Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

ABSTRACTBacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that theEscherichia colipKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning inAgrobacterium tumefaciens,Anaplasma phagocytophilum, orWolbachia pipientis. A chimeric receptor assembled fromA. tumefaciensVirD4 (VirD4At) mediated transfer of a MOBQ plasmid (pML122) andA. tumefacienseffector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4Atand the TraJ/VirD4Atchimera with their CTDs deleted supported pML122 transfer at wild-type levels, ΔCTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4At's CTD to the VirE2 effector, although other VirD4Atdomains bound this substratein vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains.IMPORTANCEBacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through theEscherichia colipKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.

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Membrane and Core Periplasmic Agrobacterium tumefaciens Virulence Type IV Secretion System Components Localize to Multiple Sites around the Bacterial Perimeter during Lateral Attachment to Plant Cells

mBio ◽

10.1128/mbio.00218-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 33

Author(s):

Julieta Aguilar ◽

Todd A. Cameron ◽

John Zupan ◽

Patricia Zambryski

Keyword(s):

Agrobacterium Tumefaciens ◽

Plant Cells ◽

Secretion System ◽

Type Iv Secretion ◽

Host Cells ◽

Type Iv Secretion System ◽

Secretion Systems ◽

Content Type ◽

Protein Substrates ◽

Type Iv

ABSTRACTType IV secretion systems (T4SS) transfer DNA and/or proteins into recipient cells. Here we performed immunofluorescence deconvolution microscopy to localize the assembled T4SS by detection of its native components VirB1, VirB2, VirB4, VirB5, VirB7, VirB8, VirB9, VirB10, and VirB11 in the C58 nopaline strain ofAgrobacterium tumefaciens, following induction of virulence (vir) gene expression. These different proteins represent T4SS components spanning the inner membrane, periplasm, or outer membrane. Native VirB2, VirB5, VirB7, and VirB8 were also localized in theA. tumefaciensoctopine strain A348. Quantitative analyses of the localization of all the above Vir proteins in nopaline and octopine strains revealed multiple foci in single optical sections in over 80% and 70% of the bacterial cells, respectively. Green fluorescent protein (GFP)-VirB8 expression followingvirinduction was used to monitor bacterial binding to live host plant cells; bacteria bind predominantly along their lengths, with few bacteria binding via their poles or subpoles.vir-induced attachment-defective bacteria or bacteria without the Ti plasmid do not bind to plant cells. These data support a model where multiplevir-T4SS around the perimeter of the bacterium maximize effective contact with the host to facilitate efficient transfer of DNA and protein substrates.IMPORTANCETransfer of DNA and/or proteins to host cells through multiprotein type IV secretion system (T4SS) complexes that span the bacterial cell envelope is critical to bacterial pathogenesis. Early reports suggested that T4SS components localized at the cell poles. Now, higher-resolution deconvolution fluorescence microscopy reveals that all structural components of theAgrobacterium tumefaciens vir-T4SS, as well as its transported protein substrates, localize to multiple foci around the cell perimeter. These results lead to a new model ofA. tumefaciensattachment to a plant cell, whereA. tumefacienstakes advantage of the multiplevir-T4SS along its length to make intimate lateral contact with plant cells and thereby effectively transfer DNA and/or proteins through thevir-T4SS. The T4SS ofA. tumefaciensis among the best-studied T4SS, and the majority of its components are highly conserved in different pathogenic bacterial species. Thus, the results presented can be applied to a broad range of pathogens that utilize T4SS.

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

mBio ◽

10.1128/mbio.03350-19 ◽

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.

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Advances in the Assembly Model of Bacterial Type IVB Secretion Systems

Applied Sciences ◽

10.3390/app8122368 ◽

2018 ◽

Vol 8 (12) ◽

pp. 2368 ◽

Cited By ~ 1

Author(s):

Shan Wang ◽

Dan Wang ◽

Dan Du ◽

Shanshan Li ◽

Wei Yan

Keyword(s):

Protein Interactions ◽

Protein Composition ◽

Effector Proteins ◽

Type Iv Secretion ◽

Assembly Model ◽

Secretion Systems ◽

Core Complex ◽

Type Iv ◽

Type Ivb Secretion ◽

Bacterial Type

Bacterial type IV secretion systems (T4SSs) are related to not only secretion of effector proteins and virulence factors, but also to bacterial conjugation systems that promote bacterial horizontal gene transfer. The subgroup T4BSS, with a unique mosaic architecture system, consists of nearly 30 proteins that are similar to those from other secretory systems. Despite being intensively studied, the secretion mechanism of T4BSS remains unclear. This review systematically summarizes the protein composition, coding gene set, core complex, and protein interactions of T4BSS. The interactions of proteins in the core complex of the system and the operation mechanism between each element needs to be further studied.

