Analysis of Surface-Exposed Outer Membrane Proteins in Helicobacter pylori

Voss, Bradley J.; Gaddy, Jennifer A.; McDonald, W. Hayes; Cover, Timothy L.

doi:10.1128/jb.01768-14

Cited by 62 publications

(80 citation statements)

References 56 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Secretion of VacA is believed to proceed via a type V or auto-transporter mechanism (6,(34)(35)(36)(37)(38). The secreted 88-kDa VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55, which remain together after proteolysis (27,36,(39)(40)(41)(42).…”

mentioning

confidence: 99%

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

et al. 2016

Self Cite

View full text Add to dashboard Cite

Helicobacter pylori secretes a pore-forming VacA toxin that has structural features and activities substantially different from those of other known bacterial toxins. VacA can assemble into multiple types of water-soluble flower-shaped oligomeric structures, and most VacA activities are dependent on its capacity to oligomerize. The 88-kDa secreted VacA protein can undergo limited proteolysis to yield two domains, designated p33 and p55. The p33 domain is required for membrane channel formation and intracellular toxic activities, and the p55 domain has an important role in mediating VacA binding to cells. Previous studies showed that the p55 domain has a predominantly ␤-helical structure, but no structural data are available for the p33 domain. We report here the purification and analysis of a nonoligomerizing mutant form of VacA secreted by H. pylori. The nonoligomerizing 88-kDa mutant protein retains the capacity to enter host cells but lacks detectable toxic activity. Analysis of crystals formed by the monomeric protein reveals that the ␤-helical structure of the p55 domain extends into the C-terminal portion of p33. Fitting the p88 structural model into an electron microscopy map of hexamers formed by wild-type VacA (predicted to be structurally similar to VacA membrane channels) reveals that p55 and the ␤-helical segment of p33 localize to peripheral arms but do not occupy the central region of the hexamers. We propose that the amino-terminal portion of p33 is unstructured when VacA is in a monomeric form and that it undergoes a conformational change during oligomer assembly. Helicobacter pylori is a Gram-negative bacterium that persistently colonizes the stomach in about half of the human population worldwide (1-3). Most people tolerate the presence of this organism for long periods without any adverse consequences, but a small subset of H. pylori-infected individuals develop gastric adenocarcinoma or peptic ulcer disease.H. pylori is a relatively noninvasive organism that lives in the gastric mucus layer overlying gastric epithelial cells, sometimes adhering to the gastric epithelium. Many H. pylori-induced alterations in the gastric mucosa are attributable to the actions of secreted bacterial proteins on host cells (4-6). One such protein is the secreted vacuolating toxin, VacA (4, 7-10). VacA causes multiple alterations in gastric epithelial cells, including swelling of endosomes (vacuolation) (11, 12), permeabilization of mitochondrial membranes, and activation of mitogen-activated protein kinases (4, 7-9). In addition to its effects on gastric epithelial cells, VacA can disrupt the functions of many types of immune cells (T cells, B cells, neutrophils, eosinophils, and monocytes) (4, 7-9, 13-16). The cellular activities of VacA are attributed mainly to its ability to form anion-selective channels in host cells (17-21). VacA lacks sequence similarity to any other known bacterial toxins.H. pylori strains isolated from unrelated individuals are genetically heterogeneous, and the risk of developing gastric...

show abstract

mentioning

confidence: 99%

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…The third ring from the outside shows the GC-skew between the genomes. The cyan lines mark the distribution of outer membrane protein (OMP) genes (as defined by Alm et al, 2000 and Voss et al, 2014). (B) A Venn diagram compares the core and pan-genomes of Nic_25A and P12 based on orthologous clustering with 0.8 identity cut-off.…”

Section: Resultsmentioning

confidence: 99%

“…As seen in the Figure 4 , the adhesion factors AlpB, HpaA, and HopQ were also found to be more abundant in the P12 strain. Based on the unique proteins identified in the triplicate runs ( Supporting File 8 ), the adhesins BabB and SabB were more abundant in the P12 strain, as were OMPs of the Hop and Hom families (Voss et al, 2014) and other virulence factors including vacuolating cytotoxic protein A (VacA), elongation factor (EF-Tu), and flagellar basal body protein FliL.…”

