Regulator of ribonuclease activity modulates the pathogenicity of Vibrio vulnificus.

RraA, a protein regulator of RNase E activity, plays a unique role in modulating the mRNA abundance in Escherichia coli. The marine pathogenic bacterium Vibrio vulnificus also possesses homologs of RNase E (VvRNase E) and RraA (VvRraA1 and VvRraA2). However, their physiological roles have not yet been investigated. In this study, we demonstrated that VvRraA1 expression levels affect the pathogenicity of V. vulnificus. Compared to the wild-type strain, the VvrraA1-deleted strain (ΔVvrraA1) showed decreased motility, invasiveness, biofilm formation ability as well as virulence in mice; these phenotypic changes of ΔVvrraA1 were restored by the exogenous expression of VvrraA1. Transcriptomic analysis indicated that VvRraA1 expression levels affect the abundance of a large number of mRNA species. Among them, the half-lives of mRNA species encoding virulence factors (e.g., smcR and htpG) that have been previously shown to affect VvrraA1 expression-dependent phenotypes were positively correlated with VvrraA1 expression levels. These findings suggest that VvRraA1 modulates the pathogenicity of V. vulnificus by regulating the abundance of a subset of mRNA species.

Wzb of Vibrio vulnificus represents a new group of low-molecular-weight protein tyrosine phosphatases with a unique insertion in the W-loop.

Protein tyrosine phosphorylation regulates the production of capsular polysaccharide, an essential virulence factor of the deadly pathogen Vibrio vulnificus. The process requires the protein tyrosine kinase Wzc and its cognate phosphatase Wzb, both of which are largely uncharacterized. Herein, we report the structures of Wzb of V. vulnificus (VvWzb) in free and ligand-bound forms. VvWzb belongs to the low-molecular-weight protein tyrosine phosphatase (LMWPTP) family. Interestingly, it contains an extra four-residue insertion in the W-loop, distinct from all known LMWPTPs. The W-loop of VvWzb protrudes from the protein body in the free structure, but undergoes significant conformational changes to fold toward the active site upon ligand binding. Deleting the four-residue insertion from the W-loop severely impaired the enzymatic activity of VvWzb, indicating its importance for optimal catalysis. However, mutating individual residues or even substituting the whole insertion with four alanine residues only modestly decreased the enzymatic activity, suggesting that the contribution of the insertion to catalysis is not determined by the sequence specificity. Furthermore, inserting the four residues into Escherichia coli Wzb at the corresponding position enhanced its activity as well, indicating that the four-residue insertion in the W-loop can act as a general activity enhancing element for other LMWPTPs. The novel W-loop type and phylogenetic analysis suggested that VvWzb and its homologs should be classified into a new group of LMWPTPs. Our study sheds new insight into the catalytic mechanism and structural diversity of the LMWPTP family and promotes the understanding of the protein tyrosine phosphorylation system in prokaryotes.

Structural snapshots of CmoB in various states during wobble uridine modification of tRNA.

CmoB utilizes carboxy-S-adenosyl-l-methionine (CxSAM) to carry out unusual carboxymethyl transfer to form 5-carboxymethoxyuridine (cmo5U) of several tRNA species in Gram-negative bacteria. In this report, we present three X-ray crystal structures of CmoB from Vibrio vulnificus representing different states in the course of the reaction pathway; i.e., apo-, substrate-bound, and product-bound forms. Especially, the crystal structure of apo-CmoB unveils a novel open state of the enzyme, capturing unprecedented conformational dynamics around the substrate-binding site. The apo-structure demonstrates that the open conformation favors the release of CxSAM thus representing an inactive form. Our crystal structures of CmoB complexed with CxSAM and S-adenosyl-l-homocysteine (SAH) and combined binding assay results support the proposed mechanism underlying the cofactor selectivity, where CmoB preferentially senses negative charge around amino acid residues Lys-91, Tyr-200, and Arg-315.

Comparative structural and mechanistic studies of 4-hydroxy-tetrahydrodipicolinate reductases from Mycobacterium tuberculosis and Vibrio vulnificus.

