scholarly journals Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Carrie L. Shaffer ◽  
James A. D. Good ◽  
Santosh Kumar ◽  
K. Syam Krishnan ◽  
Jennifer A. Gaddy ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Small Molecules ◽  
Bacterial Pathogens ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Target Cells ◽  
Effector Protein ◽  
Secretion Systems ◽  
Type Iv ◽  
H Pylori

ABSTRACT Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori , KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori , we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. IMPORTANCE Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.

scholarly journals Molecular and Structural Analysis of the Helicobacter pylori cag Type IV Secretion System Core Complex

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Arwen E. Frick-Cheng ◽  
Tasia M. Pyburn ◽  
Bradley J. Voss ◽  
W. Hayes McDonald ◽  
Melanie D. Ohi ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Bacterial Species ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Secretion Systems ◽  
Core Complex ◽  
Type Iv ◽  
Membrane Spanning ◽  
H Pylori

ABSTRACT Bacterial type IV secretion systems (T4SSs) can function to export or import DNA, and can deliver effector proteins into a wide range of target cells. Relatively little is known about the structural organization of T4SSs that secrete effector proteins. In this report, we describe the isolation and analysis of a membrane-spanning core complex from the Helicobacter pylori cag T4SS, which has an important role in the pathogenesis of gastric cancer. We show that this complex contains five H. pylori proteins, CagM, CagT, Cag3, CagX, and CagY, each of which is required for cag T4SS activity. CagX and CagY are orthologous to the VirB9 and VirB10 components of T4SSs in other bacterial species, and the other three Cag proteins are unique to H. pylori . Negative stain single-particle electron microscopy revealed complexes 41 nm in diameter, characterized by a 19-nm-diameter central ring linked to an outer ring by spoke-like linkers. Incomplete complexes formed by Δ cag3 or Δ cagT mutants retain the 19-nm-diameter ring but lack an organized outer ring. Immunogold labeling studies confirm that Cag3 is a peripheral component of the complex. The cag T4SS core complex has an overall diameter and structural organization that differ considerably from the corresponding features of conjugative T4SSs. These results highlight specialized features of the H. pylori cag T4SS that are optimized for function in the human gastric mucosal environment. IMPORTANCE Type IV secretion systems (T4SSs) are versatile macromolecular machines that are present in many bacterial species. In this study, we investigated a T4SS found in the bacterium Helicobacter pylori. H. pylori is an important cause of stomach cancer, and the H. pylori T4SS contributes to cancer pathogenesis by mediating entry of CagA (an effector protein regarded as a “bacterial oncoprotein”) into gastric epithelial cells. We isolated and analyzed the membrane-spanning core complex of the H. pylori T4SS and showed that it contains unique proteins unrelated to components of T4SSs in other bacterial species. These results constitute the first structural analysis of the core complex from this important secretion system.

scholarly journals Epistatic Interplay between Type IV Secretion Effectors Engages the Small GTPase Rab2 in the Brucella Intracellular Cycle

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Erin P. Smith ◽  
Alexis Cotto-Rosario ◽  
Elizabeth Borghesan ◽  
Kiara Held ◽  
Cheryl N. Miller ◽  
...  
Keyword(s):  
Brucella Abortus ◽  
Bacterial Pathogens ◽  
Small Gtpase ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Secretion Systems ◽  
Content Type ◽  
Type Iv ◽  
Infectious Cycle

