Molecular Analysis of the Vibrio vulnificus Genes Encoding N-acetylglucosamine Binding Protein and Phospholipase A2 Induced by Mucin

Abstract

Mucin is a glycoprotein composed of a polypeptide backbone with branched oligosaccharide side chains and a major component of mucus layer that serves as the first line of defense against many enteric pathogens. To survive and cause disease, the pathogens interact with mucin and change their transcriptomic profile to adapt to the host environment. In this study, to better understand the bacteriums interactions to the mucus layer, transcriptional response of Vibrio vulnificus to the mucin and the mucin-secreting HT29-methotrexate (MTX) cells was investigated. The growth and survival of V. vulnificus cells exposed to M9 medium supplemented with 0.6% (wt/vol) mucin (M9M) and the HT29-MTX cells were monitored, respectively. To monitor the levels of all transcripts in the bacteria grown in the M9 medium supplemented with 0.4% (wt/vol) glucose (M9G) and M9M, RNA-seq technology was used. Also, transcriptomes of the bacteria exposed to basal medium eagle (BME) and the HT29-MTX cells was analyzed using the same technology. From the analysis, 337 genes were identified to be differentially expressed with significance between M9G and M9M. In addition, the analysis and comparison of RNA-seq data identified 650 genes with altered transcript level in the bacteria exposed to the HT29-MTX cells compared to BME. Furthermore, several virulence related genes encoding metalloprotease, N-acetylglucosamine binding protein, cytolysin, and phospholipase were induced by the mucin and the mucin-secreting host cells.

Among the genes whose expression was up-regulated when V. vulnificus cells were grown in M9M, a gene encoding an N-acetylglucosamine binding protein GbpA, a homologue of Vibrio cholerae GbpA, was selected and further studied. A mutational analysis demonstrated that GbpA contributes to the ability of adherence to the HT29-MTX cells as well as the mucin. Furthermore, compared with the wild type, the gbpA mutant exhibited reduced intestinal colonization and virulence in mice. The gbpA transcription was growth-phase dependent, reaching a maximum during the exponential phase. The Fe-S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated whereas SmcR, a LuxR homologue, repressed gbpA expression at the transcriptional level. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. Primer extension analysis revealed that the transcription of gbpA begins at a single site. Direct bindings of IscR, SmcR, and CRP to PgbpA were demonstrated by EMSA. The binding sites for the regulator proteins were mapped based on a deletion analysis of the PgbpA and confirmed by DNase I protection assays. Interestingly, gbpA was induced by exposure to H2O2, and the induction appeared to be mediated by elevated intracellular levels of IscR. The combined results proposed a model in which IscR, CRP, and SmcR cooperate for precise control of the gbpA expression during infection.

Among the V. vulnificus genes specifically induced by exposure to the mucin and the mucin-secreting host cells, a gene, annotated as plp encoding a putative phospholipase Plp, was identified and further characterized. The amino acid sequences of V. vulnificus Plp (VvPlp) were 67% identical to those of Vibrio anguillarum phospholipase (VaPlp). To examine the role of VvPlp, a mutant with disruption of the plp gene was constructed by allelic exchanges, and its virulence was evaluated. Compared with the wild type, the plp mutant showed a low level of cytotoxicity toward the HT29-MTX cells and reduced virulence in mice. Genetic and biochemical analyses using the recombinant Plp protein demonstrated that Plp is a secreted phospholipase A2 essential for pathogenesis of V. vulnificus. Examination of global regulatory proteins on the expression of plp revealed that the transcription activator HlyU and CRP upregulate the plp expression. The cellular levels of HlyU and CRP were not significantly affected by one another, indicating that the regulator proteins function cooperatively to activate plp rather than sequentially in a regulatory cascade. The regulatory proteins directly bound to the upstream of the plp promoter Pplp. DNase I protection assays, together with the deletion analyses of Pplp, demonstrated that HlyU binds three distinct sequences centered at -174, -139.5, and -109.5 and CRP binds specifically to the sequences centered at -69.5 relative to the transcription start site of Pplp. Consequently, the combined results indicated that V. vulnificus plp encodes a phospholipase A2 essential for virulence and is cooperatively activated by HlyU and CRP.