OmpW is positively regulated by iron via Fur, and negatively regulated by SoxS contribution to oxidative stress resistance in Escherichia coli.

Iron plays a central role at the interface of pathogen and host. The ability to sequester iron from a host not only reduces host immune defenses but also promotes pathogen virulence, leading to the occurrence of infectious disease. Recently, outer membrane protein OmpW was shown to protect bacteria against harsh environmental conditions and to play a role in infectious disease. The expression of this versatile protein is controlled by iron, but the underlying mechanism of iron regulation has not been elucidated. In this study, the relation between OmpW expression and iron was investigated. Our results demonstrated that expression of OmpW is responsive to iron. Iron uptake analysis showed that an ompW mutant strain has a strong requirement for iron as compared to wild type and the ompW complemented strain. Moreover, ferric uptake regulation protein Fur, an iron binding transcriptional factor, was downregulated under iron limitation conditions and had a similar expression profile to OmpW in the presence or absence of iron. Based on these results, we suggest that iron regulates OmpW by binding to Fur. Furthermore, SoxS, a transcriptional factor involved in oxidative stress, was found to negatively regulate OmpW. We found that downregulating or knocking out OmpW results in bacterial resistance to oxidative stress. These findings provide new insight into the regulation of OmpW expression by iron, and may represent a new mechanism contributing to iron-mediated infectious disease.

LptD is a promising vaccine antigen and potential immunotherapeutic target for protection against Vibrio species infection.

Outer membrane proteins (OMPs) are unique to Gram-negative bacteria. Several features, including surface exposure, conservation among strains and ability to induce immune responses, make OMPs attractive targets for using as vaccine antigens and immunotherapeutics. LptD is an essential OMP that mediates the final transport of lipopolysaccharide (LPS) to outer leaflet. The protein in Vibrio parahaemolyticus was identified to have immunogenicity in our previous report. In this study, broad distribution, high conservation and similar surface-epitopes of LptD were found among the major Vibrio species. LptD was further revealed to be associated with immune responses, and it has a strong ability to stimulate antibody response. More importantly, it conferred 100% immune protection against lethal challenge by V. parahaemolyticus in mice when the mice were vaccinated with LptD, and this finding was consistent with the observation of efficient clearance of bacteria in vaccination mice. Strikingly, targeting of bacteria by the LptD antibody caused significant decreases in both the growth and LPS level and an increase in susceptibility to hydrophobic antibiotics. These findings were consistent with those previously obtained in lptD-deletion bacteria. These data demonstrated LptD is a promising vaccine antigens and a potential target for antibody-based therapy to protect against Vibrio infections.

Contribution of the outer membrane protein OmpW in Escherichia coli to complement resistance from binding to factor H.

The serum complement system is essential for innate immune defense against invading pathogenic bacteria. Some of the 8-stranded ß-barrel outer membrane proteins confer bacterial resistance to the innate host immunity. We have previously demonstrated that OmpW, also an 8-stranded ß-barrel protein that was identified a decade ago, protects bacteria against host phagocytosis. In this study, we investigated the complement resistance of OmpW. Our results indicate that the upregulation of OmpW is associated with increased survival when bacteria are exposed to normal human sera (NHS). Mutant bacteria lacking OmpW in NHS exhibited significantly lower survival rates in comparison to wild-type and ompW complemented bacteria. Furthermore, the bacterial survival significantly decreased in NHS that was supplemented with EGTA-Mg(2+) compared to that in NHS supplemented with EDTA. These results suggest that OmpW confer resistance to alternative complement pathway-mediated killing. Moreover, the binding of OmpW to factor H, a major inhibitor of alternative pathway, was found, indicating that OmpW recruitment of factor H is a mechanism for bacterial evasion of complement attack.

Immunoproteomic Approach for Screening Vaccine Candidates from Bacterial Outer Membrane Proteins.

Outer membrane proteins (OMPs) are unique to Gram-negative bacteria and have been revealed as potential vaccine candidates for conferring protection against infections in recent years. Immunoproteomics is a powerful technique that is ideally suited to screen and identify potential vaccine candidates. This chapter presents a brief outline of the screening of immunogenic OMPs from Vibrio parahaemolyticus by an immunoproteomic strategy that was based on two-dimensional electrophoresis (2-DE) and immunoblotting. The protective efficacy provided by the immunogenic OMP Vp0802 determined by active protection experiment assays is also presented in brief.

Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus.

Protein lysine acylation, including acetylation and succinylation, has been found to be a major post-translational modification (PTM) and is associated with the regulation of cellular processes that are widespread in bacteria. Vibrio parahemolyticus is a model marine bacterium that causes seafood-borne illness in humans worldwide. The lysine acetylation of V. parahemolyticus has been extensively characterized in our previous work, and here, we report the first global analysis of lysine succinylation and the overlap between the two types of acylation in this bacterium. Using high-accuracy nano liquid chromatography-tandem mass spectrometry combined with affinity purification, we identified 1931 lysine succinylated peptides matched on 642 proteins, with the quantity of the succinyl-proteins accounting for 13.3% of the total proteins in cells. Bioinformatics analysis results showed that these succinylated proteins are involved in almost every cellular process, particularly in protein biosynthesis and metabolism, and are distributed in diverse subcellular compartments. Moreover, several sequence motifs were identified, including succinyl-lysine flanked by a lysine or arginine residue at the -8, -7, or +7 position and without these residues at the -1 or +2 position, and these motifs differ from those found in other bacteria and eukaryotic cells. Furthermore, a total of 517 succinyl-lysine sites (26.7%) on 288 proteins (44.9%) were also found to be acetylated, suggesting extensive overlap between succinylation and acetylation in this bacterium. This systematic analysis provides a promising starting point for further investigations of the physiologic and pathogenic roles of lysine succinylation and acetylation in V. parahemolyticus.

Identification of a novel vaccine candidate by immunogenic screening of Vibrio parahaemolyticus outer membrane proteins.

Vibrio parahaemolyticus is an important halophilous pathogen that can cause not only a broad range of disease in aquatic animals but also serious seafood-borne illness in humans as a result of the consumption of seafood. To avoid the use of antibiotics, it is critical to identify protective antigens for developing highly effective vaccines against this pathogen. Outer membrane proteins (OMPs) have been suggested as potential vaccine candidates for conferring protection against infection. In this study, we identified novel immunogenic OMPs using an immune assay with serum antibodies from mice infected by V. parahaemolyticus combined with mass spectrometry analysis. Nine OMPs were identified to be immunogenic proteins, and four of these identified proteins with relatively low abundance in OMP profiles, LptD, VP0802, VP1243 and VP0966, were determined to have immunogenicity for the first time. One OMP of interest, VP0802, is highly conserved among major Vibrio species and was proposed to adopt a ß-barrel conformation and to be a member of the OprD protein family by bioinformatic analysis. The immunogenicity and protective efficacy of VP0802 were further evaluated by bacterial challenge postimmunization in a mouse model. VP0802 was confirmed to be highly immunogenic and to offer strong protection against V. parahaemolyticus infection, with an RPS of at least 66.7. Efficient clearance of bacteria from the blood of vaccinated mice was also observed. Moreover, upregulation of VP0802 expression was found after bacteria were exposed to fresh sera. These data, taken together, suggest that VP0802 is a promising candidate for the development of a subunit vaccine to prevent V. parahaemolyticus infection.

Systematic analysis of the lysine acetylome in Vibrio parahemolyticus.

Lysine acetylation of proteins is a major post-translational modification that plays an important regulatory role in almost every aspect of cells, both eukaryotes and prokaryotes. Vibrio parahemolyticus, a model marine bacterium, is a worldwide cause of bacterial seafood-borne illness. Here, we conducted the first lysine acetylome in this bacterium through a combination of highly sensitive immune-affinity purification and high-resolution LC-MS/MS. Overall, we identified 1413 lysine acetylation sites in 656 proteins, which account for 13.6% of the total proteins in the cells; this is the highest ratio of acetyl proteins that has so far been identified in bacteria. The bioinformatics analysis of the acetylome showed that the acetylated proteins are involved in a wide range of cellular functions and exhibit diverse subcellular localizations. More specifically, proteins related to protein biosynthesis and carbon metabolism are the preferential targets of lysine acetylation. Moreover, two types of acetylation motifs, a lysine or arginine at the +4/+5 positions and a tyrosine, histidine, or phenylalanine at the +1/+2 positions, were revealed from the analysis of the acetylome. Additionally, protein interaction network analysis demonstrates that a wide range of interactions are modulated by protein acetylation. This study provides a significant beginning for the in-depth exploration of the physiological role of lysine acetylation in V. parahemolyticus.