טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentEfrat Hartog
SubjectRegulation and Function of the MAR Proteins in Salmonella
Typhimurium
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Full Professor Yaron Sima
Full Thesis textFull thesis text - English Version


Abstract

Salmonella is an example of pathogen that adapts to antibiotics, superoxides, organic solvents, disinfectants and weak acids through mutations within chromosomal genes as the marRAB operon. This operon encodes the MarR, which represses the expression of the activator MarA. The expression is activated when MarR is inactivated, and MarA or its homologues SoxS or RobA bind the operator. Each stress signal turns on one or more of these regulators which regulate acrAB and micF for activating efflux and depressing influx of antimicrobial compounds, respectively.

The primary goal of the first part of our research was to further understand the role of each regulator in adjusting acrAB and micF transcription to the required extent suitable for the distinctive signal. We investigated the transcription of the regulatory genes marA, soxS and rob and their shared regulated genes micF and acrAB in response to salicylate (SAL), paraquat (PQ) and decanoate (DEC). The activity of the promoters was determined by qRT-PCR and GFP reporter protein.

Regulation profiles showed that all treatments elicit a defense response by stimulating micF and acrAB. marA is strongly induced by SAL, soxS by PQ, acrA and micF by all inducers while the transcription of robA is reduced by SAL and DEC. Results obtained with mutant strains demonstrate that Rob maintains low rates of basal expressions and adjusts activators’ transcription to normal level following induction in order to obtain controlled defensive response.

Since Salmonella is capable of survival and replication in the host cells, the second part of our research was aimed at testing if SAL activates the mar regulon in intracellular Salmonella and contributes to antimicrobial resistance. We quantified the transcription of marA, acrAB and micF in intracellular S. Typhimurium associated with J774 macrophages with or without SAL, and investigated the effect of SAL on the activity of ciprofloxacin against intracellular and extracellular Salmonella.

Results showed that SAL does stimulate the expression of marRAB, acrAB and micF also within intracellular cells. It was indicated that pre-exposure of extracellular Salmonella to SAL prior to ciprofloxacin treatment enhances the tolerance through induction of acrAB by MarA. Within intracellular cells, the basal expression of marRAB, acrAB and soxS is higher compared to extracellular cells; therefore, SAL exposure does not result in significant acrAB induction and subsequent resistance enhancement. Results explain the tolerance of intracellular bacteria to ciprofloxacin and show that conclusions rise from extracellular bacteria cannot be referred to intracellular bacteria.