Cag T4SS Research Articles

Correction: Subdomains of the Helicobacter pylori Cag T4SS outer membrane core complex exhibit structural independence.

Correction: Subdomains of the Helicobacter pylori Cag T4SS outer membrane core complex exhibit structural independence.

Subdomains of the Helicobacter pylori Cag T4SS outer membrane core complex exhibit structural independence.

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.

Role of the CagY antenna projection in Helicobacter pylori Cag type IV secretion system activity.

Helicobacter pylori strains containing the cag pathogenicity island (PAI) are associated with the development of gastric adenocarcinoma and peptic ulcer disease. The cag PAI encodes a secreted effector protein (CagA) and a type IV secretion system (Cag T4SS). Cag T4SS activity is required for the delivery of CagA and non-protein substrates into host cells. The Cag T4SS outer membrane core complex (OMCC) contains a channel-like domain formed by helix-loop-helix elements (antenna projections, AP) from 14 copies of the CagY protein (a VirB10 ortholog). Similar VirB10 antenna regions are present in T4SS OMCCs from multiple bacterial species and are predicted to span the outer membrane. In this study, we investigated the role of the CagY antenna region in Cag T4SS OMCC assembly and Cag T4SS function. An H. pylori mutant strain with deletion of the entire CagY AP (∆AP) retained the capacity to produce CagY and assemble an OMCC, but it lacked T4SS activity (CagA translocation and IL-8 induction in AGS gastric epithelial cells). In contrast, a mutant strain with Gly-Ser substitutions in the unstructured CagY AP loop retained Cag T4SS activity. Mutants containing CagY AP loops with shortened lengths were defective in CagA translocation and exhibited reduced IL-8-inducing activity compared to control strains. These data indicate that the CagY AP region is required for Cag T4SS activity and that Cag T4SS activity can be modulated by altering the length of the CagY AP unstructured loop.

IL-17 Receptor A protects against Helicobacter pylori-induced gastric cancer

Abstract Infection with Helicobacter pylori is the predominant risk factor for the development of gastric cancer. Risk is enhanced by specific H. pylori virulence factors, diet, and the inflammatory response. Chronic activation of T helper (Th) 1 and Th17 pathways contribute to inflammation. Previous work suggests that while IL-17 activates several antimicrobial components of the innate immune response, IL-17 is also required to prevent a hyper-inflammatory adaptive immune response. To investigate if IL-17 receptor signaling contributes to the development of carcinogenesis, the transgenic InsGAS mouse, which is susceptible to H. pylori-induced gastric cancer, was used. InsGAS mice and InsGAS Il17ra −/−mice were infected with a cag T4SS positive strain for up to 6 months. By 6 weeks post infection, IL-17RA deficiency led to increased bacterial burden and increased gastritis, concomitant with an exacerbated Th1/Th17 cytokine profile, increased B cell infiltration and enhanced production of IgA despite reduced Pigr expression. Paragastric lymph nodes of InsGAS Il17ra −/−mice, while enlarged relative to InsGAS mice, exhibited altered gene expression profiles and potential loss of fibroblastic reticular cells. Gastric pathologies, including intramucosal carcinoma and adenocarcinoma with submucosal invasion, developed more frequently by 3 and 6 months post infection in H. pylori-infected InsGASIl17ra −/−mice. These increases in severe gastric disease were accompanied by loss of parietal cells, loss of mucin-producing cells and enhanced expression of ceacam1. These data suggest that IL-17RA signaling activates a protective pathway to prevent excessive inflammation which can drive gastric carcinogenesis after H. pylori infection. This work was supported in part by Merit Review I01BX000915 (Algood) from the U.S. Department of Veterans Affairs, Biomedical Laboratory Research and Development Service. We acknowledge the Translational Pathology Shared Resource supported by NCI.NIH Cancer Center Support Grant 5P30 CA68485-19. Core Services performed through Vanderbilt University Medical Center's Digestive Disease Research Center supported by NIH grant P30DK058404.