ABSTRACT Intracellular bacterial pathogens remodel cellular functions during their infectious cycle via the coordinated actions of effector molecules delivered through dedicated secretion systems. While the function of many individual effectors is known, how they interact to promote pathogenesis is rarely understood. The zoonotic bacterium Brucella abortus, the causative agent of brucellosis, delivers effector proteins via its VirB type IV secretion system (T4SS) which mediate biogenesis of the endoplasmic reticulum (ER)-derived replicative Brucella-containing vacuole (rBCV). Here, we show that T4SS effectors BspB and RicA display epistatic interactions in Brucella replication. Defects in rBCV biogenesis and Brucella replication caused by deletion of bspB were dependent on the host GTPase Rab2a and suppressed by the deletion of ricA, indicating a role of Rab2-binding effector RicA in these phenotypic defects. Rab2a requirements for rBCV biogenesis and Brucella intracellular replication were abolished upon deletion of both bspB and ricA, demonstrating that the functional interaction of these effectors engages Rab2-dependent transport in the Brucella intracellular cycle. Expression of RicA impaired host secretion and caused Golgi fragmentation. While BspB-mediated changes in ER-to-Golgi transport were independent of RicA and Rab2a, BspB-driven alterations in Golgi vesicular traffic also involved RicA and Rab2a, defining BspB and RicA’s functional interplay at the Golgi interface. Altogether, these findings support a model where RicA modulation of Rab2a functions impairs Brucella replication but is compensated by BspB-mediated remodeling of Golgi apparatus-associated vesicular transport, revealing an epistatic interaction between these T4SS effectors. IMPORTANCE Bacterial pathogens with an intracellular lifestyle modulate many host cellular processes to promote their infectious cycle. They do so by delivering effector proteins into host cells via dedicated secretion systems that target specific host functions. While the roles of many individual effectors are known, how their modes of action are coordinated is rarely understood. Here, we show that the zoonotic bacterium Brucella abortus delivers the BspB effector that mitigates the negative effect on bacterial replication that the RicA effector exerts via modulation of the host small GTPase Rab2. These findings provide an example of functional integration between bacterial effectors that promotes proliferation of pathogens.

scholarly journals Two type IV secretion systems with different functions in Burkholderia cenocepacia K56-2

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 4005-4013 ◽  
Author(s):  
Ruifu Zhang ◽  
John J. LiPuma ◽  
Carlos F. Gonzalez
Keyword(s):  
Type Iv Secretion ◽  
Burkholderia Cenocepacia ◽  
Target Cells ◽  
Secretion Systems ◽  
Type Iv ◽  
Plasmid Mobilization ◽  
Type Iv Secretion Systems ◽  
Derivative Plasmid ◽  
Chromosome Ii ◽  
Bacterial Type

Bacterial type IV secretion systems (T4SS) perform two fundamental functions related to pathogenesis: the delivery of effector molecules to eukaryotic target cells, and genetic exchange. Two T4SSs have been identified in Burkholderia cenocepacia K56-2, a representative of the ET12 lineage of the B. cepacia complex (Bcc). The plant tissue watersoaking (Ptw) T4SS encoded on a resident 92 kb plasmid is a chimera composed of VirB/D4 and F-specific subunits, and is responsible for the translocation of effector(s) that have been linked to the Ptw phenotype. The bc-VirB/D4 system located on chromosome II displays homology to the VirB/D4 T4SS of Agrobacterium tumefaciens. In contrast to the Ptw T4SS, the bc-VirB/D4 T4SS was found to be dispensable for Ptw effector(s) secretion, but was found to be involved in plasmid mobilization. The fertility inhibitor Osa did not affect the secretion of Ptw effector(s) via the Ptw system, but did disrupt the mobilization of a RSF1010 derivative plasmid.

The Legionella pneumophila effector RavY contributes to a replication-permissive vacuolar environment during infection

2021 ◽  
Author(s):  
Luying Liu ◽  
Craig R. Roy
Keyword(s):  
Legionella Pneumophila ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Effector Protein ◽  
Intracellular Replication ◽  
Phagocytic Cells ◽  
Host Molecules ◽  
Type Iv ◽  
Legionnaires Disease

Legionella pneumophila is the causative agent of Legionnaires’ Disease and is capable replicating inside phagocytic cells such as mammalian macrophages. The Dot/Icm type IV secretion system is a L. pneumophila virulence factor that is essential for successful intracellular replication. During infection, L. pneumophila builds a replication permissive vacuole by recruiting multiple host molecules and hijacking host cellular signaling pathways, a process mediated by the coordinated functions of multiple Dot/Icm effector proteins. RavY is a predicted Dot/Icm effector protein found to be important for optimal L. pneumophila replication inside host cells. Here, we demonstrate that RavY is a Dot/Icm-translocated effector protein that is dispensable for axenic replication of L. pneumophila , but critical for optimal intracellular replication of the bacteria. RavY is not required for avoidance of endosomal maturation, nor does RavY contribute to the recruitment of host molecules found on replication-permissive vacuoles, such as ubiquitin, RAB1a, and RTN4. Vacuoles containing L. pneumophila ravY mutants promote intracellular survival but limit replication. The replication defect of the L. pneumophila ravY mutant was complemented when the mutant was in the same vacuole as wild type L. pneumophila . Thus, RavY is an effector that is essential for promoting intracellular replication of L. pneumophila once the specialized vacuole has been established.

