Degree
Doctor of Philosophy (PhD)
Department
Biological Sciences
Document Type
Dissertation
Abstract
Biofilms produced by the cholera pathogen Vibrio cholerae not only enhance its ability to survive in the environment but also allow pathogenic success of the bacterium. This organism also undergoes phase variation, switching between smooth and rugose colonial phenotypes, the latter forming much more vibrio polysaccharide (VPS) resulting in more robust biofilms and subsequently having an increased level of persistence. In a previous study, many genes were found to be upregulated in the transition from the smooth to the rugose phenotype. Here, several of these upregulated genes were investigated for their potential role in biofilm production, and an additional gene, VC1345, which encodes a key factor in the tyrosine catabolism pathway, was also studied. We mutated each of the genes individually in a rugose parent and characterized mutants, where applicable, for colonial morphology, pyomelanin pigment production, acetate assimilation and biofilm production at multiple timepoints. We found evidence that the tyrosine catabolism pathway and probably acetate assimilation contribute to biofilm production and maintenance in this species. The end products of both pathways would be funneled through gluconeogenesis, which would ultimately contribute to VPS production.
Vibrio vulnificus, is a contaminant of raw shellfish that can cause fatal septicemia, necrotizing fasciitis or gastroenteritis in the human host. The bacterium produces capsular polysaccharide (CPS), which is a major virulence factor since it provides protection against host immune responses. This organism also undergoes phase variation between opaque (Op) and translucent (Tr) colony morphotypes. CPS is produced by virulent Op strains and is only marginally produced or is absent in Tr isolates resulting in their decreased virulence. Previously it has been shown that some Tr variants are “phase locked” due a deletion of one of the CPS transport genes, wzb, which is located in the Group I CPS operon. However, another translucent variant (TR1) possesses an intact wzb gene as well as the ability to switch again to an Op phenotype. To characterize reversible phase variation in V. vulnificus in more detail, we isolated four independent lineages each consisting of the Op parent strain MO6-24/O, a Tr1 derived from the Op parent, and another Op isolate derived from the Tr1, which was denoted as the opaque derivative (OpD). Lineage members were subjected to quantification of their CPS, and assessment of their ability to undergo phenotypic switching. In an effort to elucidate an underlying mechanism of reversible phase variation, we investigated the potential role of quorum sensing, specifically of the master regulator, SmcR in this reversible process. Introduction of an intact wild-type smcR gene, behind an inducible promoter on a plasmid vector, did not restore opacity to TR1 isolates; moreover, no mutations were found in the smcR gene located in the chromosome of these isolates. Whole genome sequencing of all lineage members identified 32 different sites of mutation relative to the MO6-24/O reference genome. One mutation site that appeared in all four lineages was found in the wzy gene, which is located in the Group I CPS operon and which encodes a polysaccharide polymerase that is vital for CPS production. Mutations at this site appear to involve the addition or deletion of repeating units of the hexanucleotide sequence 5’ATTATA 3’, with each unit designating a pair of isoleucine residues. While the MO6-24/O reference genome showed four tandem copies of the repeat encoding a stretch of eight consecutive isoleucines residues in the Wzy protein, lineage members varied between four or five repeat units present. We propose the addition or deletion of isoleucine residues within Wzy could alter the activity of this critical CPS biosynthetic component and thus form the basis of an underlying mechanism for reversible CPS phase variation in this pathogen. Thus, the general premise of my dissertation research is the elucidation of aspects of polysaccharide-based phase variation in human pathogenic species of Vibrio.
Date
7-8-2025
Recommended Citation
Mukerji, Aheli S., "ELUCIDATION OF A ROLE IN BIOFILM PRODUCTION FOR THE TYROSINE CATABOLISM PATHWAY IN VIBRIO CHOLERAE AND ANALYSIS OF REVERSIBLE PHASE VARIATION IN VIBRIO VULNIFICUS" (2025). LSU Doctoral Dissertations. 6841.
https://repository.lsu.edu/gradschool_dissertations/6841
Committee Chair
Gregg Pettis
DOI
10.31390/gradschool_dissertations.6841