Functional plasticity in the type IV secretion system of Helicobacter pylori.

Helicobacter pylori causes clinical disease primarily in those individuals infected with a strain that carries the cytotoxin associated gene pathogenicity island (cagPAI). The cagPAI encodes a type IV secretion system (T4SS) that injects the CagA oncoprotein into epithelial cells and is required for induction of the pro-inflammatory cytokine, interleukin-8 (IL-8). CagY is an essential component of the H. pylori T4SS that has an unusual sequence structure, in which an extraordinary number of direct DNA repeats is predicted to cause rearrangements that invariably yield in-frame insertions or deletions. Here we demonstrate in murine and non-human primate models that immune-driven host selection of rearrangements in CagY is sufficient to cause gain or loss of function in the H. pylori T4SS. We propose that CagY functions as a sort of molecular switch or perhaps a rheostat that alters the function of the T4SS and "tunes" the host inflammatory response so as to maximize persistent infection

Bacterial Killing Via A Type Iv Secretion System.

Type IV secretion systems (T4SSs) are multiprotein complexes that transport effector proteins and protein-DNA complexes through bacterial membranes to the extracellular milieu or directly into the cytoplasm of other cells. Many bacteria of the family Xanthomonadaceae, which occupy diverse environmental niches, carry a T4SS with unknown function but with several characteristics that distinguishes it from other T4SSs. Here we show that the Xanthomonas citri T4SS provides these cells the capacity to kill other Gram-negative bacterial species in a contact-dependent manner. The secretion of one type IV bacterial effector protein is shown to require a conserved C-terminal domain and its bacteriolytic activity is neutralized by a cognate immunity protein whose 3D structure is similar to peptidoglycan hydrolase inhibitors. This is the first demonstration of the involvement of a T4SS in bacterial killing and points to this special class of T4SS as a mediator of both antagonistic and cooperative interbacterial interactions.6645

Identification of Anaplasma marginale Type IV Secretion System Effector Proteins

Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS). The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.Published copyLockwood, S., D. E. Voth, K. A. Brayton, P. A. Beare, W. C. Brown, R. A. Heinzen, and S. L. Broschat, Identification of Anaplasma marginale type IV secretion system effector proteins, PLoS ONE, Vol. 6, No. 11, e7724, Nov. 2011. DOI: 10.1371/journal.pone.0027724

An Anomalous Type IV Secretion System in Rickettsia Is Evolutionarily Conserved

Bacterial type IV secretion systems (T4SSs) comprise a diverse transporter family functioning in conjugation, competence, and effector molecule (DNA and/or protein) translocation. Thirteen genome sequences from Rickettsia, obligate intracellular symbionts/pathogens of a wide range of eukaryotes, have revealed a reduced T4SS relative to the Agrobacterium tumefaciens archetype (vir). However, the Rickettsia T4SS has not been functionally characterized for its role in symbiosis/virulence, and none of its substrates are known.Superimposition of T4SS structural/functional information over previously identified Rickettsia components implicate a functional Rickettsia T4SS. virB4, virB8 and virB9 are duplicated, yet only one copy of each has the conserved features of similar genes in other T4SSs. An extraordinarily duplicated VirB6 gene encodes five hydrophobic proteins conserved only in a short region known to be involved in DNA transfer in A. tumefaciens. virB1, virB2 and virB7 are newly identified, revealing a Rickettsia T4SS lacking only virB5 relative to the vir archetype. Phylogeny estimation suggests vertical inheritance of all components, despite gene rearrangements into an archipelago of five islets. Similarities of Rickettsia VirB7/VirB9 to ComB7/ComB9 proteins of epsilon-proteobacteria, as well as phylogenetic affinities to the Legionella lvh T4SS, imply the Rickettsiales ancestor acquired a vir-like locus from distantly related bacteria, perhaps while residing in a protozoan host. Modern modifications of these systems likely reflect diversification with various eukaryotic host cells.We present the rvh (Rickettsiales vir homolog) T4SS, an evolutionary conserved transporter with an unknown role in rickettsial biology. This work lays the foundation for future laboratory characterization of this system, and also identifies the Legionella lvh T4SS as a suitable genetic model

Investigating the protein subcomplexes from a conjugative Type IV Secretion System

Type IV secretion system (T4SS) are versatile nanomachines that enable the efficient transport of substrates in bacteria. In general, they are formed from two major membrane embedded subassemblies: an outer membrane core complex (OMCC) and an inner membrane complex (IMC). The conjugative T4SS encoded by the F plasmid is of particular interest due to its clinical relevance as it facilitates the spread of antibiotic resistance amongst bacterial population. Despite its importance, atomic details of the F-T4SS structure and protein-protein interactions were rudimentary which in turn precludes thorough understanding of how conjugation is orchestrated. Therefore, this thesis aimed to improve knowledge on the F-T4SS by studying the structure of the F-OMCC and investigating other proteins the complex may interact with. After optimising the detergent solubilisation of the F-OMCC expressed from the pED208 F-like plasmid, and improving the purification of the complex, a cryo-EM dataset was collected. Using single particle analysis, the structure was solved with an overall resolution of 3.3 Å. The F-OMCC is formed from two concentric rings which have two distinct symmetries. The outer ring adopts 13-fold symmetry whereas the inner ring showed 17-fold symmetry, together they form a 2.1 MDa complex. The atomic models of TraB, TraK and TraV were built into the structure, and they revealed a unique stoichiometric arrangement. Interestingly, TraV and TraK proteins were found to adopt two different conformations within the outer ring. TraV and TraB were found to accommodate the symmetry mismatch by existing in both F-OMCC rings, and also appeared to confer flexibility. This makes the F-OMCC a dynamic complex which is likely to have important implications in the pilus and T4SS activity during conjugation. The interactions between the F-OMCC and other Tra/Trb proteins were also investigated to decipher how the concerted dynamics of the pilus may be connected to the complex. A potential interaction between F-OMCC and the proteins TraH and TraN was observed by pull-down assays. Furthermore, initial work on TraG found that it seems to assemble as a high order oligomer in solution. The results are reminiscent of a hexameric protein which may be functionally important. Together, the findings of this thesis reveal novel insights into the F-T4SS and its subassemblies. The approach used to purify the F-OMCC and study the interactions will act as the basis of future work on the F-T4SS and is directly applicable to the other protein complexes within the conjugative nanomachine.Open Acces

