The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE. We previously demonstrated that vvpE expression is differentially directed by two different promoters in a growth phase-dependent manner. The activity of the stationaryphase promoter (promoter S (PS)) is dependent on RpoS and is also under the positive control of cyclic AMP receptor protein (CRP). In this study, primer extension analyses revealed that SmcR, the Vibrio harveyi LuxR homolog, is also involved in the regulation of vvpE transcription by activating PS. Although the influence of CRP on PS is mediated by SmcR, the level of PS activity observed when CRP and SmcR function together was found to be greater than the sum of the PS activities achieved by each activator alone. Western blot analyses demonstrated that the cellular levels of RpoS, CRP, and SmcR were not significantly affected by one other, indicating that CRP and SmcR function cooperatively to activate PS rather than sequentially in a regulatory cascade. The binding sites for CRP and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro DNase I protection assays. The binding sites for CRP and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of PS, respectively. Accordingly, these results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoSdependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase.The pathogenic marine bacterium Vibrio vulnificus is the causative agent of food-borne diseases, including life-threatening septicemia and possibly gastroenteritis, in individuals with underlying predisposed conditions such as liver damage, excess levels of iron, and immunocompromised conditions. Wound infections also result from exposure to seawater or from the handling of shellfish contaminated with V. vulnificus. The mortality from septicemia is very high (Ͼ50%), and death can occur within 1-2 days after the first signs of illness. Several potential virulence factors, including an endotoxin, polysaccharide capsule, iron-sequestering systems, cytolytic hemolysin, elastase, phospholipase A 2 , and other exotoxins, have been identified in V. vulnificus (for recent reviews, see Refs. 1 and 2).The elastase activity is from a neutral metalloprotease and represents the major proteolytic activity of V. vulnificus (3, 4). Our previous study revealed the existence of at least two proteases that are produced by V. vulnificus (5). Therefore, vvpE was designed to differentiate the elastase gene from other genes encoding other potential proteases of V. vulnificus (5). As such, a gene encoding the V. vulnificus elastase was recently cloned and sequenced by us (5) and by others (6). The deduced gene product was predicted to be a 609-amino acid polypeptide, and the mature elastase is a 45-kDa protein consisting of 413 amino acids generated by the deletion of the N-termi...

Since both myocardium and vasculature in the heart are excessively damaged following myocardial infarction (MI), therapeutic strategies for treating MI hearts should concurrently target both so as to achieve true cardiac repair. Here we demonstrate a concomitant method that exploits the advantages of cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) and human mesenchymal stem cell-loaded patch (hMSC-PA) to amplify cardiac repair in a rat MI model. Epicardially implanted hMSC-PA provide a complimentary microenvironment which enhances vascular regeneration through prolonged secretion of paracrine factors, but more importantly it significantly improves the retention and engraftment of intramyocardially injected hiPSC-CMs which ultimately restore the cardiac function. Notably, the majority of injected hiPSC-CMs display adult CMs like morphology suggesting that the secretomic milieu of hMSC-PA constitutes pleiotropic effects in vivo. We provide compelling evidence that this dual approach can be a promising means to enhance cardiac repair on MI hearts.

The extracellular tissue penetrating protozoan parasite Entamoeba histolytica has been known to induce host cell apoptosis. However, the intracellular signaling mechanism used by the parasite to trigger apoptosis is poorly understood. In this study, we investigated the roles of reactive oxygen species (ROS), and of MAPKs in the Entamoeba-induced apoptosis of human neutrophils. The neutrophils incubated with live trophozoites of E. histolytica revealed a marked increase of receptor shedding of CD16 as well as phosphatidylserine (PS) externalization on the cell surface. The Entamoeba-induced apoptosis was effectively blocked by pretreatment of cells with diphenyleneiodonium chloride (DPI), a flavoprotein inhibitor of NADPH oxidase. A large amount of intracellular ROS was detected after exposure to viable trophozoites, and the treatment with DPI strongly inhibited the Entamoeba-induced ROS generation. However, a mitochondrial inhibitor rotenone did not attenuate the Entamoeba-induced ROS generation and apoptosis. Although E. histolytica strongly induced activation of ERK1/2 and p38 MAPK in neutrophils, the activation of ERK1/2 was closely associated with ROS-mediated apoptosis. Pretreatment of neutrophils with MEK1 inhibitor PD98059, but not p38 MAPK inhibitor SB202190, prevented Entamoeba-induced apoptosis. Moreover, DPI almost completely inhibited Entamoeba-induced phosphorylation of ERK1/2, but not phosphorylation of p38 MAPK. These results strongly suggest that NADPH oxidase-derived ROS-mediated activation of ERK1/2 is required for the Entamoeba-induced neutrophil apoptosis.

