Joon Haeng Rhee

Generation of regulatory dendritic cells and CD4+Foxp3+ T cells by probiotics administration suppresses immune disorders.

The beneficial effects of probiotics have been described in many diseases, but the mechanism by which they modulate the immune system is poorly understood. In this study, we identified a mixture of probiotics that up-regulates CD4+Foxp3+ regulatory T cells (Tregs). Administration of the probiotics mixture induced both T-cell and B-cell hyporesponsiveness and down-regulated T helper (Th) 1, Th2, and Th17 cytokines without apoptosis induction. It also induced generation of CD4+Foxp3+ Tregs from the CD4+CD25− population and increased the suppressor activity of naturally occurring CD4+CD25+ Tregs. Conversion of T cells into Foxp3+ Tregs is directly mediated by regulatory dendritic cells (rDCs) that express high levels of IL-10, TGF-β, COX-2, and indoleamine 2,3-dioxygenase. Administration of probiotics had therapeutical effects in experimental inflammatory bowel disease, atopic dermatitis, and rheumatoid arthritis. The therapeutical effect of the probiotics is associated with enrichment of CD4+Foxp3+ Tregs in the inflamed regions. Collectively, the administration of probiotics that enhance the generation of rDCs and Tregs represents an applicable treatment of inflammatory immune disorders.

Molecular characterization of vulnibactin biosynthesis in Vibrio vulnificus indicates the existence of an alternative siderophore

Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicemia and necrotizing wound infections in humans. Virulent V. vulnificus isolates produce a catechol siderophore called vulnibactin, made up of one residue of 2, 3-dihydroxybenzoic acid (2, 3-DHBA) and two residues of salicylic acid (SA). Vulnibactin biosynthetic genes (VV2_0828 to VV2_0844) are clustered at one locus of chromosome 2, expression of which is significantly up-regulated in vivo. In the present study, we decipher the biosynthetic network of vulnibactin, focusing specifically on genes around SA and 2, 3-DHBA biosynthetic steps. Deletion mutant of isochorismate pyruvate lyase (VV2_0839) or 2, 3-dihydroxybenzoate-2, 3-dehydrogenase (VV2_0834) showed retarded growth under iron-limited conditions though the latter showed more significant growth defect than the former, suggesting a dominant role of 2, 3-DHBA in the vulnibactin biosynthesis. A double deletion mutant of VV2_0839 and VV2_0834 manifested additional growth defect under iron limitation. Though the growth defect of respective single deletion mutants could be restored by exogenous SA or 2, 3-DHBA, only 2, 3-DHBA could rescue the double mutant when supplied alone. However, double mutant could be rescued with SA only when hydrogen peroxide was supplied exogenously, suggesting a chemical conversion of SA to 2, 3-DHBA. Assembly of two SA and one 2, 3-DHBA into vulnibactin was mediated by two AMP ligase genes (VV2_0836 and VV2_0840). VV2_0836 deletion mutant showed more significant growth defect under iron limitation, suggesting its dominant function. In conclusion, using molecular genetic analytical tools, we confirm that vulnibactin is assembled of both 2, 3-DHBA and SA. However, conversion of SA to 2, 3-DHBA in presence of hydrogen peroxide and growth profile of AMP ligase mutants suggest a plausible existence of yet unidentified alternative siderophore that may be composed solely of 2, 3-DHBA.

Characterization and Pathogenic Significance of Vibrio vulnificus Antigens Preferentially Expressed in Septicemic Patients

ABSTRACT Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.