Reconstitution and localisation studies of a type IV secretion system

Bacterial conjugation is the transport of a DNA molecule from a donor cell to a recipient. Since bacteria do not reproduce sexually, conjugation is a major contributor to prokaryotic genome plasticity and the spread of antibiotic resistance genes. A Type IV Secretion System (T4SS) mediates the DNA transport during conjugation. T4SSs are large macromolecular assemblies embedded in the membrane of bacteria, and are associated with pathogenesis, bacterial conjugation and natural transformation. They are a versatile family of secretion systems, who transport a wide variety of substrates, such as virulence proteins, DNA––protein complexes as well as only DNA. Here, we investigate the minimal requirements for conjugation, and the T4SS’s localisation within the cell. We show that the conjugative T4SS of the plasmid pR388 requires a total of 14 genes to efficiently mobilise DNA from a donor cell to a recipient cell and is arranged around the cell circumfence in a helical array. Our study of two reconstituted and fully functional conjugative T4SSs opens doors for further structural and functional analysis

Role of type IV secretion systems in trafficking of virulence determinants of Burkholderia cenocepacia

Type IV secretion systems have been identified in several human pathogens including Bordetella pertussis, Helicobacter pylori, and Legionella pneumophila. These systems are responsible for the translocation of virulence proteins and/or DNA, thereby playing an important role in the pathogenesis of infection and plasticity of genomes. Burkholderia cenocepacia is an important opportunistic human pathogen, particularly in persons with cystic fibrosis (CF). Respiratory tract infection by B. cenocepacia in CF patients is often associated with a decline in respiratory function, and can result in acute systemic infection. Burkholderia cenocepacia strain K56-2 is part of the epidemic and clinically problematic ET12 lineage. Two type IV secretion systems have been identified in this strain; one system is plasmid encoded (designated the Ptw type IV secretion system) whereas the other is chromosomally encoded (designated the VirB/D type IV secretion system) and shows homology to the Agrobacterium tumefaciens VirB/D4 type IV secretion system. It was determined that the plasmid encoded Ptw system is a chimeric type IV secretion system composed of VirB/D4-like elements and F-specific subunits. More recently, it was found that this system translocates a protein effector (PtwE1) that is cytotoxic to plant cells. It was also determined that the positively charged C-terminal region of PtwE1 is important for translocation via the Ptw type IV secretion system. Strains of the epidemic B. cenocepacia PHDC lineage contain only a chromosomal VirB/D4-like type IV secretion system (designated BcVirB/D); and a putative effector protein associated with this system has been identified that has C-terminal transport signal and sequences different from the effectors of the Ptw type IV secretion system. It has also been shown that a competing plasmid substrate and a plasmid fertility inhibition factor act to render B. cenocepacia of the PHDC lineage incapable of expressing a plant phenotype. Thus, three type IV secretion systems have been identified in epidemic B. cenocepacia lineages. From two of these, an effector has been identified that has cytotoxic effects on eukaryotic cells, and at least one of these type IV secretion systems is able to translocate DNA substrates

In Vivo Structures of the Helicobacter pylori cag Type IV Secretion System

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

Adhesion and host cell modulation: critical pathogenicity determinants of Bartonella henselae

Bartonella henselae, the agent of cat scratch disease and the vasculoproliferative disorders bacillary angiomatosis and peliosis hepatis, contains to date two groups of described pathogenicity factors: adhesins and type IV secretion systems. Bartonella adhesin A (BadA), the Trw system and possibly filamentous hemagglutinin act as promiscous or specific adhesins, whereas the virulence locus (Vir)B/VirD4 type IV secretion system modulates a variety of host cell functions. BadA mediates bacterial adherence to endothelial cells and extracellular matrix proteins and triggers the induction of angiogenic gene programming. The VirB/VirD4 type IV secretion system is responsible for, e.g., inhibition of host cell apoptosis, bacterial persistence in erythrocytes, and endothelial sprouting. The Trw-conjugation system of Bartonella spp. mediates host-specific adherence to erythrocytes. Filamentous hemagglutinins represent additional potential pathogenicity factors which are not yet characterized. The exact molecular functions of these pathogenicity factors and their contribution to an orchestral interplay need to be analyzed to understand B. henselae pathogenicity in detail

Polar delivery of Legionella type IV secretion system substrates is essential for virulence

A recurrent emerging theme is the targeting of proteins to subcellular microdomains within bacterial cells, particularly to the poles. In most cases, it has been assumed that this localization is critical to the protein’s function. Legionella pneumophila uses a type IVB secretion system (T4BSS) to export a large number of protein substrates into the cytoplasm of host cells. Here we show that the Legionella export apparatus is localized to the bacterial poles, as is consistent with many T4SS substrates being retained on the phagosomal membrane adjacent to the poles of the bacterium. More significantly, we were able to demonstrate that polar secretion of substrates is critically required for Legionella’s alteration of the host endocytic pathway, an activity required for this pathogen’s virulence