BACKGROUND: The products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are essential for bacterial survival. This paper focuses on the structural and mechanistic characterization of 4-hydroxy-tetrahydrodipicolinate reductase (DapB), which is one of the enzymes from the lysine biosynthesis pathway. DapB catalyzes the conversion of (2S, 4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate (HTPA) to 2,3,4,5-tetrahydrodipicolinate in an NADH/NADPH dependent reaction. Genes coding for DapBs were identified as essential for many pathogenic bacteria, and therefore DapB is an interesting new target for the development of antibiotics. METHODS: We have combined experimental and computational approaches to provide novel insights into mechanism of the DapB catalyzed reaction. RESULTS: Structures of DapBs originating from Mycobacterium tuberculosis and Vibrio vulnificus in complexes with NAD+, NADP+, as well as with inhibitors, were determined and described. The structures determined by us, as well as currently available structures of DapBs from other bacterial species, were compared and used to elucidate a mechanism of reaction catalyzed by this group of enzymes. Several different computational methods were used to provide a detailed description of a plausible reaction mechanism. CONCLUSIONS: This is the first report presenting the detailed mechanism of reaction catalyzed by DapB. GENERAL SIGNIFICANCE: Structural data in combination with information on the reaction mechanism provide a background for development of DapB inhibitors, including transition-state analogues.

Recombinant expression and characterization of the glycogen branching enzyme from Vibrio vulnificus and its application in starch modification.

Resistant starch (RS) is helpful in controlling and preventing metabolic syndrome relevant diseases. However, the RS content of natural starch and modified starch produced by enzymatic method is generally low. To solve this problem, we selected the glycogen branching enzyme from Vibrio vulnificus (VvGBE) and investigated its application. Firstly, it was expressed in E. coli with the enzyme activity was 53.33 U/mL, and its optimum temperature and pH was 35 °C and 7.5, respectively. The half-life of VvGBE at 35 °C was 10 h, and the enzyme was most stable at pH 9.5. When we used the recombinant enzyme to treat corn starch, the content of RS increased by 19.41%, which was higher than that achieved with other enzymes. More specially, the conversion of slowly digestible starch to RS, which was only demonstrated in chemical modification, was accomplished. The fine structure of the modified starch was further investigated. Results showed that the number of short chains (DP < 13) increased to 90.58%, and the α-1,6 linkages ratio increased from 7.19% to 15.64%. The increase of short chains and α-1,6 linkages may contribute to high RS content. This study can provide a reference for the development of modified starch with lower digestibility.

A cofactor consumption screen identifies promising NfsB family nitroreductases for dinitrotoluene remediation.

OBJECTIVES: To survey a library of over-expressed nitroreductases to identify those most active with 2,4- and 2,6-dinitrotoluene substrates, as promising candidates for phytoremediation of soils and groundwater contaminated with poly-nitro toluene pollutants. RESULTS: To indirectly monitor dinitrotoluene reduction we implemented a nitroblue tetrazolium dye screen to compare relative rates of NADPH consumption for 58 nitroreductase candidates, over-expressed in a nitroreductase-deleted strain of Escherichia coli. Although the screen only provides activity data at a single substrate concentration, by altering the substrate concentration and duration of incubation we showed we could first distinguish between more-active and less-active enzymes and then discriminate between the relative rates of reduction exhibited by the most active nitroreductases in the collection. We observed that members of the NfsA and NfsB nitroreductase families were the most active with 2,4-dinitrotoluene, but that only members of the NfsB family reduced 2,6-dinitrotoluene effectively. Two NfsB family members, YfkO from Bacillus subtilis and NfsB from Vibrio vulnificus, appeared especially effective with these substrates. Purification of both enzymes as His6-tagged recombinant proteins enabled in vitro determination of Michaelis-Menten kinetic parameters with each dinitrotoluene substrate. CONCLUSIONS: Vibrio vulnificus NfsB is a particularly promising candidate for bioremediation applications, being ca. fivefold more catalytically efficient with 2,4-dinitrotoluene and over 26-fold more active with 2,6-dinitrotoluene than the benchmark E. coli nitroreductases NfsA and NfsB.

Characterization of temperature-dependent hemin uptake receptors HupA and HvtA in Vibrio vulnificus.