Mechanism of regulation of the Helicobacter pylori Cagβ ATPase by CagZ

The transport of the CagA effector into gastric epithelial cells by the Cag Type IV secretion system (Cag T4SS) of Helicobacter pylori (H. pylori) is critical for pathogenesis. CagA is recruited to Cag T4SS by the Cagβ ATPase. CagZ, a unique protein in H. pylori, regulates Cagβ-mediated CagA transport, but the underlying mechanisms remain unclear. Here we report the crystal structure of the cytosolic region of Cagβ, showing a typical ring-like hexameric assembly. The central channel of the ring is narrow, suggesting that CagA must unfold for transport through the channel. Our structure of CagZ in complex with the all-alpha domain (AAD) of Cagβ shows that CagZ adopts an overall U-shape and tightly embraces Cagβ. This binding mode of CagZ is incompatible with the formation of the Cagβ hexamer essential for the ATPase activity. CagZ therefore inhibits Cagβ by trapping it in the monomeric state. Based on these findings, we propose a refined model for the transport of CagA by Cagβ.

Molecular architecture of bacterial type IV secretion systems.

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.

Positive Selection of Mutations in the Helicobacter pylori katA 5' Untranslated Region in a Mongolian Gerbil Model of Gastric Disease.

To evaluate potential effects of gastric inflammation on Helicobacter pylori diversification and evolution within the stomach, we experimentally infected Mongolian gerbils with an H. pylori strain in which Cag type IV secretion system (T4SS) activity is controlled by a TetR/tetO system. Gerbils infected with H. pylori under conditions in which Cag T4SS activity was derepressed had significantly higher levels of gastric inflammation than gerbils infected under conditions with repressed Cag T4SS activity. Mutations in the 5' untranslated region (UTR) of katA (encoding catalase) were detected in strains cultured from 8 of the 17 gerbils infected with Cag T4SS-active H. pylori and none of the strains from 17 gerbils infected with Cag T4SS-inactive H. pylori. Catalase enzymatic activity, steady-state katA transcript levels, and katA transcript stability were increased in strains with these single nucleotide polymorphisms (SNPs) compared to strains in which these SNPs were absent. Moreover, strains harboring these SNPs exhibited increased resistance to bactericidal effects of hydrogen peroxide, compared to control strains. Experimental introduction of the SNPs into the wild-type katA 5' UTR resulted in increased katA transcript stability, increased katA steady-state levels, and increased catalase enzymatic activity. Based on site-directed mutagenesis and modeling of RNA structure, increased katA transcript levels were correlated with higher predicted thermal stability of the katA 5' UTR secondary structure. These data suggest that high levels of gastric inflammation positively select for H. pylori strains producing increased levels of catalase, which may confer survival advantages to the bacteria in an inflammatory gastric environment.

Unique TLR9 Activation by Helicobacter pylori Depends on the cag T4SS, But Not on VirD2 Relaxases or VirD4 Coupling Proteins

The genomes of the gastric bacterial pathogen Helicobacter pylori harbor multiple type-IV secretion systems (T4SSs). Here we analyzed components of three T4SSs, the cytotoxin-associated genes (cag) T4SS, TFS3 and TFS4. The cag T4SS delivers the effector protein CagA and the LPS-metabolite ADP-heptose into gastric epithelial cells, which plays a pivotal role in chronic infection and development of gastric disease. In addition, the cag T4SS was reported to facilitate conjugative transport of chromosomal bacterial DNA into the host cell cytoplasm, where injected DNA activates intracellular toll-like receptor 9 (TLR9) and triggers anti-inflammatory signaling. Canonical DNA-delivering T4SSs in a variety of bacteria are composed of 11 VirB proteins (VirB1-11) which assemble and engage VirD2 relaxase and VirD4 coupling proteins that mediate DNA processing and guiding of the covalently bound DNA through the T4SS channel. Nevertheless, the role of the latter components in H. pylori is unclear. Here, we utilized isogenic knockout mutants of various virB (virB9 and virB10, corresponding to cagX and cagY), virD2 (rlx1 and rlx2), virD4 (cag5, traG1/2) and xerD recombinase genes in H. pylori laboratory strain P12 and studied their role in TLR9 activation by reporter assays. While inactivation of the structural cag T4SS genes cagX and cagY abolished TLR9 activation, the deletion of rlx1, rlx2, cag5, traG or xerD genes had no effect. The latter mutants activated TLR9 similar to wild-type bacteria, suggesting the presence of a unique non-canonical T4SS-dependent mechanism of TLR9 stimulation by H. pylori that is not mediated by VirD2, VirD4 and XerD proteins. These findings were confirmed by the analysis of TLR9 activation by H. pylori strains of worldwide origin that possess different sets of T4SS genes. The exact mechanism of TLR9 activation should be explored in future studies.

