Blair, Jessica Mary Alice (2010)
Ph.D. thesis, University of Birmingham.
AcrA is the periplasmic adaptor protein component of the major efflux system AcrB-TolC of \(Salmonella\) Typhimurium. AcrA of S. \(Typhimurium\) SL1344 was inactivated and the mutant phenotype characterised. RT-PCR and western blotting were used to confirm expression of \(acrB/AcrB\) was retained in the acrA mutant. The AcrA mutant was hyper-susceptible to a range of antimicrobials and was more susceptible to some agents than strains lacking AcrB and TolC. This is partially explained by the increase in accumulation of Hoechst H33342, a fluorescent substrate of AcrAB-TolC, indicating that the inactivation of \(acrA\) resulted in reduced efflux activity. Lack of AcrA also attenuated the ability of S. Typhimurium to infect INT-407 and RAW 264.7 cells \(in vitro\) as previously published for AcrB and TolC mutants. The virulence defect of the mutants could not be rescued by addition of supernatant from an infection of INT-407 cells with SL1344 or addition of media conditioned by uninfected INT 407 cells. However, addition of media conditioned by overnight growth of SL1344 was able to ameliorate the virulence defect of the mutants. This suggests that AcrAB-TolC of SL1344 exports a factor/s required for virulence which the mutants are unable to export and that exogenous addition of this factor can restore the virulent phenotype. Inactivation of \(acrA\) conferred a phenotype distinct to that of inactivation of \(acrB\) or \(tolC\) indicating a role for AcrA distinct to that of other protein partners in both efflux of substrates and virulence
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