How bacterial pathogens use type III and type IV secretion systems to facilitate their transmission

2017 ◽  
Vol 35 ◽  
pp. 1-7 ◽  
Author(s):  
Mariana X Byndloss ◽  
Fabian Rivera-Chávez ◽  
Renée M Tsolis ◽  
Andreas J Bäumler
Keyword(s):  
Bacterial Pathogens ◽  
Type Iv Secretion ◽  
Secretion Systems ◽  
Type Iii ◽  
Type Iv ◽  
Type Iv Secretion Systems

scholarly journals Temporal Control of the Helicobacter pylori Cag Type IV Secretion System in a Mongolian Gerbil Model of Gastric Carcinogenesis

mBio ◽  
2020 ◽  
Vol 11 (3) ◽  
Author(s):  
Aung Soe Lin ◽  
Mark S. McClain ◽  
Amber C. Beckett ◽  
Rhonda R. Caston ◽  
M. Lorena Harvey ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Infectious Agent ◽  
Gastric Carcinogenesis ◽  
Type Iv Secretion ◽  
Effector Protein ◽  
Content Type ◽  
Infected Control ◽  
Type Iv ◽  
Hit And Run ◽  
H Pylori

ABSTRACT The Helicobacter pylori Cag type IV secretion system (T4SS) translocates the effector protein CagA and nonprotein bacterial constituents into host cells. In this study, we infected Mongolian gerbils with an H. pylori strain in which expression of the cagUT operon (required for Cag T4SS activity) is controlled by a TetR/tetO system. Transcript levels of cagU were significantly higher in gastric tissue from H. pylori-infected animals receiving doxycycline-containing chow (to derepress Cag T4SS activity) than in tissue from infected control animals receiving drug-free chow. At 3 months postinfection, infected animals receiving doxycycline had significantly increased gastric inflammation compared to infected control animals. Dysplasia (a premalignant histologic lesion) and/or invasive gastric adenocarcinoma were detected only in infected gerbils receiving doxycycline, not in infected control animals. We then conducted experiments in which Cag T4SS activity was derepressed during defined stages of infection. Continuous Cag T4SS activity throughout a 3-month time period resulted in higher rates of dysplasia and/or gastric cancer than observed when Cag T4SS activity was limited to early or late stages of infection. Cag T4SS activity for the initial 6 weeks of infection was sufficient for the development of gastric inflammation at the 3-month time point, with gastric cancer detected in a small proportion of animals. These experimental results, together with previous studies of cag mutant strains, provide strong evidence that Cag T4SS activity contributes to gastric carcinogenesis and help to define the stages of H. pylori infection during which Cag T4SS activity causes gastric alterations relevant for cancer pathogenesis. IMPORTANCE The “hit-and-run model” of carcinogenesis proposes that an infectious agent triggers carcinogenesis during initial stages of infection and that the ongoing presence of the infectious agent is not required for development of cancer. H. pylori infection and actions of CagA (an effector protein designated a bacterial oncoprotein, secreted by the Cag T4SS) are proposed to constitute a paradigm for hit-and-run carcinogenesis. In this study, we report the development of methods for controlling H. pylori Cag T4SS activity in vivo and demonstrate that Cag T4SS activity contributes to gastric carcinogenesis. We also show that Cag T4SS activity during an early stage of infection is sufficient to initiate a cascade of cellular alterations leading to gastric inflammation and gastric cancer at later time points.

scholarly journals Biological Diversity of Prokaryotic Type IV Secretion Systems

2009 ◽  
Vol 73 (4) ◽  
pp. 775-808 ◽  
Author(s):  
Cristina E. Alvarez-Martinez ◽  
Peter J. Christie
Keyword(s):  
Target Cell ◽  
Biological Diversity ◽  
Cell Interaction ◽  
Type Iv Secretion ◽  
Target Cells ◽  
Secretion Systems ◽  
Extracellular Milieu ◽  
Type Iv ◽  
Type Iv Secretion Systems ◽  
Cell Envelopes

