Mark S. McClain

Type 1 fimbriae mutants of Escherichia coli K12: characterization of recognized afimbriate strains and construction of new fim deletion mutants

We have used Southern hybridization analysis to characterize the extent of fim homology in recognized type 1 fimbriae mutants of Escherichia coli K12, including strains HB101, P678‐54, and VL584. We have found extensive homology in strain HB101, and confirm that P678‐54 lacks the majority of fim DNA. Strain VL584 contains a deletion of the entire fim region. We have used a new allelic exchange procedure to generate novel fim deletion derivatives of strains MG1655, MM294, and YMC9. To increase the utility of the new deletion strains we also isolated recA derivatives of each mutant. These strains facilitate the isolation, characterization, and manipulation of cloned fimbriae genes from diverse sources.

Essential role of a GXXXG motif for membrane channel formation by Helicobacter pylori vacuolating toxin

Helicobacter pylori secretes a toxin, VacA, that can form anion-selective membrane channels. Within a unique amino-terminal hydrophobic region of VacA, there are three tandem GXXXG motifs (defined by glycines at positions 14, 18, 22, and 26), which are characteristic of transmembrane dimerization sequences. The goals of the current study were to investigate whether these GXXXG motifs are required for membrane channel formation and cytotoxicity and to clarify the role of membrane channel formation in the biological activity of VacA. Six different alanine substitution mutations (P9A, G13A, G14A, G18A, G22A, and G26A) were introduced into the unique hydrophobic region located near the amino terminus of VacA. The effects of these mutations were first analyzed using the TOXCAT system, which permits the study of transmembrane oligomerization of proteins in a natural membrane environment. None of the mutations altered the capacity of ToxR-VacA-maltose-binding protein fusion proteins to insert into a membrane, but G14A and G18A mutations markedly diminished the capacity of the fusion proteins to oligomerize. We then introduced the six alanine substitution mutations into the vacA chromosomal gene of H. pylori and analyzed the properties of purified mutant VacA proteins. VacA-G13A, VacA-G22A, and VacA-G26A induced vacuolation of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. Subsequent experiments examined the capacity of each mutant toxin to form membrane channels. In a planar lipid bilayer assay, VacA proteins containing G13A, G22A, and G26A mutations formed anion-selective membrane channels, whereas VacA proteins containing P9A, G14A, and G18A mutations did not. Similarly, VacA-G13A, VacA-G22A, and VacA-G26A induced depolarization of HeLa cells, whereas VacA-P9A, VacA-G14A, and VacA-G18A did not. These data indicate that an intact proline residue and an intact G14 XXXG18 motif within the amino-terminal hydrophobic region of VacA are essential for membrane channel formation, and they also provide strong evidence that membrane channel formation is essential for VacA cytotoxicity.

Crystal structure of the Helicobacter pylori vacuolating toxin p55 domain

Helicobacter pylori VacA, a pore-forming toxin secreted by an autotransporter pathway, causes multiple alterations in human cells, contributes to the pathogenesis of peptic ulcer disease and gastric cancer, and is a candidate antigen for inclusion in an H. pylori vaccine. Here, we present a 2.4-Å crystal structure of the VacA p55 domain, which has an important role in mediating VacA binding to host cells. The structure is predominantly a right-handed parallel β-helix, a feature that is characteristic of autotransporter passenger domains but unique among known bacterial protein toxins. Notable features of VacA p55 include disruptions in β-sheet contacts that result in five β-helix subdomains and a C-terminal domain that contains a disulfide bond. Analysis of VacA protein sequences from unrelated H. pylori strains, including m1 and m2 forms of VacA, allows us to identify structural features of the VacA surface that may be important for interactions with host receptors. Docking of the p55 structure into a 19-Å cryo-EM map of a VacA dodecamer allows us to propose a model for how VacA monomers assemble into oligomeric structures capable of membrane channel formation.

A dominant negative mutant of Helicobacter pylori vacuolating toxin (VacA) inhibits VacA-induced cell vacuolation.

