-containing toxins target ADP-ribosylation factor proteins ubiquitously expressed in cells to activate and disseminate effectors across subcellular compartments simultaneously, eventually leading to systemic pathogenicity. Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.
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46
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Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli. Proc Natl Acad Sci U S A 2019; 116:14222-14227. [PMID: 31239340 DOI: 10.1073/pnas.1904428116] [Citation(s) in RCA: 74] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or "mating" channel, the channel-pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs.
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47
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Sgro GG, Oka GU, Souza DP, Cenens W, Bayer-Santos E, Matsuyama BY, Bueno NF, dos Santos TR, Alvarez-Martinez CE, Salinas RK, Farah CS. Bacteria-Killing Type IV Secretion Systems. Front Microbiol 2019; 10:1078. [PMID: 31164878 PMCID: PMC6536674 DOI: 10.3389/fmicb.2019.01078] [Citation(s) in RCA: 98] [Impact Index Per Article: 16.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2019] [Accepted: 04/29/2019] [Indexed: 01/25/2023] Open
Abstract
Bacteria have been constantly competing for nutrients and space for billions of years. During this time, they have evolved many different molecular mechanisms by which to secrete proteinaceous effectors in order to manipulate and often kill rival bacterial and eukaryotic cells. These processes often employ large multimeric transmembrane nanomachines that have been classified as types I-IX secretion systems. One of the most evolutionarily versatile are the Type IV secretion systems (T4SSs), which have been shown to be able to secrete macromolecules directly into both eukaryotic and prokaryotic cells. Until recently, examples of T4SS-mediated macromolecule transfer from one bacterium to another was restricted to protein-DNA complexes during bacterial conjugation. This view changed when it was shown by our group that many Xanthomonas species carry a T4SS that is specialized to transfer toxic bacterial effectors into rival bacterial cells, resulting in cell death. This review will focus on this special subtype of T4SS by describing its distinguishing features, similar systems in other proteobacterial genomes, and the nature of the effectors secreted by these systems and their cognate inhibitors.
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Affiliation(s)
- Germán G. Sgro
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Gabriel U. Oka
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Diorge P. Souza
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - William Cenens
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Ethel Bayer-Santos
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Bruno Y. Matsuyama
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Natalia F. Bueno
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | | | - Cristina E. Alvarez-Martinez
- Departamento de Genética, Evolução, Microbiologia e Imunologia, Instituto de Biologia, University of Campinas (UNICAMP), Campinas, Brazil
| | - Roberto K. Salinas
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
| | - Chuck S. Farah
- Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil
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48
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Abstract
Helicobacter pylori colonizes about half of humans worldwide, and its presence in the gastric mucosa is associated with an increased risk of gastric adenocarcinoma, gastric lymphoma, and peptic ulcer disease. H. pylori strains carrying the cag pathogenicity island (cagPAI) are associated with increased risk of disease progression. The cagPAI encodes the Cag type IV secretion system (CagT4SS), which delivers the CagA oncoprotein and other effector molecules into human gastric epithelial cells. We visualized structures of native and mutant CagT4SS machines on the H. pylori cell envelope by cryoelectron tomography. Individual H. pylori cells contain multiple CagT4SS nanomachines, each composed of a wheel-shaped outer membrane complex (OMC) with 14-fold symmetry and an inner membrane complex (IMC) with 6-fold symmetry. CagX, CagY, and CagM are required for assembly of the OMC, whereas strains lacking Cag3 and CagT produce outer membrane complexes lacking peripheral components. The IMC, which has never been visualized in detail, is configured as six tiers in cross-section view and three concentric rings surrounding a central channel in end-on view. The IMC contains three T4SS ATPases: (i) VirB4-like CagE, arranged as a hexamer of dimers at the channel entrance; (ii) a hexamer of VirB11-like Cagα, docked at the base of the CagE hexamer; and (iii) VirD4-like Cagβ and other unspecified Cag subunits, associated with the stacked CagE/Cagα complex and forming the outermost rings. The CagT4SS and recently solved Legionella pneumophila Dot/Icm system comprise new structural prototypes for the T4SS superfamily.IMPORTANCE Bacterial type IV secretion systems (T4SSs) have been phylogenetically grouped into two subfamilies. The T4ASSs, represented by the Agrobacterium tumefaciens VirB/VirD4T4SS, include "minimized" machines assembled from 12 VirB- and VirD4-like subunits and compositionally larger systems such as the Helicobacter pylori CagT4SS T4BSSs encompass systems closely related in subunit composition to the Legionella pneumophila Dot/IcmT4SS Here, we present structures of native and mutant H. pylori Cag machines determined by in situ cryoelectron tomography. We identify distinct outer and inner membrane complexes and, for the first time, visualize structural contributions of all three "signature" ATPases of T4SSs at the cytoplasmic entrance of the translocation channel. Despite their evolutionary divergence, the CagT4SS aligns structurally much more closely to the Dot/IcmT4SS than an available VirB/VirD4 subcomplex. Our findings highlight the diversity of T4SSs and suggest a structural classification scheme in which T4SSs are grouped as minimized VirB/VirD4-like or larger Cag-like and Dot/Icm-like systems.
