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.

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Bacterial cancer therapy relies on the fact that several bacterial species are capable of targeting tumor tissue and that bacteria can be genetically engineered to selectively deliver therapeutic proteins of interest to the targeted tumors. However, the challenge of bacterial cancer therapy is the release of the therapeutic proteins from the bacteria and entry of the proteins into tumor cells. This study employed an attenuated Salmonella typhimurium to selectively deliver the mitochondrial targeting domain of Noxa (MTD) as a potential therapeutic cargo protein, and examined its anti-cancer effect. To release MTD from the bacteria, a novel bacterial lysis system of phage origin was deployed. To facilitate the entry of MTD into the tumor cells, the MTD was fused to DS4.3, a novel cell-penetrating peptide (CPP) derived from a voltage-gated potassium channel (Kv2.1). The gene encoding DS4.3-MTD and the phage lysis genes were placed under the control of PBAD, a promoter activated by L-arabinose. We demonstrated that DS4.3-MTD chimeric molecules expressed by the Salmonellae were anti-tumoral in cultured tumor cells and in mice with CT26 colon carcinoma.

dVibrio vulnificus is a halophilic pathogenic bacterium that is motile due to the presence of a single polar flagellum. V. vulnificus possesses a total of six flagellin genes organized into two loci (flaFBA and flaCDE). We proved that all six of the flagellin genes were transcribed, whereas only five (FlaA, -B, -C, -D, and -F) of the six flagellin proteins were detected. To understand roles of the six V. vulnificus flagellins in motility and virulence, mutants with single and multiple flagellin deletions were constructed. Mutations in flaB or flaC or the flaCDE locus resulted in a significant decrease in motility, adhesion, and cytotoxicity, whereas single mutations in the other flagellin genes or the flaFBA locus showed little or no effect. The motility was completely abolished only in the mutant lacking all six flagellin genes (flaFBA flaCDE). Surprisingly, a double mutation of flaB and flaD, a gene sharing 99% identity with the flaB at the amino acid level, resulted in the largest decrease in motility, adhesion, and cytotoxicity except for the mutant in which all six genes were deleted (the hexa mutant). Additionally, the 50% lethal doses (LD 50 s) of the flaB flaD and the flaFBA flaCDE mutants increased 23-and 91-fold in a mouse model, respectively, and the in vitro and in vivo invasiveness of the mutants was significantly decreased compared to that of the wild type. Taken together, the multiple flagellin subunits differentially contribute to the flagellum biogenesis and the pathogenesis of V. vulnificus, and among the six flagellin genes, flaB, flaD, and flaC were the most influential components.

Chronic periodontitis is caused by interactions between the oral polymicrobial community and host factors. Periodontal diseases are associated with dysbiotic shift in oral microbiota. Vaccination against periodontopathic bacteria could be a fundamental therapeutic to modulate polymicrobial biofilms. Because oral cavity is the site of periodontopathic bacterial colonization, mucosal vaccines should provide better protection than vaccines administered systemically. We previously reported that bacterial flagellin is an excellent mucosal adjuvant. In this study, we investigated whether mucosal immunization with a flagellin-adjuvanted polypeptide vaccine induces protective immune responses using a Porphyromonas gingivalis infection model. We used the Hgp44 domain polypeptide of Arg-gingipain A (RgpA) as a mucosal antigen. Intranasal (IN) immunization induced a significantly higher Hgp44-specific IgG titer in the serum of mice than sublingual (SL) administration. The co-administration of flagellin potentiated serum IgG responses for both the IN and SL vaccinations. On the other hand, the anti-Hgp44-specific IgA titer in the saliva was comparable between IN and SL vaccinations, suggesting SL administration as more compliant vaccination route for periodontal vaccines. The co-administration of flagellin significantly potentiated the secretory IgA response in saliva also. Furthermore, mice administered a mixture of Hgp44 and flagellin via the IN and SL routes exhibited significant reductions in alveolar bone loss induced by live P. gingivalis infections. An intranasally administered Hgp44-flagellin fusion protein induced a comparable level of Hgp44-specific antibody responses to the mixture of Hgp44 and flagellin. Overall, a flagellin-adjuvanted Hgp44 antigen would serve an important component for a multivalent mucosal vaccine against polymicrobial periodontitis.