Search

Helicobacter pylori colonizes the stomach in about half of the world's population. H. pylori strains containing the cag pathogenicity island ( cag PAI) are associated with a higher risk of gastric adenocarcinoma or peptic ulcer disease than cag PAI-negative strains. The cag PAI encodes a type IV secretion system (T4SS) that mediates delivery of the CagA effector protein as well as nonprotein bacterial constituents into gastric epithelial cells. H. pylori -induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and interleukin-8 (IL-8) secretion are attributed to T4SS-dependent delivery of lipopolysaccharide metabolites and peptidoglycan into host cells, and Toll-like receptor 9 (TLR9) activation is attributed to delivery of bacterial DNA. In this study, we analyzed the bacterial energetic requirements associated with these cellular alterations. Mutant strains lacking Cagα, Cagβ, or CagE (putative ATPases corresponding to VirB11, VirD4, and VirB4 in prototypical T4SSs) were capable of T4SS core complex assembly but defective in CagA translocation into host cells. Thus, the three Cag ATPases are not functionally redundant. Cagα and CagE were required for H. pylori -induced NF-κB activation, IL-8 secretion, and TLR9 activation, but Cagβ was dispensable for these responses. We identified putative ATP-binding motifs (Walker-A and Walker-B) in each of the ATPases and generated mutant strains in which these motifs were altered. Each of the Walker box mutant strains exhibited properties identical to those of the corresponding deletion mutant strains. These data suggest that Cag T4SS-dependent delivery of nonprotein bacterial constituents into host cells occurs through mechanisms different from those used for recruitment and delivery of CagA into host cells., (Copyright © 2020 American Society for Microbiology.)

Helicobacter pylori infection and a high salt diet are each risk factors for gastric cancer. In this study, we tested the hypothesis that environmental salt concentration influences the composition of the H. pylori exoproteome. H. pylori was cultured in media containing varying concentrations of sodium chloride, and aliquots were fractionated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified proteins that were selectively released into the extracellular space, and we identified selectively released proteins that were differentially abundant in culture supernatants, depending on the environmental salt concentration. We also used RNA-seq analysis to identify genes that were differentially expressed in response to environmental salt concentration. The salt-responsive proteins identified by proteomic analysis and salt-responsive genes identified by RNA-seq analysis were mostly non-concordant, but the secreted toxin VacA was salt-responsive in both analyses. Western blot analysis confirmed that VacA levels in the culture supernatant were increased in response to high salt conditions, and quantitative RT-qPCR experiments confirmed that vacA transcription was upregulated in response to high salt conditions. These results indicate that environmental salt concentration influences the composition of the H. pylori exoproteome, which could contribute to the increased risk of gastric cancer associated with a high salt diet. SIGNIFICANCE: Helicobacter pylori-induced alterations in the gastric mucosa have been attributed, at least in part, to the actions of secreted H. pylori proteins. In this study, we show that H. pylori growth in high salt concentrations leads to increased levels of a secreted VacA toxin. Salt-induced alterations in the composition of the H. pylori exoproteome is relevant to the increased risk of gastric cancer associated with consumption of a high salt diet., (Published by Elsevier B.V.)

Helicobacter pylori colonizes the human stomach and is associated with an increased risk of gastric cancer and peptic ulcer disease. Analysis of H. pylori protein secretion is complicated by the occurrence of bacterial autolysis. In this study, we analyzed the exoproteome of H. pylori at multiple phases of bacterial growth and identified 74 proteins that are selectively released into the extracellular space. These include proteins known to cause alterations in host cells, antigenic proteins, and additional proteins that have not yet been studied in any detail. The composition of the H. pylori exoproteome is dependent on the phase of bacterial growth. For example, the proportional abundance of the vacuolating toxin VacA in culture supernatant is higher during late growth phases than early growth phases, whereas the proportional abundance of many other proteins is higher during early growth phases. We detected marked variation in the subcellular localization of putative secreted proteins within soluble and membrane fractions derived from intact bacteria. By providing a comprehensive view of the H. pylori exoproteome, these results provide new insights into the array of secreted H. pylori proteins that may cause alterations in the gastric environment., (Published by Elsevier B.V.)

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.