Cover Feature: The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System (ChemBioChem 18/2021)

High-resolution light microscopy of Helicobacter pylori-infected gastric tissue (400× magnification): The picture shows a composite fluorescent image of gastric tissue stained with eosin (pseudo-colored blue) producing lactoferrin (pseudocolored overlay cyan), with the structure of lactoferrin in an inset (circular panel). H. pylori is a Gram-negative bacterial pathogen that infects the stomach and causes ulcers and gastric cancer. The host innate immune system produces the glycoprotein lactoferrin as part of the antimicrobial response to bacterial infection, and this molecule can repress the oncogenic activity of the H. pylori cag T4SS in an iron-dependent manner. More information can be found in the Full Paper by S. D. Townsend, J. A. Gaddy et al.

Tracking bacterial effector protein delivery into host cells.

Bacterial Type IV secretion systems (T4SSs) are a functionally heterogeneous group of nanomachines that can deliver substrates into a wide range of target cells. The Helicobacter pylori Cag T4SS has an important role in the pathogenesis of gastric cancer. CagA, the only effector protein known to be secreted by the H. pylori Cag T4SS, enters human gastric cells and causes alterations in intracellular signaling that are linked to cancer pathogenesis. Understanding the molecular mechanisms by which CagA is delivered into gastric cells has been hindered by the lack of robust methods for monitoring this process. A publication in this issue of Molecular Microbiology describes a split luciferase assay for monitoring T4SS-mediated translocation of CagA into host cells. The use of this translocation reporter allowed the quantification of CagA translocation in real-time assays, thereby facilitating the analysis of the kinetics of CagA delivery. This system also allowed the tracking of several types of CagA fusion proteins and confirmed that protein unfolding is important for secretion by the Cag T4SS. This commentary discusses T4SS-dependent delivery of H. pylori CagA into host cells and the use of the split luciferase system for monitoring bacterial protein secretion and delivery into target cells.

The Innate Immune Glycoprotein Lactoferrin Represses the Helicobacter pylori cag Type IV Secretion System.

Chronic infection with Helicobacter pylori increases risk of gastric diseases including gastric cancer. Despite development of a robust immune response, H. pylori persists in the gastric niche. Progression of gastric inflammation to serious disease outcomes is associated with infection with H. pylori strains which encode the cag Type IV Secretion System (cag T4SS). The cag T4SS is responsible for translocating the oncogenic protein CagA into host cells and inducing pro-inflammatory and carcinogenic signaling cascades. Our previous work demonstrated that nutrient iron modulates the activity of the T4SS and biogenesis of T4SS pili. In response to H. pylori infection, the host produces a variety of antimicrobial molecules, including the iron-binding glycoprotein, lactoferrin. Our work shows that apo-lactoferrin exerts antimicrobial activity against H. pylori under iron-limited conditions, while holo-lactoferrin enhances bacterial growth. Culturing H. pylori in the presence of holo-lactoferrin prior to co-culture with gastric epithelial cells, results in repression of the cag T4SS activity. Concomitantly, a decrease in biogenesis of cag T4SS pili at the host-pathogen interface was observed under these culture conditions by high-resolution electron microscopy analyses. Taken together, these results indicate that acquisition of alternate sources of nutrient iron plays a role in regulating the pro-inflammatory activity of a bacterial secretion system and present novel therapeutic targets for the treatment of H. pylori-related disease.

Cryo-EM reveals species-specific components within the Helicobacter pylori Cag type IV secretion system core complex.

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.

Four Chromosomal Type IV Secretion Systems in Helicobacter pylori: Composition, Structure and Function.