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Christie PJ, Gomez Valero L, Buchrieser C. Biological Diversity and Evolution of Type IV Secretion Systems. Curr Top Microbiol Immunol 2019; 413:1-30. [PMID: 29536353 PMCID: PMC5912172 DOI: 10.1007/978-3-319-75241-9_1] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
The bacterial type IV secretion systems (T4SSs) are a highly functionally and structurally diverse superfamily of secretion systems found in many species of Gram-negative and -positive bacteria. Collectively, the T4SSs can translocate DNA and monomeric and multimeric protein substrates to a variety of bacterial and eukaryotic cell types. Detailed phylogenomics analyses have established that the T4SSs evolved from ancient conjugation machines whose original functions were to disseminate mobile DNA elements within and between bacterial species. How members of the T4SS superfamily evolved to recognize and translocate specific substrate repertoires to prokaryotic or eukaryotic target cells is a fascinating question from evolutionary, biological, and structural perspectives. In this chapter, we will summarize recent findings that have shaped our current view of the biological diversity of the T4SSs. We focus mainly on two subtypes, designated as the types IVA (T4ASS) and IVB (T4BSS) systems that respectively are represented by the paradigmatic Agrobacterium tumefaciens VirB/VirD4 and Legionella pneumophila Dot/Icm T4SSs. We present current information about the composition and architectures of these representative systems. We also describe how these and a few related T4ASS and T4BSS members evolved as specialized nanomachines through acquisition of novel domains or subunits, a process that ultimately generated extensive genetic and structural mosaicism among this secretion superfamily. Finally, we present new phylogenomics information establishing that the T4BSSs are much more broadly distributed than initially envisioned.
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Affiliation(s)
- Peter J Christie
- Department of Microbiology and Molecular Genetics, McGovern Medical School, 6431 Fannin St, Houston, TX, 77030, USA.
| | - Laura Gomez Valero
- Institut Pasteur, Biologie des Bactéries Intracellulaires, 75724, Paris, France
- CNRS, UMR 3525, 75724, Paris, France
| | - Carmen Buchrieser
- Institut Pasteur, Biologie des Bactéries Intracellulaires, 75724, Paris, France
- CNRS, UMR 3525, 75724, Paris, France
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50
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Boudaher E, Shaffer CL. Inhibiting bacterial secretion systems in the fight against antibiotic resistance. MEDCHEMCOMM 2019; 10:682-692. [PMID: 31741728 PMCID: PMC6677025 DOI: 10.1039/c9md00076c] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/04/2019] [Accepted: 04/22/2019] [Indexed: 12/11/2022]
Abstract
Antimicrobial resistance is a mounting global health crisis that threatens a resurgence of life-threatening bacterial infections. Despite intensive drug discovery efforts, the rate of antimicrobial resistance outpaces the discovery of new antibiotic agents. One of the major mechanisms driving the rapid propagation of antibiotic resistance is bacterial conjugation mediated by the versatile type IV secretion system (T4SS). The search for therapeutic compounds that prevent the spread of antibiotic resistance via T4SS-dependent mechanisms has identified several promising molecular scaffolds that disrupt resistance determinant dissemination. In this brief review, we highlight the progress and potential of conjugation inhibitors and anti-virulence compounds that target diverse T4SS machineries. These studies provide a solid foundation for the future development of potent, dual-purpose molecular scaffolds that can be used as biochemical tools to probe type IV secretion mechanisms and target bacterial conjugation in clinical settings to prevent the dissemination of antibiotic resistance throughout microbial populations.
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Affiliation(s)
- Elizabeth Boudaher
- University of Kentucky , Department of Veterinary Science , Gluck Equine Research Center , 1400 Nicholasville Road , Lexington , KY , USA . ; Tel: +1 (859) 218 1168
| | - Carrie L Shaffer
- University of Kentucky , Department of Veterinary Science , Gluck Equine Research Center , 1400 Nicholasville Road , Lexington , KY , USA . ; Tel: +1 (859) 218 1168
- University of Kentucky , Department of Microbiology, Immunology, and Molecular Genetics , 800 Rose Street , Lexington , KY , USA
- University of Kentucky , Department of Pharmaceutical Sciences , 789 South Limestone Street , Lexington , KY , USA
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