Regulation of Vibrio vulnificus virulence by the LuxS quorum-sensing system

Vibrio vulnificus is a halophilic estuarine bacterium that causes fatal septicaemia and necrotizing wound infections. We tested whether V. vulnificus produces signalling molecules (autoinducer 1 and/or 2) stimulating Vibrio harveyi quorum‐sensing system 1 and/or 2. Although there was no evidence for signalling system 1, we found that V. vulnificus produced a signalling activity in the culture supernatant that induced luminescence expression in V. harveyi through signalling system 2. Maximal autoinducer 2 (AI‐2) activity was observed during mid‐exponential to early stationary phase and disappeared in the late stationary phase when V. vulnificus was grown in heart infusion broth containing 2.5% NaCl. V. vulnificus showed increased signalling activity when it was cultured in the presence of glucose (0.5%) and at low pH (pH 6.0). From a cosmid library of V. vulnificus type strain ATCC 29307, we have identified the AI‐2 synthase gene (luxSVv) showing 80% identity with that of V. harveyi (luxSVh) at the amino acid level. To investigate the pathogenic role of luxSVv, a deletion mutant of the clinical isolate V. vulnificus MO6‐24/O was constructed. The luxSVv mutant showed a significant delay in protease production and an increase in haemolysin production. The decreased protease and increased haemolysin activities were restored to the isogenic wild‐type level by complementation with the wild‐type luxSVv allele. The change in phenotypes was also complemented by logarithmic phase spent media produced by the wild‐type bacteria. Transcriptional activities of the haemolysin gene (vvhA) and protease gene (vvpE) were also observed in the mutant using chromosomal PvvhA::lacZ and PvvpE::lacZ transcriptional reporter constructs: transcription of vvhA was increased and of vvpE decreased by the mutation. The mutation resulted in an attenuation of lethality to mice. Intraperitoneal LD50 of the luxSVv mutant increased by 10‐ and 750‐fold in ferric ammonium citrate‐non‐overloaded and ferric ammonium citrate‐overloaded mice respectively. The time required for the death of mice was also significantly delayed in the luxSVv mutant. Cytotoxic activity of the organism against HeLa cells, measured by lactate dehydrogenase (LDH) release assay, was also decreased significantly by the mutation. Taken together, the V. vulnificus LuxS quorum‐sensing system seems to play an important role in co‐ordinating the expression of virulence factors.

A bacterial flagellin, Vibrio vulnificus FlaB, has a strong mucosal adjuvant activity to induce protective immunity

ABSTRACT Flagellin, the structural component of flagellar filament in various locomotive bacteria, is the ligand for Toll-like receptor 5 (TLR5) of host cells. TLR stimulation by various pathogen-associated molecular patterns leads to activation of innate and subsequent adaptive immune responses. Therefore, TLR ligands are considered attractive adjuvant candidates in vaccine development. In this study, we show the highly potent mucosal adjuvant activity of a Vibrio vulnificus major flagellin (FlaB). Using an intranasal immunization mouse model, we observed that coadministration of the flagellin with tetanus toxoid (TT) induced significantly enhanced TT-specific immunoglobulin A (IgA) responses in both mucosal and systemic compartments and IgG responses in the systemic compartment. The mice immunized with TT plus FlaB were completely protected from systemic challenge with a 200× minimum lethal dose of tetanus toxin. Radiolabeled FlaB administered into the nasal cavity readily reached the cervical lymph nodes and systemic circulation. FlaB bound directly to human TLR5 expressed on cultured epithelial cells and consequently induced NF-κB and interleukin-8 activation. Intranasally administered FlaB colocalized with CD11c as patches in putative dendritic cells and caused an increase in the number of TLR5-expressing cells in cervical lymph nodes. These results indicate that flagellin would serve as an efficacious mucosal adjuvant inducing protective immune responses through TLR5 activation.

Vibrio vulnificus RTX toxin kills host cells only after contact of the bacteria with host cells.

Vibrio vulnificus causes acute cell death and a fatal septicaemia. In this study, we show that contact with host cells is a prerequisite to the acute cytotoxicity. We screened transposon mutants defective in the contact‐dependent cytotoxicity. Two mutants had insertions within two open reading frames in a putative RTX toxin operon, the rtxA1 or rtxD encoding an RTX toxin (4701 amino acids) or an ABC type transporter (467 amino acids). An rtxA1 mutation resulted in a cytotoxicity defect, which was fully restored by in trans complementation. The expression of RtxA1 toxin increased after host cell contact in a time‐dependent manner. The RtxA1 toxin induced cytoskeletal rearrangements and plasma membrane blebs, which culminated in a necrotic cell death. RtxA1 colocalized with actin and caused actin aggregation coinciding with a significant decrease in the F/G actin ratio. The RtxA1 toxin caused haemolysis through pore formation (radius 1.63 nm). The rtxA1 deletion mutant was defective in invading the blood stream from ligated ileal loops of CD1 mice. The rtxA1 null mutation resulted in over 100‐fold increase in both intragastric and intraperitoneal LD50s against mice. Overall, these results show that the RtxA1 toxin is a multifunctional cytotoxin and plays an essential role in the pathogenesis of V. vulnificus infections.

