Investigating Vibrio parahaemolyticus interactions with the Pacific oyster, Crassostrea gigas

Vibrio parahaemolyticus is a Gram-negative, halophilic, human pathogenic bacterium ubiquitous in the marine environment. Like many Vibrio species, V. parahaemolyticus commonly associates with shellfish, particularly oysters. Ingestion of a raw or under cooked oysters contaminated with V. parahaemolyticus can cause gastroenteritis, which is typically self-limiting and rarely causes death. Globally, oyster production is highly lucrative, especially on the West Coast of the United States where approximately 60% of oyster production occurs each year. Outbreaks of V. parahaemolyticus can result in a significant public health problem as well as an economic burden for the oyster farms implicated in the outbreak. With the increase in overall V. parahaemolyticus outbreaks, improved post-harvest processing strategies have been developed to reduce this natural contaminant. Depuration was developed to allow shellfish to purge contaminants from their tissues into the clean, flowing seawater where they are held. This post-harvest processing technique can typically reduce fecal contaminants from the oyster tissues but is relatively ineffective at eliminating V. parahaemolyticus and other Vibrio species.. Thus, improved methods for reducing this and other human pathogenic Vibrio are needed to effectively produce safer oysters for the consumer. To develop more effective and novel V. parahaemolyticus intervention strategies, first we must identify the factors that are involved in V. parahaemolyticus colonization of the oyster, allowing them toresist depuration. This study sought to investigate specific factors utilized by V. parahaemolyticus and, in the process, determined that various strains of V. parahaemolyticus have different alleles of the Type IV pili, mannose-sensitive hemagglutinin (MSHA)and chitin-regulated pilus (PilA). In addition, we expanded our investigations into the allelic diversity of MSHA and PilA from Vibrio cholerae and Vibrio vulnificus and found that V. cholerae strains that possess the Type IV toxin co-regulated pilus (TCP) maintained highly conserved MSHA and PilA sequences while strains of V. cholerae without TCP, and all of the V. vulnificus and V. parahaemolyticus strains examined, had highly divergent sequences with no discernable connection to isolation source or observed phenotype. Following that discovery, we determined that Type I, and Type IV pili, as well as polar and lateral flagellar systems contribute to V. parahaemolyticus persistence in the Pacific oyster during depuration, while Type III secretion systems and phase variation do not. Overall, we have identified factors involved in colonization of the Pacific oyster by V. parahaemolyticus. Future studies investigating conditions that affect pili and flagella production in V. parahaemolyticus may provide novel depuration conditions that could easily and effectively increase the efficiency of oyster depuration, ultimately reducing the risk of seafood-borne illness by V. parahaemolyticus associated with oysters

Roles of the Sodium-Translocating NADH:Quinone Oxidoreductase (Na⁺-NQR) on Vibrio cholerae Metabolism, Motility and Osmotic Stress Resistance

The Na⁺ translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na⁺-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na⁺-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na⁺-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na⁺ pump(s) can compensate for Na⁺ pumping activity of Na⁺-NQR. Overall, our study provides important insights into the contribution of Na⁺-NQR to V. cholerae physiology

Mycobacterium tuberculosis Resists Stress by Regulating PE19 Expression

Mycobacterium tuberculosis requires the phosphate-sensing signal transduction system Pst/SenX3-RegX3 to resist host immune responses. A Delta pstA1 mutant lacking a Pst phosphate uptake system component is hypersensitive to diverse stress conditions in vitro and is attenuated in vivo due to constitutive expression of the phosphate starvation-responsive RegX3 regulon. Transcriptional profiling of the Delta pstA1 mutant revealed aberrant expression of certain pe and ppe genes. PE and PPE proteins, defined by conserved N-terminal domains containing Pro-Glu (PE) or Pro-Pro-Glu (PPE) motifs, account for a substantial fraction of the M. tuberculosis genome coding capacity, but their functions are largely uncharacterized. Because some PE and PPE proteins localize to the cell wall, we hypothesized that overexpression of these proteins sensitizes M. tuberculosis to stress by altering cell wall integrity. To test this idea, we deleted pe and ppe genes that were overexpressed by Delta pstA1 bacteria. Deletion of a single pe gene, pe19, suppressed hypersensitivity of the Delta pstA1 mutant to both detergent and reactive oxygen species. Ethidium bromide uptake assays revealed increased envelope permeability of the Delta pstA1 mutant that was dependent on PE19. The replication defect of the Delta pstA1 mutant in NOS2(-/-) mice was partially reversed by deletion of pe19, suggesting that increased membrane permeability due to PE19 overexpression sensitizes M. tuberculosis to host immunity. Our data indicate that PE19, which comprises only a 99-amino-acid PE domain, has a unique role in the permeability of the M. tuberculosis envelope that is regulated to resist stresses encountered in the host

Roles of the sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) on vibrio cholerae metabolism, motility and osmotic stress resistance.

The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology

FassioSaraMicrobiologyRolesSodium-Translocating.pdf

The Na⁺ translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na⁺-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na⁺-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na⁺-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na⁺ pump(s) can compensate for Na⁺ pumping activity of Na⁺-NQR. Overall, our study provides important insights into the contribution of Na⁺-NQR to V. cholerae physiology

FassioSaraMicrobiologyRolesSodium-Translocating_SupportingInformation.xlsx

The Na⁺ translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na⁺-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na⁺-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na⁺-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na⁺ pump(s) can compensate for Na⁺ pumping activity of Na⁺-NQR. Overall, our study provides important insights into the contribution of Na⁺-NQR to V. cholerae physiology

Effect of Δ<i>nqrA-F</i> mutation on swarming activity.

<p>Swarming assays were performed in LB medium supplemented with 100 mM NaCl and buffered to pH6.5 either with or without the addition of 33 mM D, L-lactate. Mean values and standard error from 16 experiments are presented. P values were calculated using Student's t test.</p

Genes down-regulated in the Δ<i>nqrA-F</i> mutant based on microarray analysis.

<p>Genes down-regulated in the Δ<i>nqrA-F</i> mutant based on microarray analysis.</p

Metabolites changed in the Δ<i>nqrA-F</i> mutant based on metabolomics analysis.

<p>Metabolites changed in the Δ<i>nqrA-F</i> mutant based on metabolomics analysis.</p

Changes in central metabolism in <i>V. cholerae</i> Δ<i>nqrA-F</i> mutant.

<p>Red solid squares show metabolites that are increased in the Δ<i>nqrA-F</i> mutant. Blue solid squares show metabolites that are decreased in the Δ<i>nqrA-F</i> mutant. Red solid arrows show metabolic pathways that are expected to be decreased in the Δ<i>nqrA-F</i> mutant. Blue solid arrows show metabolic pathways that are expected to be increased in the Δ<i>nqrA-F</i> mutant. AcP, acetyl phosphate. RP, Ribose phosphate.</p