To assess the role of the flagellum which was detected by immunoscreening of surface proteins of Vibrio vulnificus, an flgE-deleted mutant was constructed and tested for its pathogenicity. The ability of this nonmotile mutant to adhere to INT-407 cells and its role in biofilm were decreased, as was its lethality to mice.Vibrio vulnificus is a gram-negative bacterium that causes gastroenteritis and primary septicemia, especially in immunocompromised humans (11). Several virulence factors have been discovered in V. vulnificus, including the expression of lipopolysaccharide (1), capsular polysaccharide (21), elastase (6), and a phospholipase A 2 (20), as well as iron availability (22). Motility could be proposed to be another virulence determinant in addition to the aforementioned factors (3). V. vulnificus is a highly motile organism by virtue of a polar flagellum, as are the closely related vibrios. In V. cholerae, nonmotile mutants have been shown to accumulate less fluid in rabbit ligated ileal loops (17). Recently, a V. vulnificus mutant showing a decreased cytotoxicity to HeLa cells was found to have a transposon insertion at the flgC gene encoding a flagellar basal body (8).We performed an experiment to identify bacterial surface molecules, which are required for the initiation of pathogenic interactions of V. vulnificus with a host. From an extensive screening process, a clone containing the flgDEF operon which encodes the components of the flagellum was obtained. Having constructed a knockout mutant of the flgE gene, we made a flagellum-deficient V. vulnificus mutant, and we then went on to investigate the role of flagellum-derived motility in the virulence of this pathogen to host cells.Isolation of the flgDEF clone from immunoscreening of surface proteins of V. vulnificus. The strains and plasmids used in this study are listed in Table 1. To prepare whole-cell lysate, exponential-phase V. vulnificus ATCC 29307 was resuspended in 10 mM Tris-HCl (pH 7.4) and disrupted with an ultrasonic liquid processor (model XL2020 sonicator; Misonix). After ultracentrifugation (100,000 ϫ g) for 1 h at 4°C, the pellet was added to 0.1% sodium lauryl sarkosinate in 7 mM EDTA. A sarkosyl-insoluble fraction (140 g) was used for three consecutive immunizations of a rabbit. Ten days after the last injection, the blood of the immunized rabbit was collected and used for immunoscreening of the ZAPII-based expression library of V. vulnificus.Approximately 20,000 plaques from the expression library were screened for clones interacting with the anti-surface protein serum described above. Five of the 11 candidate plaques showed reproducible immune reactions with the serum during further purification steps. The plasmids derived from the excision of these five clones, pBKH1 to pBKH5, were found to contain the identical DNA fragment; therefore, pBKH1 was used for further studies.Restriction analysis of pBKH1 by using BamHI showed that it had an insert of 2.7 kb (Fig. 1A). The DNA insert of pBKH1 was found to contain a partial sequence ...

SummaryVibrio vulnificus has been shown to require a global transcription factor, NtrC for mature biofilm development via controlling the biosyntheses of lipopolysaccharide and exopolysaccharide (EPS). Biofilm formation and EPS production were dramatically increased in a medium including a tricarboxylic acid cycle-intermediate as a carbon source. These phenotypes required functional NtrC and were abolished by the addition of ammonium chloride. During the initial stage of biofilm formation, both expression of the ntrC gene and the cellular content of NtrC protein increased. Thus, the regulatory roles of NtrC in EPS biosynthesis were studied with three gene clusters for EPS biosyntheses. Transcriptions of the three clusters were positively controlled by NtrC and showed maximal expression at the early stage of biofilm development. Mutants deficient in one of the genes (VV1_2661, VV2_1579 and VV1_2305) in each cluster showed decreased production of EPS, attenuated ability to form biofilm and lowered cytoadherence to human epithelial cells. However, mutations in VV2_1579 and VV1_2305 resulted in lower cytotoxicity to human cells and mortality to mice than the mutation in VV1_2661. These results demonstrate that NtrC-regulated EPS are crucial in biofilm formation of V. vulnificus, and some EPS components play important roles in interacting with hosts.