The Gram-negative pathogen Vibrio vulnificus produces several iron-sequestration systems including a hemin uptake system in response to iron limitation as a means to acquire this essential element. Strains of this organism are capable of causing serious septicemia in humans and eels, where hemin is abundant and an advantageous source of iron. Vibrio vulnificus hemin uptake systems consist of HupA, a well studied outer membrane protein, and a recently identified HvtA protein receptor. In this study, we confirmed that the expression of the hvtA gene is iron-regulated in a fur-dependent manner. When analyzed for virulence in a hemin-overloaded murine model system, the hupA gene was more important for establishing infection than the hvtA gene. Transcriptional profiling of these genes using strains of two different biotypes, biotype 1 (human pathogen) and biotype 2 (eel pathogen), showed that the expression of the two receptors was also regulated in response to temperature. The expression of hupA was highly induced in elevated temperatures in the human pathogenic strain when tested in iron-depleted conditions. Conversely, hvtA expression was induced significantly in the eel pathogenic strain at a lower temperature, a condition where the hupA locus was relatively repressed. Our results indicate that although both hupA and hvtA are involved for optimal hemin uptake in V. vulnificus, their expression is dually regulated by the environmental cues of iron concentration and temperature. Together, these data suggest that the virulence genes hupA and hvtA are tightly regulated and strictly induced during iron limitation combined with the physiological temperature of the host organism.

Unveiling the acid stress response of clinical genotype Vibrio vulnificus isolated from the marine environments of Mangaluru coast, India.

Gastric acidity is one of the earliest host defences faced by ingested organisms, and successful pathogens need to overcome this hurdle. The objective of this study was the systematic assessment of acid-stress response of Vibrio vulnificus isolated from coastal regions of Mangaluru. Acid-shock experiments were carried out at pH 4.0 and pH 4.5, with different experimental conditions expected to produce a varied acid response. Exposure to mild acid before the acid shock was favourable to the bacteria but was dependent on cell population and pH of the media and was independent of the strains tested. Lysine-dependent acid response was demonstrated with reference to the previously identified lysine decarboxylase system. Additionally, the results showed that inoculation into oysters provided some level of protection against acid stress. Increased expression of lysine/cadaverine genes was observed upon the addition of ground oyster and was confirmed by quantitative real-time PCR. The potential role of ornithine was analyzed with regard to acid stress, but no change in the survival pattern was observed. These findings highlight the physiology of bacteria in acid stress.

Structural analysis of a Vibrio phospholipase reveals an unusual Ser-His-chloride catalytic triad.

Phospholipases can disrupt host membranes and are important virulence factors in many pathogens. VvPlpA is a phospholipase A2 secreted by Vibrio vulnificus and essential for virulence. Its homologs, termed thermolabile hemolysins (TLHs), are widely distributed in Vibrio bacteria, but no structural information for this virulence factor class is available. Herein, we report the crystal structure of VvPlpA to 1.4-Å resolution, revealing that VvPlpA contains an N-terminal domain of unknown function and a C-terminal phospholipase domain and that these two domains are packed closely together. The phospholipase domain adopts a typical SGNH hydrolase fold, containing the four conserved catalytic residues Ser, Gly, Asn, and His. Interestingly, the structure also disclosed that the phospholipase domain accommodates a chloride ion near the catalytic His residue. The chloride is five-coordinated in a distorted bipyramid geometry, accepting hydrogen bonds from a water molecule and the amino groups of surrounding residues. This chloride substitutes for the most common Asp/Glu residue and forms an unusual Ser-His-chloride catalytic triad in VvPlpA. The chloride may orient the catalytic His and stabilize the charge on its imidazole ring during catalysis. Indeed, VvPlpA activity depended on chloride concentration, confirming the important role of chloride in catalysis. The VvPlpA structure also revealed a large hydrophobic substrate-binding pocket that is capable of accommodating a long-chain acyl group. Our results provide the first structure of the TLH family and uncover an unusual Ser-His-chloride catalytic triad, expanding our knowledge on the biological role of chloride.

Purification and biochemical characterization of FrsA protein from Vibrio vulnificus as an esterase.