The pathogenic bacterium Helicobacter pylori is genetically highly diverse and a major risk factor for the development of peptic ulcer disease and gastric adenocarcinoma in humans. During evolution, H. pylori has acquired multiple type IV secretion systems (T4SSs), and then adapted for various purposes. These T4SSs represent remarkable molecular transporter machines, often associated with an extracellular pilus structure present in many bacteria, which are commonly composed of multiple structural proteins spanning the inner and outer membranes. By definition, these T4SSs exhibit central functions mediated through the contact-dependent conjugative transfer of mobile DNA elements, the contact-independent release and uptake of DNA into and from the extracellular environment as well as the secretion of effector proteins in mammalian host target cells. In recent years, numerous features on the molecular functionality of these T4SSs were disclosed. H. pylori encodes up to four T4SSs on its chromosome, namely the Cag T4SS present in the cag pathogenicity island (cagPAI), the ComB system, as well as the Tfs3 and Tfs4 T4SSs, some of which exhibit unique T4SS functions. The Cag T4SS facilitates the delivery of the CagA effector protein and pro-inflammatory signal transduction through translocated ADP-heptose and chromosomal DNA, while various structural pilus proteins can target host cell receptors such as integrins or TLR5. The ComB apparatus mediates the import of free DNA from the extracellular milieu, whereas Tfs3 may accomplish the secretion or translocation of effector protein CtkA. Both Tfs3 and Tfs4 are furthermore presumed to act as conjugative DNA transfer machineries due to the presence of tyrosine recombinases with cognate recognition sequences, conjugational relaxases, and potential origins of transfer (oriT) found within the tfs3 and tfs4 genome islands. In addition, some extrachromosomal plasmids, transposons and phages have been discovered in multiple H. pylori isolates. The genetic exchange mediated by DNA mobilization events of chromosomal genes and plasmids combined with recombination events could account for much of the genetic diversity found in H. pylori. In this review, we highlight our current knowledge on the four T4SSs and the involved mechanisms with consequences for H. pylori adaptation to the hostile environment in the human stomach.

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

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.

Lipoprotein Processing and Sorting in Helicobacter pylori.

Our current understanding of lipoprotein synthesis and localization in Gram-negative bacteria is based primarily on studies of Escherichia coli Newly synthesized E. coli prolipoproteins undergo posttranslational modifications catalyzed by three essential enzymes (Lgt, LspA, and Lnt). The mature lipoproteins are then sorted to the inner or outer membrane via the Lol system (LolABCDE). Recent studies suggested that this paradigm may not be universally applicable among different classes of proteobacteria. In this study, we conducted a systematic analysis of lipoprotein processing and sorting in Helicobacter pylori, a member of the Epsilonproteobacteria that colonizes the human stomach. We show that H. pylorilgt, lspA, and lnt homologs can complement conditionally lethal E. coli mutant strains in which expression of these genes is conditionally regulated. Mutagenesis studies and analyses of conditionally lethal H. pylori mutant strains indicate that lgt and lspA are essential for H. pylori growth but lnt is dispensable. H. pylorilolA and the single lolC (or lolE) homolog are also essential genes. We then explored the role of lipoproteins in H. pylori Cag type IV secretion system (Cag T4SS) activity. Comparative analysis of the putative VirB7 homolog CagT in wild-type and lnt mutant H. pylori strains indicates that CagT undergoes amino-terminal modifications consistent with lipidation, and we show that CagT lipidation is essential for CagT stability and Cag T4SS function. This work demonstrates that lipoprotein synthesis and localization in H. pylori diverge from the canonical pathways and that lipidation of a T4SS component is necessary for H. pylori Cag T4SS activity.IMPORTANCE Bacterial lipoproteins have diverse roles in multiple aspects of bacterial physiology, antimicrobial resistance, and pathogenesis. Dedicated pathways direct the posttranslational lipidation and localization of lipoproteins, but there is considerable variation in these pathways among the proteobacteria. In this study, we characterized the proteins responsible for lipoprotein synthesis and localization in Helicobacter pylori, a member of the Epsilonproteobacteria that contributes to stomach cancer pathogenesis. We also provide evidence suggesting that lipidation of CagT, a component of the H. pylori Cag T4SS, is required for delivery of the H. pylori CagA oncoprotein into human gastric cells. Overall, these results constitute the first systematic analysis of H. pylori lipoprotein production and localization pathways and reveal how these processes in H. pylori differ from corresponding pathways in model proteobacteria.

The Helicobacter pylori Cag Type IV Secretion System.

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.

Association between polymorphisms in HLA-A, HLA-B, HLA-DR, and DQ genes from gastric cancer and duodenal ulcer patients and cagL among cagA-positive Helicobacter pylori strains: The first study in a Turkish population