Section: Resultsmentioning

confidence: 99%

Comparative Analysis of Two Helicobacter pylori Strains using Genomics and Mass Spectrometry-Based Proteomics

et al. 2016

View full text Add to dashboard Cite

Helicobacter pylori, a gastroenteric pathogen believed to have co-evolved with humans over 100,000 years, shows significant genetic variability. This motivates the study of different H. pylori strains and the diseases they cause in order to identify determinants for disease evolution. In this study, we used proteomics tools to compare two H. pylori strains. Nic25_A was isolated in Nicaragua from a patient with intestinal metaplasia, and P12 was isolated in Europe from a patient with duodenal ulcers. Differences in the abundance of surface proteins between the two strains were determined with two mass spectrometry-based methods, label-free quantification (MaxQuant) or the use of tandem mass tags (TMT). Each approach used a lipid-based protein immobilization (LPITM) technique to enrich peptides of surface proteins. Using the MaxQuant software, we found 52 proteins that differed significantly in abundance between the two strains (up- or downregulated by a factor of 1.5); with TMT, we found 18 proteins that differed in abundance between the strains. Strain P12 had a higher abundance of proteins encoded by the cag pathogenicity island, while levels of the acid response regulator ArsR and its regulatory targets (KatA, AmiE, and proteins involved in urease production) were higher in strain Nic25_A. Our results show that differences in protein abundance between H. pylori strains can be detected with proteomic approaches; this could have important implications for the study of disease progression.

show abstract

“…Bacterial cultures were harvested by centrifugation at 3,500 ϫ g, and the pellets were resuspended in phosphate-buffered saline. Following a second centrifugation at 3,500 ϫ g, pellets were resuspended in Tris buffer containing cOmplete protease inhibitor cocktail (Roche) as described previously (29). Bacteria were lysed by sonication, and lysates were clarified by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…Bacteria were lysed by sonication, and lysates were clarified by centrifugation. These preparations were then analyzed by multidimensional protein identification technology (MudPIT) as described previously (29)(30)(31). Peptide tandem mass spectra were queried with SEQUEST (full tryptic specificity) against an H. pylori strain B8 (a closely related strain for which a complete genome sequence is available) (32) database to which both common contaminants and reversed versions of the proteins had been appended.…”

Section: Methodsmentioning

confidence: 99%

Helicobacter pylori Adaptation In Vivo in Response to a High-Salt Diet

et al. 2015

Self Cite

View full text Add to dashboard Cite

e Helicobacter pylori exhibits a high level of intraspecies genetic diversity. In this study, we investigated whether the diversification of H. pylori is influenced by the composition of the diet. Specifically, we investigated the effect of a high-salt diet (a known risk factor for gastric adenocarcinoma) on H. pylori diversification within a host. We analyzed H. pylori strains isolated from Mongolian gerbils fed either a high-salt diet or a regular diet for 4 months by proteomic and whole-genome sequencing methods. Compared to the input strain and output strains from animals fed a regular diet, the output strains from animals fed a highsalt diet produced higher levels of proteins involved in iron acquisition and oxidative-stress resistance. Several of these changes were attributable to a nonsynonymous mutation in fur (fur-R88H). Further experiments indicated that this mutation conferred increased resistance to high-salt conditions and oxidative stress. We propose a model in which a high-salt diet leads to high levels of gastric inflammation and associated oxidative stress in H. pylori-infected animals and that these conditions, along with the high intraluminal concentrations of sodium chloride, lead to selection of H. pylori strains that are most fit for growth in this environment. Helicobacter pylori is a Gram-negative bacterium that persistently colonizes the human stomach in half of the world's population (1, 2). H. pylori exhibits a high level of intraspecies genetic diversity, characterized by marked variation among strains in gene content, as well as a high level of variation in the nucleotide sequences of individual genes (3, 4). A high rate of allelic diversity is attributable to both a high mutation rate and intraspecies recombination (5, 6).Most H. pylori-infected persons remain asymptomatic, but the presence of this bacterium increases the risk of peptic ulcer disease and gastric cancer (1, 2, 7, 8). The clinical outcome of H. pylori infection is determined by a combination of bacterial, host, and environmental factors. For example, H. pylori strains containing the cag pathogenicity island (PAI) are associated with a higher risk of symptomatic disease than are strains that lack the cag PAI (9). One of the proteins encoded by the cag PAI, CagA, enters host cells and causes numerous cellular alterations linked to malignant transformation (10). The cag PAI also encodes multiple protein components of a type IV secretion system required for entry of CagA into host cells (11). H. pylori strains that secrete specific types of the VacA toxin are also linked to adverse disease outcome, in comparison to strains that secrete relatively nontoxic forms of the VacA protein (12, 13).One of the environmental factors associated with increased gastric cancer risk is a high-salt diet (14). Epidemiologic studies have demonstrated a link between high dietary salt consumption and increased gastric cancer risk in many parts of the world (15-17), and a high-salt diet also increases the gastric cancer risk in animal models (...

show abstract

Analysis of Surface-Exposed Outer Membrane Proteins in Helicobacter pylori

Cited by 62 publications

References 56 publications

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

A Nonoligomerizing Mutant Form of Helicobacter pylori VacA Allows Structural Analysis of the p33 Domain

Comparative Analysis of Two Helicobacter pylori Strains using Genomics and Mass Spectrometry-Based Proteomics

Helicobacter pylori Adaptation In Vivo in Response to a High-Salt Diet

Contact Info

Product

Resources

About