Fermentation-respiration switch protein (FrsA) was thought to play an important role in controlling the metabolic flux between respiration and fermentation pathways, whereas the biochemical function of FrsA was unclear yet. A gene coding for FrsA protein from Vibrio vulnificus was chemically synthesized. The recombinant VvFrsA was expressed as a soluble protein and purified by Ni-NTA affinity chromatography. The protein had a subunit molecular weight of ca. 45 kDa by SDS-PAGE and preferred short-chain esters when p-nitrophenyl alkanoate esters were used as substrates. Optimum condition for VvFrsA was found to be at pH 9.0 and 50 °C. The protein retained high esterase activity at alkaline condition and would denature slowly at over 50 °C. With p-nitrophenyl acetate as the substrate, the Km and kcat were determined to be 18.6 mM and 0.67 s-1, respectively, by steady-state kinetic assay. Molecular dynamics simulation and docking model structure revealed that p-nitrophenyl acetate could be the substrate of VvFrsA. In conclusion our results demonstrated that the protein was able to catalyze the hydrolysis of esters, especially p-nitrophenyl acetate, for the first time.

Structural characterization of 1-deoxy-D-xylulose 5-phosphate Reductoisomerase from Vibrio vulnificus.

Vibrio vulnificus, a gram-negative bacterium, is the leading cause of seafood-borne illnesses and mortality in the United States. Previous studies have identified metabolites 2-C-methylerythritol 4-phosphate (MEP) as being essential for V. vulnificus growth and function. It was shown that 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr) is a critical enzyme in the viability of V. vulnificus, and many other bacteria, as it catalyzes the rearrangement of 1-deoxy-D-xylulose-5-phosphate (Dxp) to 2-C-methylerythritol 4-phosphate (MEP) within the MEP pathway, found in plants and bacteria. The MEP pathway produces the isoprenoids, isopentenyl diphosphate and dimethylallyl pyrophosphate. In this study, we produced and structurally characterized V. vulnificus Dxr. The enzyme forms a dimeric assembly and contains a metal ion in the active site. Protein produced in Escherichia coli co-purifies with Mg2+ ions, however the Mg2+ cations may be substituted with Mn2+, as both of these metals may be utilized by Dxrs. These findings will provide a basis for the design of Dxr inhibitors that may find application as antimicrobial compounds.

Structural basis of inactivation of Ras and Rap1 small GTPases by Ras/Rap1-specific endopeptidase from the sepsis-causing pathogen Vibrio vulnificus.

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site-coordinating core ß-sheet-containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.

Structural and Biochemical Characterization of the Curcumin-Reducing Activity of CurA from Vibrio vulnificus.

Curcumin is a yellow-colored ingredient in dietary spice turmeric ( Curcuma longa Linn). This nontoxic polyphenol has antitumor, anti-inflammatory, apoptotic, and antioxidant activities. The ingested curcumin is reduced to multihydrated forms with more potent therapeutic potentials by the curcumin reductase (CurA) from commensal Escherichia coli. In this study, we demonstrated that Vibrio vulnificus CurA ( VvCurA) with 87% sequence similarity to the E. coli CurA exhibits the curcumin-reducing activity through spectrophotometric detection of NADPH oxidation and high performance liquid chromatographic analysis of curcumin consumption and product generation. Afterward, we determined the crystal structures of VvCurA and the VvCurA/NADPH complex, and made the in silico model of the VvCurA/NADPH/curcumin ternary complex through induced fit docking. Based on structural information, active site residues that play critical roles in catalysis have been identified and characterized by mutational and kinetic studies, leading us to propose the reaction mechanism of CurA.

Biochemical characterization of protoporphyrinogen dehydrogenase and protoporphyrin ferrochelatase of Vibrio vulnificus and the critical complex formation between these enzymes.