Bacterial phosphopentomutases (PPMs) are alkaline phosphatase superfamily members that interconvert α-D-ribose 5-phosphate (ribose 5-phosphate) and α-D-ribose 1-phosphate (ribose 1-phosphate). We investigated the reaction mechanism of Bacillus cereus PPM using a combination of structural and biochemical studies. Four high resolution crystal structures of B. cereus PPM revealed the active site architecture, identified binding sites for the substrate ribose 5-phosphate and the activator α-D-glucose 1,6-bisphosphate (glucose 1,6-bisphosphate), and demonstrated that glucose 1,6-bisphosphate increased phosphorylation of the active site residue Thr-85. The phosphorylation of Thr-85 was confirmed by Western and mass spectroscopic analyses. Biochemical assays identified Mn(2+)-dependent enzyme turnover and demonstrated that glucose 1,6-bisphosphate treatment increases enzyme activity. These results suggest that protein phosphorylation activates the enzyme, which supports an intermolecular transferase mechanism. We confirmed intermolecular phosphoryl transfer using an isotope relay assay in which PPM reactions containing mixtures of ribose 5-[(18)O(3)]phosphate and [U-(13)C(5)]ribose 5-phosphate were analyzed by mass spectrometry. This intermolecular phosphoryl transfer is seemingly counter to what is anticipated from phosphomutases employing a general alkaline phosphatase reaction mechanism, which are reported to catalyze intramolecular phosphoryl transfer. However, the two mechanisms may be reconciled if substrate encounters the enzyme at a different point in the catalytic cycle.

The tremendous success of Staphylococcus aureus as a pathogen is due to the controlled expression of a diverse array of virulence factors. The effects of host environments on the expression of virulence factors and the mechanisms by which S. aureus adapts to colonize distinct host tissues are largely unknown. Vertebrates have evolved to sequester nutrient iron from invading bacteria, and iron availability is a signal that alerts pathogenic microorganisms when they enter the hostile host environment. Consistent with this, we report here that S. aureus senses alterations in the iron status via the ferric uptake regulator (Fur) and alters the abundance of a large number of virulence factors. These Fur-mediated changes protect S. aureus against killing by neutrophils, and Fur is required for full staphylococcal virulence in a murine model of infection. A potential mechanistic explanation for the impact of Fur on virulence is provided by the observation that Fur coordinates the reciprocal expression of cytolysins and a subset of immunomodulatory proteins. More specifically, S. aureus lacking fur exhibits decreased expression of immunomodulatory proteins and increased expression of cytolysins. These findings reveal that Fur is involved in initiating a regulatory program that organizes the expression of virulence factors during the pathogenesis of S. aureus pneumonia.

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.

A profile likelihood algorithm is proposed for quantitative shotgun proteomics to infer the abundance ratios of proteins from the abundance ratios of isotopically labeled peptides derived from proteolysis. Previously, we have shown that the estimation variability and bias of peptide abundance ratios can be predicted from their profile signal-to-noise ratios. Given multiple quantified peptides for a protein, the profile likelihood algorithm probabilistically weighs the peptide abundance ratios by their inferred estimation variability, accounts for their expected estimation bias, and suppresses contribution from outliers. This algorithm yields maximum likelihood point estimation and profile likelihood confidence interval estimation of protein abundance ratios. This point estimator is more accurate than an estimator based on the average of peptide abundance ratios. The confidence interval estimation provides an "error bar" for each protein abundance ratio that reflects its estimation precision and statistical uncertainty. The accuracy of the point estimation and the precision and confidence level of the interval estimation were benchmarked with standard mixtures of isotopically labeled proteomes. The profile likelihood algorithm was integrated into a quantitative proteomics program, called ProRata, freely available at www.MSProRata.org.

Rhodopseudomonas palustris is a purple nonsulfur anoxygenic phototrophic bacterium that is ubiquitous in soil and water. R. palustris is metabolically versatile with respect to energy generation and carbon and nitrogen metabolism. We have characterized and compared the baseline proteome of a R. palustris wild-type strain grown under six metabolic conditions. The methodology for proteome analysis involved protein fractionation by centrifugation, subsequent digestion with trypsin, and analysis of peptides by liquid chromatography coupled with tandem mass spectrometry. Using these methods, we identified 1664 proteins out of 4836 predicted proteins with conservative filtering constraints. A total of 107 novel hypothetical proteins and 218 conserved hypothetical proteins were detected. Qualitative analyses revealed over 311 proteins exhibiting marked differences between conditions, many of these being hypothetical or conserved hypothetical proteins showing strong correlations with different metabolic modes. For example, five proteins encoded by genes from a novel operon appeared only after anaerobic growth with no evidence of these proteins in extracts of aerobically grown cells. Proteins known to be associated with specialized growth states such as nitrogen fixation, photoautotrophic, or growth on benzoate, were observed to be up-regulated under those states.