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Acinetobacter baumannii: Resistance and Virulence mediated through bacterial type IV secretion system

Revista Estomatología ◽

10.25100/re.v21i2.5765 ◽

2017 ◽

Vol 21 (2) ◽

pp. 37-45

Author(s):

Andrés Zúñiga-Bahamon ◽

Fabián Tobar ◽

Juan Fernando Duque ◽

Pedro Moreno

Keyword(s):

Bacterial Resistance ◽

Genetic Material ◽

Secretion System ◽

Effector Proteins ◽

Type Iv Secretion ◽

Host Cells ◽

Secretion Systems ◽

Type Iv ◽

Bacterial Secretion ◽

Bacterial Type

Introduction: Type IV Bacterial Secretion Systems (TFSS) have a variety of biological functions such as the exchange of genetic material with other bacteria and virulent translocation of DNA with its effector proteins into host cells. A. baumannii is a pathogen that causes infections in humans and exhibits high rates of multidrug resistance to drugs. Objective: To relate how type IV secretion systems is associated with patterns of resistance and virulence in A. baumannii. Materials and Methods: Exhaustive search in PMC (NCBI) using a set of keywords was performed. Results: The search yielded 133 articles. Fourteen articles were analysed to determine the bacterial secretion system and the resistant and virulence of AA. baumannii. Conclusions: Systems of bacterial type IV secretion present in A. baumannii are crucial in understanding the patterns of virulence and resistance. Key words: Pathogenicity, type four secretion system (T4SS), A. baumannii, virulence factors, multidrug bacterial resistance (MDR), horizontal gene transfer (HGT).

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

Microbiology ◽

10.1099/mic.0.033043-0 ◽

2009 ◽

Vol 155 (12) ◽

pp. 4005-4013 ◽

Cited By ~ 13

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.

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Identification of ElpA, a Coxiella burnetii Pathotype-Specific Dot/Icm Type IV Secretion System Substrate

Infection and Immunity ◽

10.1128/iai.02855-14 ◽

2015 ◽

Vol 83 (3) ◽

pp. 1190-1198 ◽

Cited By ~ 11

Author(s):

Joseph G. Graham ◽

Caylin G. Winchell ◽

Uma M. Sharma ◽

Daniel E. Voth

Keyword(s):

Coxiella Burnetii ◽

Q Fever ◽

Parasitophorous Vacuole ◽

Secretion System ◽

Terminal Region ◽

Effector Proteins ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Content Type ◽

Type Iv

Coxiella burnetiicauses human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages,C. burnetiiuses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100C. burnetiiDot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in allC. burnetiiisolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizingproteinA). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function duringC. burnetiiinfection.

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

mBio ◽

10.1128/mbio.00221-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 22

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.

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Cooperative Immune Suppression by Escherichia coli and Shigella Effector Proteins

Infection and Immunity ◽

10.1128/iai.00560-17 ◽

2018 ◽

Vol 86 (4) ◽

Cited By ~ 6

Author(s):

Maarten F. de Jong ◽

Neal M. Alto

Keyword(s):

Escherichia Coli ◽

Defense Mechanisms ◽

Immune Defense ◽

Effector Proteins ◽

Innate Immune ◽

Host Cells ◽

Secretion Systems ◽

Content Type ◽

E Coli ◽

Type Iii Secretion Systems

ABSTRACT The enteric attaching and effacing (A/E) pathogens enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) and the invasive pathogens enteroinvasive E. coli (EIEC) and Shigella encode type III secretion systems (T3SS) used to inject effector proteins into human host cells during infection. Among these are a group of effectors required for NF-κB-mediated host immune evasion. Recent studies have identified several effector proteins from A/E pathogens and EIEC/ Shigella that are involved in suppression of NF-κB and have uncovered their cellular and molecular functions. A novel mechanism among these effectors from both groups of pathogens is to coordinate effector function during infection. This cooperativity among effector proteins explains how bacterial pathogens are able to effectively suppress innate immune defense mechanisms in response to diverse classes of immune receptor signaling complexes (RSCs) stimulated during infection.

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