BACKGROUND: Protoporphyrin IX (PPn), an intermediate in the heme biosynthesis reaction, generates singlet oxygen upon exposure to UV light. It has been proposed that PPn is channeled directly to ferrochelatase within a protoporphyrinogen dehydrogenase (PgdH1)-protoporphyrin ferrochelatase (PpfC) complex as a way to avoid this damaging side reaction. However, the PgdH1-PpfC complex has not been characterized, and the question of how heme affects the activities of PgdH1 has not been addressed. METHODS: Protein interactions were explored through pull-down assays and western blotting, and the importance of this complex in vivo was examined using inter-species combinations of the two proteins. The purified PgdH1-PpfC complex was characterized kinetically and used for heme binding studies. RESULTS: In Vibrio vulnificus, PgdH1 and PpfC formed an 8:8 heterohexadecameric complex that was important for maintaining PPn at low levels. PpfC catalyzed PPn efficiently whether or not it was part of the complex. Notably, heme was a noncompetitive inhibitor of V. vulnificus PgdH1, but a competitive inhibitor of the human protoporphyrinogen oxidase PgoX. CONCLUSION: The PdgH1-PpfC complex is important for protective channeling of PPn and for efficient catalysis of free PPn. The production of PPn by PgdH1 is regulated by feedback inhibition by heme. GENERAL SIGNIFICANCE: Both proteobacteria and eukaryotes have evolved mechanisms to prevent the harmful accumulation of the heme biosynthesis intermediate PPn. The data presented here suggest two previously unknown mechanisms: the channeling of PPn through the PgdH1-PpfC complex, and the direct inhibition of PgdH1 activity (PgoX activity as well) by heme.

Cyclo-(l-Phe-l-Pro), a Quorum-Sensing Signal of Vibrio vulnificus, Induces Expression of Hydroperoxidase through a ToxR-LeuO-HU-RpoS Signaling Pathway To Confer Resistance against Oxidative Stress.

Vibrio vulnificus, an opportunistic human pathogen, produces cyclo-(l-Phe-l-Pro) (cFP), which serves as a signaling molecule controlling the ToxR-dependent expression of innate bacterial genes, and also as a virulence factor eliciting pathogenic effects on human cells by enhancing intracellular reactive oxygen species levels. We found that cFP facilitated the protection of V. vulnificus against hydrogen peroxide. At a concentration of 1 mM, cFP enhanced the level of the transcriptional regulator RpoS, which in turn induced expression of katG, encoding hydroperoxidase I, an enzyme that detoxifies H2O2 to overcome oxidative stress. We found that cFP upregulated the transcription of the histone-like proteins vHUα and vHUß through the cFP-dependent regulator LeuO. LeuO binds directly to upstream regions of vhuA and vhuB to enhance transcription. vHUα and vHUß then enhance the level of RpoS posttranscriptionally by stabilizing the mRNA. This cFP-mediated ToxR-LeuO-vHUαß-RpoS pathway also upregulates genes known to be members of the RpoS regulon, suggesting that cFP acts as a cue for the signaling pathway responsible for both the RpoS and the LeuO regulons. Taken together, this study shows that cFP plays an important role as a virulence factor, as well as a signal for the protection of the cognate pathogen.

Functional analysis of N-terminal propeptide in the precursor of Vibrio vulnificus metalloprotease by using cell-free translational system.

Vibrio vulnificus is a human pathogen causing fatal septicemia with edematous and hemorrhagic skin damage. Among multiple virulence factors, an extracellular metalloprotease termed as V. vulnificus protease (VVP) is known to play a crucial role in eliciting the skin damage. The mature VVP (413 aa) is composed of two domains, the N-terminal core domain with proteolytic activity and the C-terminal domain mediates efficient attachment to protein substrates. However, VVP is produced as an inactive precursor (609 aa) with a signal peptide (24 aa) and propeptide (172 aa). In order to clarify the function of propeptide, a series of DNA fragments encoding the VVP precursor and its various domains were designed and the proteins were expressed in vitro by using cell-free translational system. The results indicated that the propeptide might function as an intramolecular chaperon to promote the proper folding of both N-terminal and C-terminal domains. The obtained results also suggest that the propeptide, itself was unstable and thus digested easily by the enzymes present in cell lysate used for cell-free system. Additionally, the C-terminal domain in VVP found to inhibit the folding of the N-terminal domain in absence of propeptide.

Repression of VvpM Protease Expression by Quorum Sensing and the cAMP-cAMP Receptor Protein Complex in Vibrio vulnificus.

