The chitin-binding protein GbpA of Vibrio cholerae has been recently described as a common adherence factor for chitin and intestinal surface. Using an isogenic in-frame gbpA deletion mutant, we first show that V. cholerae O1 El Tor interacts with mouse intestinal mucus quickly, using GbpA in a specific manner. The gbpA mutant strain showed a significant decrease in intestinal adherence, leading to less colonization and fluid accumulation in a mouse in vivo model. Purified recombinant GbpA (rGbpA) specifically bound to N-acetyl-D-glucosamine residues of intestinal mucin in a dose-dependent, saturable manner with a dissociation constant of 11.2 M. Histopathology results from infected mouse intestine indicated that GbpA binding resulted in a time-dependent increase in mucus secretion. We found that rGbpA increased the production of intestinal secretory mucins (MUC2, MUC3, and MUC5AC) in HT-29 cells through upregulation of corresponding genes. The upregulation of MUC2 and MUC5AC genes was dependent on NF-B nuclear translocation. Interestingly, mucin could also increase GbpA expression in V. cholerae in a dose-dependent manner. Thus, we propose that there is a coordinated interaction between GbpA and mucin to upregulate each other in a cooperative manner, leading to increased levels of expression of both of these interactive factors and ultimately allowing successful intestinal colonization and pathogenesis by V. cholerae.Vibrio cholerae is the causative agent of the potentially lethal disease cholera. V. cholerae strains belonging to serogroups O1 and O139 are mainly responsible for cholera epidemics, while strains of other serogroups may cause sporadic outbreaks of the disease. Although the O139 strain has evolved recently, V. cholerae O1 biotype El Tor strains have still been responsible for most of the epidemics in recent years (20,26). In order to cause the disease, V. cholerae must adhere to the intestinal mucus barrier (52). The ability of V. cholerae to adhere to animal cells has been studied before (26,42), and various adherence factors have been proposed, including the virulence-associated toxin-coregulated pilus (5), outer membrane proteins (26, 42), and lipopolysaccharide (LPS) (11). Attachment of V. cholerae to abiotic surfaces has also been recently described (50). However, there is still no information about the factor(s) responsible for initial adherence of the bacteria to the intestine and whether the host plays any role in aiding the colonization of the intestine by the bacteria.Vibrios are marine organisms that adhere to chitin in the environment (12, 33) and utilize chitin as the sole source of nitrogen and carbon by using a family of glycosyl hydrolases, called chitinases (21). Genome analysis of V. cholerae O1 El Tor has revealed the presence of seven such chitinase genes (7), some of which have been characterized (27,37). One of these genes is the putative chitinase gene with locus number VCA0811, the product of which has been recently identified as a common adhesion molecule for both chitinou...

The microphthalmia-associated transcription factor Mitf plays a critical role in regulating many aspects of melanocyte biology. It is required for melanoblast and postnatal melanocyte survival, regulates proliferation, and activates genes associated with differentiation such as tyrosinase and related genes involved in melanogenesis. Identifying the signals that regulate Mitf expression is crucial if we are to understand how cells of the melanocyte lineage respond to environmental cues. Here we show that the Mitf promoter is induced by lipid signalling via the p38 stress-activated kinase pathway that is also activated by a wide range of receptors as well as UV irradiation. Signalling via p38 leads to increased phosphorylation and activation of cyclic adenosine monophosphate response element-binding (CREB) that binds and activates the Mitf promoter via the cyclic adenosine monophosphate (cAMP) response element. Moreover, we also show that activation of p38 mediated by lipids is potentiated by inhibition of the PI3kinase pathway but not by inhibition of protein kinase A (PKA). The results identify a mechanism in which stress signalling via p38 leads to activation of CREB, enhanced Mitf expression and consequently increased tyrosinase expression. The results are relevant for the regulation of melanocytes by Mitf, but also raise the possibility that lipid mediated activation of p38 signalling may represent a potential therapy for vitiligo.

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.Vibrio cholerae is known to be an autochthonous inhabitant of brackish waters and estuarine systems (4, 13). Among the 193 currently recognized O serogroups of V. cholerae (43), only serogroups O1 and O139 have caused epidemics of cholera. The other serogroups of V. cholerae, collectively referred to as non-O1 non-O139 serogroups, have not been associated with epidemics but can cause sporadic diarrhea (30) and are ubiquitously distributed in the aquatic environment (22,26). This sharp distinction between serogroups which can cause cholera and those which are not associated with cholera is related to the observation that more than 95% of the strains belonging to serogroups O1 and O139 produce cholera toxin (CT), which is central to the disease process. In contrast, more than 95% of the strains belonging to non-O1 non-O139 serogroups do not produce CT (15). Another important virulence factor of V. cholerae is the toxin-coregulated pilus (TCP), which is an adhesin that is coordinately regulated with CT production (39). TCP is the only V. cholerae pilus that has been demonstrated to date to have a role in colonization of the gut mucosa of humans (9) and of infant mice (39), the latter being an experimental cholera model.It has been presumed that CT and TCP are exclusively associated with clinical strains of V. cholerae, notably those belonging to serogroups O1 and O139, whereas reports on the incidence of CT among environmental strains of V. cholerae are rare (24). Sim...

SummaryRelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli, V. cholerae DrelA DspoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae. By repeated subculturing of a V. cholerae DrelA DspoT mutant, a suppressor strain with (p)ppGpp 0 phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene (VC1224), which codes for a small protein (~29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV. Using E. coli DrelA or DrelA DspoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (DrelA DspoT relV676), which seems to be responsible for the (p)ppGpp 0 phenotype. Analysis of a V. cholerae DrelA DspoT DrelV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.

The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA VCH ) was cloned and sequenced. The relA VCH gene encodes a 738-aminoacid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A ⌬relA::kan allele was generated by replacing ϳ31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.Vibrio cholerae is a facultative anaerobic gram-negative bacterium and the causative agent of the severe diarrheal disease cholera. In addition to residing temporarily in the intestinal lumen of humans during the diseased state, V. cholerae has its natural niche in the aquatic environment, residing in the freeliving aquatic flora found in estuarine areas and in association with crustaceans and mollusks (25). The strains of V. cholerae that cause epidemic cholera belong to serogroups O1 and O139 (3, 4, 28, 41, 50). The O1 serogroup is again divided into two biotypes, classical and El Tor (28). Strains other than O1 and O139 are known as non-O1/non-O139 vibrios.A pathogen in its natural environment and host-associated state is subjected to a plethora of stresses, such as fluctuations in pH, salinity, osmolarity, oxygen tension, temperature, and nutritional availability. These offer selective pressure to a bacterium, eliciting various adaptive responses for its survival. The adaptive response to nutritional stre...