Most Helicobacter pyloristrains secrete a toxin (VacA) that causes structural and functional alterations in epithelial cells and is thought to play an important role in the pathogenesis of H. pylori-associated gastroduodenal diseases. The amino acid sequence, ultrastructural morphology, and cellular effects of VacA are unrelated to those of any other known bacterial protein toxin, and the VacA mechanism of action remains poorly understood. To analyze the functional role of a unique strongly hydrophobic region near the VacA amino terminus, we constructed an H. pylori strain that produced a mutant VacA protein (VacA-(Δ6–27)) in which this hydrophobic segment was deleted. VacA-(Δ6–27) was secreted by H. pylori, oligomerized properly, and formed two-dimensional lipid-bound crystals with structural features that were indistinguishable from those of wild-type VacA. However, VacA-(Δ6–27) formed ion-conductive channels in planar lipid bilayers significantly more slowly than did wild-type VacA, and the mutant channels were less anion-selective. Mixtures of wild-type VacA and VacA-(Δ6–27) formed membrane channels with properties intermediate between those formed by either isolated species. VacA-(Δ6–27) did not exhibit any detectable defects in binding or uptake by HeLa cells, but this mutant toxin failed to induce cell vacuolation. Moreover, when an equimolar mixture of purified VacA-(Δ6–27) and purified wild-type VacA were added simultaneously to HeLa cells, the mutant toxin exhibited a dominant negative effect, completely inhibiting the vacuolating activity of wild-type VacA. A dominant negative effect also was observed when HeLa cells were co-transfected with plasmids encoding wild-type and mutant toxins. We propose a model in which the dominant negative effects of VacA-(Δ6–27) result from protein-protein interactions between the mutant and wild-type VacA proteins, thereby resulting in the formation of mixed oligomers with defective functional activity.

Helicobacter pylori exploits a unique repertoire of type IV secretion system components for pilus assembly at the bacteria-host cell interface.

Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS.

Genome sequence analysis of Helicobacter pylori strains associated with gastric ulceration and gastric cancer

BackgroundPersistent colonization of the human stomach by Helicobacter pylori is associated with asymptomatic gastric inflammation (gastritis) and an increased risk of duodenal ulceration, gastric ulceration, and non-cardia gastric cancer. In previous studies, the genome sequences of H. pylori strains from patients with gastritis or duodenal ulcer disease have been analyzed. In this study, we analyzed the genome sequences of an H. pylori strain (98-10) isolated from a patient with gastric cancer and an H. pylori strain (B128) isolated from a patient with gastric ulcer disease.ResultsBased on multilocus sequence typing, strain 98-10 was most closely related to H. pylori strains of East Asian origin and strain B128 was most closely related to strains of European origin. Strain 98-10 contained multiple features characteristic of East Asian strains, including a type s1c vacA allele and a cagA allele encoding an EPIYA-D tyrosine phosphorylation motif. A core genome of 1237 genes was present in all five strains for which genome sequences were available. Among the 1237 core genes, a subset of alleles was highly divergent in the East Asian strain 98-10, encoding proteins that exhibited <90% amino acid sequence identity compared to corresponding proteins in the other four strains. Unique strain-specific genes were identified in each of the newly sequenced strains, and a set of strain-specific genes was shared among H. pylori strains associated with gastric cancer or premalignant gastric lesions.ConclusionThese data provide insight into the diversity that exists among H. pylori strains from diverse clinical and geographic origins. Highly divergent alleles and strain-specific genes identified in this study may represent useful biomarkers for analyzing geographic partitioning of H. pylori and for identifying strains capable of inducing malignant or premalignant gastric lesions.

Acid activation of Helicobacter pylori vacuolating cytotoxin (VacA) results in toxin internalization by eukaryotic cells

Helicobacter pylori VacA is a secreted toxin that induces multiple structural and functional alterations in eukaryotic cells. Exposure of VacA to either acidic or alkaline pH (‘activation’) results in structural changes in the protein and a marked enhancement of its cell‐vacuolating activity. However, the mechanism by which activation leads to increased cytotoxicity is not well understood. In this study, we analysed the binding and internalization of [125I]‐VacA by HeLa cells. We detected no difference in the binding of untreated and activated [125I]‐VacA to cells. Binding of acid‐activated [125I]‐VacA to cells at 4°C was not saturable, and was only partially inhibited by excess unlabelled toxin. These results suggest that VacA binds either non‐specifically or to an abundant, low‐affinity receptor on HeLa cells. To study internalization of VacA, we used a protease protection assay. Analysis by SDS–PAGE and autoradiography indicated that the intact 87 kDa toxin was internalized in a time‐dependent process at 37°C but not at 4°C. Furthermore, internalization of the intact toxin was detected only if VacA was acid or alkaline activated before being added to cells. The internalization of activated [125I]‐VacA was not substantially inhibited by the presence of excess unlabelled toxin, but was blocked if cells were depleted of cellular ATP by the addition of sodium azide and 2‐deoxy‐d‐glucose. These results indicate that acid or alkaline pH‐induced structural changes in VacA are required for VacA entry into cells, and that internalization of the intact 87 kDa toxin is required for VacA cytotoxicity.