Septicemia-causing Vibrio vulnificus produces at least three exoproteases, VvpE, VvpS, and VvpM, all of which participate in interactions with human cells. Expression of VvpE and VvpS is induced in the stationary phase by multiple transcription factors, including sigma factor S, SmcR, and the cAMP-cAMP receptor protein (cAMP-CRP) complex. Distinct roles of VvpM, such as induction of apoptosis, lead us to hypothesize VvpM expression is different from that of the other exoproteases. Its transcription, which was found to be independent of sigma S, is induced at the early exponential phase and then becomes negligible upon entry into the stationary phase. SmcR and CRP were studied regarding the control of vvpM expression. Transcription of vvpM was repressed by SmcR and cAMP-CRP complex individually, which specifically bound to the regions -2 to +20 and +6 to +27, respectively, relative to the vvpM transcription initiation site. Derepression of vvpM gene expression was 10- to 40-fold greater in an smcR crp double mutant than in single-gene mutants. Therefore, these results show that the expression of V. vulnificus exoproteases is differentially regulated, and in this way, distinct proteases can engage in specific interactions with a host.IMPORTANCE An opportunistic human pathogen, Vibrio vulnificus produces multiple extracellular proteases that are involved in diverse interactions with a host. The total exoproteolytic activity is detected mainly in the supernatants of the high-cell-density cultures. However, some proteolytic activity derived from a metalloprotease, VvpM, was present in the supernatants of the low-cell-density cultures sampled at the early growth period. In this study, we present the regulatory mechanism for VvpM expression via repression by at least two transcription factors. This type of transcriptional regulation is the exact opposite of those for expression of the other V. vulnificus exoproteases. Differential regulation of each exoprotease's production then facilitates the pathogen's participation in the distinct interactions with a host.

Identification and characterization of Vibrio vulnificus plpA encoding a phospholipase A2 essential for pathogenesis.

The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA Molecular biological analyses revealed that plpA expression is controlled via the promoter, P plpA , and that HlyU and CRP directly bind to P plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.

Biochemical characterization of a catalase from Vibrio vulnificus, a pathogen that causes gastroenteritis.

Vibrio vulnificus is a virulent human pathogen causing gastroenteritis and possibly life threatening septicemia in patients. Most V. vulnificus are catalase positive and can deactivate peroxides, thus allowing them to survive within the host. In the study presented here, a catalase from V. vulnificus (CAT-Vv) was purified to homogeneity after expression in Escherichia coli. The kinetics and function of CAT-Vv were examined. CAT-Vv catalyzed the reduction of H2O2 at an optimal pH of 7.5 and temperature of 35°C. The Vmax and Km values were 65.8±1.2 U/mg and 10.5±0.7 mM for H2O2, respectively. Mutational analysis suggests that amino acids involved in heme binding play a key role in the catalysis. Quantitative reverse transcription-PCR revealed that in V. vulnificus, transcription of CAT-Vv was upregulated by low salinity, heat, and oxidative stresses. This research gives new clues to help inhibit the growth of, and infection by V. vulnificus.

Substrate Recognition of MARTX Ras/Rap1-Specific Endopeptidase.

Ras/Rap1-specific endopeptidase (RRSP) is a cytotoxic effector domain of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of highly virulent strains of Vibrio vulnificus. RRSP blocks RAS-MAPK kinase signaling by cleaving Ras and Rap1 within the switch I region between Y32 and D33. Although the RRSP processing site is highly conserved among small GTPases, only Ras and Rap1 have been identified as proteolytic substrates. Here we report that residues Y32 and D33 at the scissile bond play an important role in RRSP substrate recognition, while the nucleotide state of Ras has an only minimal effect. In addition, substrate specificity is generated by residues across the entire switch I region. Indeed, swapping the Ras switch I region into either RalA or RhoA, GTPases that are not recognized by RRSP, generated chimeras that are substrates of RRSP. However, a difference in the processing efficiency of Ras switch I in the context of Ras, RalA, or RhoA indicates that protein regions outside Ras switch I also contribute to efficient RRSP substrate recognition. Moreover, we show that synthetic peptides corresponding to the Ras and Rap1, but not RalA, switch I regions are cleaved by RRSP, demonstrating sequence-specific substrate recognition. In conclusion, this work demonstrates that the GTPase recognition of RRSP is independent of the nucleotide state and is mainly driven by the Ras and Rap1 switch I loop and also influenced by additional protein-protein interactions, increasing the substrate specificity of RRSP.