Regulation of the quorum sensing pathway in the human pathogen Vibrio parahaemolyticus
Date
2022
Authors
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Publisher
University of Delaware
Abstract
Quorum sensing (QS) is a cell-to-cell communication mechanism that bacteria use to regulate global gene expression in response to cell density. QS controls many different phenotypes required for individual cell behaviors and group or social behaviors. Bacteria accomplish this by secreting signaling molecules called autoinducers (AI), such as N-acylhomoserine lactone (AHL), that are sensed by cells using cell surface histidine kinase receptors. Each bacterial species produces unique types of AI that allow for intraspecies communication as well as common AI that allow interspecies sensing. Vibrio harveyi is a well-studied model system for QS, and in this species, amongst others, it has been shown that at low cell density (LCD), AI concentrations are low and are not bound to the cell surface receptors. At LCD, a phosphorylation cascade leads to the activation of the response regulator, LuxO. Activated LuxO binds to the regulatory regions of five non-coding regulatory small RNAs (sRNAs), called the quorum regulatory RNA (Qrr) sRNAs. LuxO is an activator of the transcription sigma factor, sigma-54 (RpoN), leading to transcription of the qrr genes. The Qrr sRNAs enhance expression of the LCD master regulator aphA, which inhibits expression of the high cell density (HCD) master regulator luxR. At HCD, AI concentration is high and is bound to the membrane receptors inducing phosphatase activity, which results in deactivation of LuxO. Deactivated LuxO cannot activate RpoN resulting in loss of transcription of the qrr genes. ☐ Vibrio parahaemolyticus, is a marine species and a human pathogen that causes severe gastroenteritis worldwide. In this dissertation work, we investigated the V. parahaemolyticus QS pathway to determine its role in biofilm production and cell surface sensing, two group or social behaviors. In Chapter 2, we examined individual components of the V. parahaemolyticus QS pathway and demonstrate that sigma-54 (encoded by rpoN) was not an essential component of the pathway as previously suggested. Our work showed that a single Qrr sRNA, Qrr2, could be transcribed independent of sigma-54 and that Qrr2 sRNA could solely regulate expression of opaR (the luxR homolog). Using bioinformatics analysis, we identified a sigma-70 (RpoD) consensus promoter region overlapping the sigma-54 promoter within the regulatory region of qrr2. We showed that this promoter was required for activation of transcription of qrr2 in the absence of sigma-54. This previously unrecognized dual regulation of Qrr sRNAs demonstrates that the Qrr2 sRNA is regulated outside of the QS pathway. Expression analysis of all five qrr genes in luxO and rpoN mutant strains also suggested that other qrr genes are expressed outside of the QS pathway. Using quadruple qrr deletion mutants, we showed that Qrr2 was the only Qrr that could act solely to repress OpaR in V. parahaemolyticus to modulate capsule polysaccharide production essential for biofilm formation. These findings demonstrated significant differences between the QS pathways of V. harveyi and V. parahaemolyticus and that model species should only be used as a guide to understand other species and strains. ☐ In Chapter 3, we investigated the global regulator Fis, a nucleoid associated protein that binds and bends DNA. In V. harveyi, Fis was shown to positively regulate all five qrr genes. By combining biochemical and bioinformatics approaches, we determined that Fis bound to the regulatory regions of all five qrr genes in V. parahaemolyticus. More specifically, using genetics assays, we showed that Fis was a direct activator of qrr2, qrr3, and qrr4, but did not significantly control qrr1 or qrr5 expression. Our work showed for the first time that Fis reciprocally controlled two signaling pathways, QS and surface sensing pathways, that oppositely control biofilm and swarming behaviors. Fis positively regulated the qrr sRNAs that negatively regulate CPS production through OpaR regulation and Fis positively regulated both the laf and the scrABC operons required for swarming behavior. Thus, Fis played a positive role in directing cells towards swarming behavior and inhibiting biofilm formation. In addition, we demonstrated that in V. parahaemolyticus Fis was a positive regulator of metabolism, through direct regulation of metabolic gene clusters required for arabinose, glucoronate and N-acetylglucosamine metabolism. ☐ The goal of Chapter 4 was to investigate the role of the cAMP receptor protein (CRP) in V. parahaemolyticus, a global regulator of metabolism in many species. Specifically, we investigated how CRP and OpaR converge to regulate capsule polysaccharide production, since both single deletion mutants are CPS deficient. Using genetic complementation assays, we determined that OpaR overexpression can significantly rescue the ∆crp mutant negative biofilm phenotype. Further, GFP reporter assays in both V. parahaemolyticus and E. coli backgrounds indicated that CRP is a repressor of opaR expression. Additional work is needed to fully understand the relationship between CRP and OpaR and capsule production, and how cell density and metabolism regulation converge.
Description
Keywords
Biofilm, Motility, Noncoding sRNA, Quorum sensing, Sigma factor-54, Vibrio parahaemolyticus