Effect on human cells of environmental Vibrio parahaemolyticus strains carrying type III secretion system 2.

Vibrio parahaemolyticus is an inhabitant of estuarine and marine environments that causes seafood-borne gastroenteritis worldwide. Recently, a type 3 secretion system (T3SS2) able to secrete and translocate virulence factors into the eukaryotic cell has been identified in a pathogenicity island (VP-PAI) located on the smaller chromosome. These virulence-related genes have previously been detected only in clinical strains. Classical virulence genes for this species (tdh, trh) are rarely detected in environmental strains, which are usually considered to lack virulence potential. However, during screening of a collection of environmental V. parahaemolyticus isolates obtained in the North Adriatic Sea in Italy, a number of marine strains carrying virulence-related genes, including genes involved in the T3SS2, were detected. In this study, we investigated the pathogenic potential of these marine V. parahaemolyticus strains by studying their adherence ability, their cytotoxicity, their effect on zonula occludin protein 1 (ZO-1) of the tight junctions, and their effect on transepithelial resistance (TER) in infected Caco-2 cells. By performing a reverse transcription-PCR, we also tested the expression of the T3SS2 genes vopT and vopB2, encoding an effector and a translocon protein, respectively. Our results indicate that, similarly to clinical strains, marine V. parahaemolyticus strains carrying vopT and vopB2 and that other genes included in the VP-PAI are capable of adhering to human cells and of causing cytoskeletal disruption and loss of membrane integrity in infected cells. On the basis of data presented here, environmental V. parahaemolyticus strains should be included in coastal water surveillance plans, as they may represent a risk for human health.

Development of a live oral attaching and effacing Escherichia coli vaccine candidate using Vibrio cholerae CVD 103-HgR as antigen vector.

Attaching and effacing Escherichia coli (AEEC) share the ability to induce pedestal formation and intimate adherence of the bacteria to the intestinal epithelial cell and effacement of microvilli of epithelial tissue. The Locus of Enterocyte Effacement (LEE) pathogenicity island encodes the ability to induce attaching and effacing (A/E) lesions and contains the gene eae, which encodes intimin, an outer membrane protein that is an adhesin for A/E lesion formation. Here we show the utility of using intimin as a vaccine to protect rabbits from challenge with rabbit Enteropathogenic E. coli (REPEC), a member of the AEEC family. The C-terminal portion of intimin was delivered by the attenuated Vibrio cholerae vaccine strain CVD 103-HgR. To export intimin, a fusion was engineered with ClyA, a secreted protein from Salmonella enterica serovar Typhi. After immunization, antibodies specific to intimin from serum and bile samples were detected and moderate protection against challenge with a virulent REPEC strain was observed. Compared to animals immunized with vector alone, intimin-immunized rabbits exhibited reduced fecal bacterial shedding, milder diarrheal symptoms, lower weight loss, and reduced colonization of REPEC in the cecum. V. cholerae CVD 103-HgR shows promise as a vector to deliver antigens and confer protection against AEEC pathogens.

An in vivo expression technology screen for Vibrio cholerae genes expressed in human volunteers.

In vivo expression technology (IVET) has been widely used to study gene expression of human bacterial pathogens in animal models, but has heretofore not been used in humans to our knowledge. As part of ongoing efforts to understand Vibrio cholerae pathogenesis and develop improved V. cholerae vaccines, we have performed an IVET screen in humans for genes that are preferentially expressed by V. cholerae during infection. A library of 8,734 nontoxigenic V. cholerae strains carrying transcriptional fusions of genomic DNA to a resolvase gene was ingested by five healthy adult volunteers. Transcription of the fusion leads to resolvase-dependent excision of a sacB-containing cassette and thus the selectable phenotype of sucrose resistance (Suc(R)). A total of approximately 20,000 Suc(R) isolates, those carrying putative in vivo-induced fusions, were recovered from volunteer stool samples. Analysis of the fusion junctions from >7,000 Suc(R) isolates from multiple samples from multiple volunteers identified 217 candidate genes for preferential expression during human infection. Of genes or operons induced in three or more volunteers, the majority of those tested (65%) were induced in an infant mouse model. VC0201 (fhuC), which encodes the ATPase of a ferrichrome ABC transporter, is one of the identified in vivo-induced genes and is required for virulence in the mouse model.

Capsular polysaccharide phase variation in Vibrio vulnificus.

Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.