In vitro regulation of quorum sensing in Pseudomonas aeruginosa using synthetic autoinducers By Hrag Dilabazian A thesis

Abstract

Quorum sensing is a remarkable type of cell-to-cell communication that is directly involved in the regulation of virulence, biofilm formation and other significant characteristics of bacteria. This process, which occurs in most pathogenic bacteria, is mainly governed by the release of significant concentrations of chemical signaling molecules, called autoinducers, by the local bacterial cell density. Pseudomonas aeruginosa, an opportunistic human pathogen, known for its complex quorum sensing system, also possesses its own autoinducers, that mediate cellular cross talk and regulate the expression of certain virulence genes. Among these genes is the lasB elastase gene. The lasI and rhlI gene products respectively drive the synthesis of N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butyryl homoserine lactone (BHL), the autoinducers of Pseudomonas aeruginosa. Forty eight clinical Pseudomonas aeruginosa isolates, from patients in LAUMC-RH, were included in this study to determine the activity of the two synthetic autoinducers, OdDHL and BHL, on the induction of the lasB gene, during early stages of bacterial growth. The strains were definitively identified and screened for their elastolytic activity. They were classified into two distinct groups: elastase positive and elastase negative. The possession of the quorum sensing controlled gene lasB and the regulatory genes lasR, lasI, rhlR and rhlI was determined by the polymerase chain reaction. One out of the four elastase negative isolates was lasR and lasI deficient. The inducing ability of OdDHL and BHL on the lasB gene was tested under different conditions: by increasing cell density or manipulating the concentration of or time of exposure to autoinducers, revealed that induction was not possible in elastase negative strains and had no detectable effect on the elastase positive strains. Our observations suggest that, in vitro autoinducer directed, early induction of the elastase gene in the clinical isolates was not possible. A plausible explanation could be the presence of a defective lasB gene, the absence of or truncated transcriptional regulatory proteins, or unusual environmental factors affecting the activity of the autoinducers on the elastase negative strains.