Helicobacter pylori VacA Induces Programmed Necrosis in Gastric Epithelial Cells

ABSTRACT Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to the development of peptic ulcer disease and gastric cancer. The secreted pore-forming toxin VacA is one of the major virulence factors of H. pylori. In the current study, we show that AZ-521 human gastric epithelial cells are highly susceptible to VacA-induced cell death. Wild-type VacA causes death of these cells, whereas mutant VacA proteins defective in membrane channel formation do not. Incubation of AZ-521 cells with wild-type VacA results in cell swelling, poly(ADP-ribose) polymerase (PARP) activation, decreased intracellular ATP concentration, and lactate dehydrogenase (LDH) release. VacA-induced death of these cells is a caspase-independent process that results in cellular release of histone-binding protein high mobility group box 1 (HMGB1), a proinflammatory protein. These features are consistent with the occurrence of cell death through a programmed necrosis pathway and suggest that VacA can be included among the growing number of bacterial pore-forming toxins that induce cell death through programmed necrosis. We propose that VacA augments H. pylori-induced mucosal inflammation in the human stomach by causing programmed necrosis of gastric epithelial cells and subsequent release of proinflammatory proteins and may thereby contribute to the pathogenesis of gastric cancer and peptic ulceration.

Protein-protein interactions among Helicobacter pylori cag proteins.

Many Helicobacter pylori isolates contain a 40-kb region of chromosomal DNA known as the cag pathogenicity island (PAI). The risk for development of gastric cancer or peptic ulcer disease is higher among humans infected with cag PAI-positive H. pylori strains than among those infected with cag PAI-negative strains. The cag PAI encodes a type IV secretion system that translocates CagA into gastric epithelial cells. To identify Cag proteins that are expressed by H. pylori during growth in vitro, we compared the proteomes of a wild-type H. pylori strain and an isogenic cag PAI deletion mutant using two-dimensional difference gel electrophoresis (2D-DIGE) in multiple pH ranges. Seven Cag proteins were identified by this approach. We then used a yeast two-hybrid system to detect potential protein-protein interactions among 14 Cag proteins. One heterotypic interaction (CagY/7 with CagX/8) and two homotypic interactions (involving H. pylori VirB11/ATPase and Cag5) were similar to interactions previously reported to occur among homologous components of the Agrobacterium tumefaciens type IV secretion system. Other interactions involved Cag proteins that do not have known homologues in other bacterial species. Biochemical analysis confirmed selected interactions involving five of the proteins that were identified by 2D-DIGE. Protein-protein interactions among Cag proteins are likely to have an important role in the assembly of the H. pylori type IV secretion apparatus.

Functional Properties of the p33 and p55 Domains of the Helicobacter pylori Vacuolating Cytotoxin

Helicobacter pylori secretes an 88-kDa vacuolating cytotoxin (VacA) that may contribute to the pathogenesis of peptic ulcer disease and gastric cancer. VacA cytotoxic activity requires assembly of VacA monomers into oligomeric structures, formation of anion-selective membrane channels, and entry of VacA into host cells. In this study, we analyzed the functional properties of recombinant VacA fragments corresponding to two putative VacA domains (designated p33 and p55). Immunoprecipitation experiments indicated that these two domains can interact with each other to form protein complexes. In comparison to the individual VacA domains, a mixture of the p33 and p55 proteins exhibited markedly enhanced binding to the plasma membrane of mammalian cells. Furthermore, internalization of the VacA domains was detected when cells were incubated with the p33/p55 mixture but not when the p33 and p55 proteins were tested individually. Incubation of cells with the p33/p55 mixture resulted in cell vacuolation, whereas the individual domains lacked detectable cytotoxic activity. Interestingly, sequential addition of p55 followed by p33 resulted in VacA internalization and cell vacuolation, whereas sequential addition in the reverse order was ineffective. These results indicate that both the p33 and p55 domains contribute to the binding and internalization of VacA and that both domains are required for vacuolating cytotoxic activity. Reconstitution of toxin activity from two separate domains, as described here for VacA, has rarely been described for pore-forming bacterial toxins, which suggests that VacA is a pore-forming toxin with unique structural properties.