SUMMARY Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

scholarly journals Postreplication Roles of theBrucellaVirB Type IV Secretion System Uncovered via Conditional Expression of the VirB11 ATPase

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Erin P. Smith ◽  
Cheryl N. Miller ◽  
Robert Child ◽  
Jennifer A. Cundiff ◽  
Jean Celli
Keyword(s):  
Brucella Abortus ◽  
Secretion System ◽  
Effector Proteins ◽  
Type Iv Secretion ◽  
Host Cells ◽  
Type Iv Secretion System ◽  
Secretion Systems ◽  
Type Iv ◽  
Conditional Expression ◽  
Bacterial Replication

ABSTRACTBrucella abortus, the bacterial agent of the worldwide zoonosis brucellosis, primarily infects host phagocytes, where it undergoes an intracellular cycle within a dedicated membrane-bound vacuole, theBrucella-containing vacuole (BCV). Initially of endosomal origin (eBCV), BCVs are remodeled into replication-permissive organelles (rBCV) derived from the host endoplasmic reticulum, a process that requires modulation of host secretory functions via delivery of effector proteins by theBrucellaVirB type IV secretion system (T4SS). Following replication, rBCVs are converted into autophagic vacuoles (aBCVs) that facilitate bacterial egress and subsequent infections, arguing that the bacterium sequentially manipulates multiple cellular pathways to complete its cycle. The VirB T4SS is essential for rBCV biogenesis, as VirB-deficient mutants are stalled in eBCVs and cannot mediate rBCV biogenesis. This has precluded analysis of whether the VirB apparatus also drives subsequent stages of theBrucellaintracellular cycle. To address this issue, we have generated aB. abortusstrain in which VirB T4SS function is conditionally controlled via anhydrotetracycline (ATc)-dependent complementation of a deletion of thevirB11gene encoding the VirB11 ATPase. We show in murine bone marrow-derived macrophages (BMMs) that early VirB production is essential for optimal rBCV biogenesis and bacterial replication. Transient expression ofvirB11prior to infection was sufficient to mediate normal rBCV biogenesis and bacterial replication but led to T4SS inactivation and decreased aBCV formation and bacterial release, indicating that these postreplication stages are also T4SS dependent. Hence, our findings support the hypothesis of additional, postreplication roles of type IV secretion in theBrucellaintracellular cycle.IMPORTANCEMany intracellular bacterial pathogens encode specialized secretion systems that deliver effector proteins into host cells to mediate the multiple stages of their intracellular cycles. Because these intracellular events occur sequentially, classical genetic approaches cannot address the late roles that these apparatuses play, as secretion-deficient mutants cannot proceed past their initial defect. Here we have designed a functionally controllable VirB type IV secretion system (T4SS) in the bacterial pathogenBrucella abortusto decipher its temporal requirements during the bacterium’s intracellular cycle in macrophages. By controlling production of the VirB11 ATPase, which energizes the T4SS, we show not only that this apparatus is required early to generate theBrucellareplicative organelle but also that it contributes to completion of the bacterium’s cycle and bacterial egress. Our findings expand upon the pathogenic functions of theBrucellaVirB T4SS and illustrate targeting of secretion ATPases as a useful strategy to manipulate the activity of bacterial secretion systems.

scholarly journals Structural and Biochemical Analysis of OrfG: The VirB8-like Component of the Conjugative Type IV Secretion System of ICESt3 From Streptococcus thermophilus

2021 ◽  
Vol 8 ◽  
Author(s):  
Julien Cappele ◽  
Abbas Mohamad Ali ◽  
Nathalie Leblond-Bourget ◽  
Sandrine Mathiot ◽  
Tiphaine Dhalleine ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Structural Analysis ◽  
Pathogenic Bacteria ◽  
Quaternary Structure ◽  
Streptococcus Thermophilus ◽  
Type Iv Secretion ◽  
Conjugative Transfer ◽  
Structural Level ◽  
Secretion Systems ◽  
Type Iv

Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.

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