John E. Gustafson, PhD New Mexico State University jgustafs@nmsu.edu Phone:(505) 646-5660 Personal Website

Title: Regulation of Multidrug Resistance in S. aureus

Project Description:

Most of the efflux pumps that transport drugs out of Gram-positive bacteria are members of the major facilitator superfamily (MFS). S. aureus possesses 18 MFS paralogues (including the well characterized multidrug efflux pump NorA) that may act as multidrug efflux pumps. We now provide data demonstrating that inactivation of the staphylococcal accessory regulator SarA leads to increased susceptibility to multiple drugs. Furthermore, we demonstrate that SarA inactivation increases ciprofloxacin and ethidium accumulation, indicating a potential role for SarA in the regulation of drug efflux pumps. We hypothesize that the increase in drug accumulation and drug susceptibility in sarA::kan mutants occurs because of altered regulation of one or more MFS paralogues found within the S. aureus genome. Using the Bioinformatics Core of the INBRE/BRIN at NMSU we have also discovered conserved or perfect SarA preferred-binding motifs in 16 MFS gene promoter regions. We also hypothesize that SarA mediates its activity on the multidrug resistance phenotype of S. aureus by directly binding to MFS paralogue promoters.

Specific Aims:
AIM 1: Determine if SarA inactivation leads to altered transcription of MFS paralogues by probing a MFS paralogue DNA array with labeled cDNA prepared from sarA::kan inactivated mutants and parent strains.
AIM 2: Determine if SarA binds directly to MFS paralogue promoter regions using purified SarA and electrophoretic mobility shift assays.
AIM 3: Identify proteins binding to select MFS paralogue promoters using a promoter-affinity purification technique coupled with mass spectrometry at the proposed INBRE/BRIN mass spectrometry facility at UNM. This research will decipher any role SarA plays in controlling the MFS paralogue network of S. aureus. We also intend to dentify other proteins that bind to the effected MFS paralogues in order to identify other factors that play a role in the response of S. aureus to multiple drugs.