Colonization of the human gastric mucosa by H. pylori may cause peptic and duodenal ulcers (DUs), gastric lymphomas, and gastric cancers. The cagL gene is a component of cag T4SS and is involved in cagA translocation into host. An association between the risk of gastric cancer and the type of HLA class II (DR and/or DQ) was suggested in different populations. The aim of this study was to investigate, the clinical association of the cagL gene with host HLA alleles in H. pylori strains that were isolated from patients with gastric cancer, DU, and non-ulcer dyspepsia (NUD) and to determine the HLA allele that confers susceptibility or resistance for the risk of gastric cancer and DU development in Turkish patients.A total of 94 patients (44 gastric cancer and 50 DU patients; 58 male, 36 female; mean age, 49.6 years), and 86 individuals (50 NUD patients and 36 persons with normal gastrointestinal system [NGIS]; 30 male, 56 female; mean age, 47.3 years) were included as the patient and the control groups, respectively. CagA and cagL were determined by PCR method. DNA from peripheral blood samples was obtained by EZ-DNA extraction kit. For HLA SSO typing, LIFECODES SSO Typing kits (HLA-A, HLA-B HLA-C, HLA-DRB1 and HLA-DQA1/B1 kits) were used.The CagL/CagA positivity distribution in the groups were as follows: 42 (95.4%) gastric cancer, 46 (92%) DU and, 34 (68%) NUD and no NGIS cases. The HLA-DQA1*01 (OR: 3.82) allele was significantly different, suggesting that these individuals with H. pylori strains harbouring the CagL/CagA positivity are susceptible to the risk of gastric cancer and DU, and the HLA-DQA1*05 (OR, 0.318) allele was suggested as a protective allele for the risk of gastric cancer and DU using univariate analyses. HLA-DQA1*01 (OR, 2.21), HLA-DQB1*06 (OR, 2.67), sex (male, OR, 2.27), and CagL/CagA/(<2) EPIYA C repeats (OR, 5.72) were detected independent risk factors that increased the risk of gastric cancer and DU using multivariate analyses. However, the HLA-DRB1*04 (OR, 0.28) allele was shown to be a protective allele, which decreased the risk of gastric cancer and DU.Gastric pathologies result from an interaction between bacterial virulence factors, host epigenetic and environmental factors, and H. pylori strain heterogeneity, such as genotypic variation among strains and variations in H. pylori populations within an individual host.

Structure of the Helicobacter pylori Cag type IV secretion system.

Bacterial type IV secretion systems (T4SSs) are molecular machines that can mediate interbacterial DNA transfer through conjugation and delivery of effector molecules into host cells. The Helicobacter pylori Cag T4SS translocates CagA, a bacterial oncoprotein, into gastric cells, contributing to gastric cancer pathogenesis. We report the structure of a membrane-spanning Cag T4SS assembly, which we describe as three sub-assemblies: a 14-fold symmetric outer membrane core complex (OMCC), 17-fold symmetric periplasmic ring complex (PRC), and central stalk. Features that differ markedly from those of prototypical T4SSs include an expanded OMCC and unexpected symmetry mismatch between the OMCC and PRC. This structure is one of the largest bacterial secretion system assemblies ever reported and illustrates the remarkable structural diversity that exists among bacterial T4SSs.

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

SUMMARYThe 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.

Functional interaction and structural characteristics of unique components of Helicobacter pylori T4SS.

The Helicobacter pylori infection of the human gastric mucosa causes chronic active gastritis and peptic ulcers and is associated with the development of gastric cancer. Epidemiological studies show that these gastric diseases are related to virulent H. pylori strains that harbor the cytotoxin-associated gene pathogenicity island (cag PAI). The cag PAI is a DNA insertion in the H. pylori chromosome that encodes ~ 27 proteins, including the oncoprotein CagA. Approximately 20 of these proteins have been designated as cag type IV secretion system (T4SS) components. However, only 11 of these proteins share function, structure, and/or sequence similarities with the prototypical VirB/VirD4 T4SS of Agrobacterium tumefaciens. The VirB/VirD4 orthologs of the cag T4SS of H. pylori are required for CagA translocation and stimulate the gastric epithelial cells to produce and secrete interleukin-8 (IL-8). The cag PAI encodes eight additional proteins, such as Cag3 (Cagδ/HP0522), CagM (Cag16/HP0537), CagU (Cag11/HP0531), CagI (Cag19/HP0540), and CagH (Cag20/HP0541), which are also required for the translocation of CagA and IL-8 secretion, meanwhile CagF (Cag22/HP0543), CagG (Cag21/HP0542), and CagZ (Cag6/HP0526) are just required for the translocation of CagA. However, relatively little is known about their functions and structural organization because they exhibit a nondetectable sequence similarity with T4SS components in the current databases. In this review, we conducted an exhaustive analysis of the literature to present the biochemistry, putative role, localization, and interactions of each of these eight additional cag T4SS components.