Integrative genome-scale metabolic analysis of Vibrio vulnificus for drug targeting and discovery

Although the genomes of many microbial pathogens have been studied to help identify effective drug targets and novel drugs, such efforts have not yet reached full fruition. In this study, we report a systems biological approach that efficiently utilizes genomic information for drug targeting and discovery, and apply this approach to the opportunistic pathogen Vibrio vulnificus CMCP6. First, we partially re‐sequenced and fully re‐annotated the V. vulnificus CMCP6 genome, and accordingly reconstructed its genome‐scale metabolic network, VvuMBEL943. The validated network model was employed to systematically predict drug targets using the concept of metabolite essentiality, along with additional filtering criteria. Target genes encoding enzymes that interact with the five essential metabolites finally selected were experimentally validated. These five essential metabolites are critical to the survival of the cell, and hence were used to guide the cost‐effective selection of chemical analogs, which were then screened for antimicrobial activity in a whole‐cell assay. This approach is expected to help fill the existing gap between genomics and drug discovery.

Construction and Phenotypic Evaluation of a Vibrio vulnificus vvpE Mutant for Elastolytic Protease

ABSTRACT Vibrio vulnificus is an opportunistic gram-negative pathogen that commonly contaminates oysters. Predisposed individuals who consume raw oysters can die within days from sepsis, and even otherwise healthy people are susceptible to serious wound infection after contact with contaminated seafood or seawater. Numerous secreted and cell-associated virulence factors have been proposed to account for the fulminating and destructive nature of V. vulnificusinfections. Among the putative virulence factors is an elastolytic metalloprotease. We cloned and sequenced the vvpE gene encoding an elastase of V. vulnificus ATCC 29307. The functions of the elastase were assessed by constructingvvpE insertional knockout mutants and evaluating phenotypic changes in vitro and in mice. Although other types of protease activity were still observed in vvpE mutants, elastase activity was completely absent in the mutants and was restored by reintroducing the recombinant vvpE gene. In contrast to previous characterization of elastase as a potential virulence factor, which was demonstrated by injecting the purified protein into animals, inactivation of the V. vulnificus vvpE gene did not affect the ability of the bacteria to infect mice and cause damage, either locally in subcutaneous tissues or systemically in the liver, in both iron-treated and normal mice. Furthermore, a vvpE mutant was not affected with regard to cytolytic activity toward INT407 epithelial cells or detachment of INT407 cells from culture dishes in vitro. Therefore, it appears that elastase is less important in the pathogenesis of V. vulnificus than would have been predicted by examining the effects of administering purified proteins to animals. However, V. vulnificus utilizes a variety of virulence factors; hence, the effects of inactivation of elastase alone could be masked by other compensatory virulence factors.

CAS agar diffusion assay for the measurement of siderophores in biological fluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.

Flagellar basal body flg operon as a virulence determinant of Vibrio vulnificus

Vibrio vulnificus, a halophilic estuarine bacterium causing a rapidly progressing fatal septicemia, is highly cytotoxic to eukaryotic cells. To identify new virulence factors associated with cytotoxicity, we constructed a mariner-based transposon (Tn Himar1) library of the highly virulent clinical isolate MO6-24/O having a double mutation in the hemolysin and protease genes. The Himar1 mutant library was extensively screened for the mutants showing decreased cytotoxicity to HeLa cells. We selected a cytotoxicity defective mutant having a Himar1 insertion in an open reading frame showing 96% identity to Vibrio parahaemolyticus FlgC, a flagella basal body rod protein. The Tn Himar1 insertion mutation also resulted in a significant decrease in motility, adhesion, cytotoxicity, and lethality to mice. This is the first report showing that flg genes, which are components of the flagellum biogenesis gene cluster, might play an important role in the virulence of V. vulnificus.