F2-380. Efflux-Mediated Resistance to an Experimental Nucleoside-Based Inhibitor of Vibriobactin Biosynthesis in Vibrio cholerae
Session: Poster Session: Screening Methods and Novel Targets
Saturday, October 25, 2008: 12:00 AM
Room: Hall C
Background: Aryl-capped siderophores produced by bacterial pathogens are potential targets for new therapeutics since these small-molecule iron chelators are responsible for obtaining iron from the human host, a process that is often associated with their virulence. Vibrio cholerae N16961 synthesizes the siderophore vibrobactin on a norspermidine scaffold with the addition of two units of threonine and three units of an aromatic acid, 2,3-dihyroxybenzoate (2,3-DHB).
Methods and Results: We cloned and purified VibE, a 2,3-DHB adenylating enzyme responsible for the first step in vibriobactin biosynthesis, and determined steady-state kinetic parameters (Kmapp of 366 μM for ATP and 3 μM for 2,3-DHB) using a [32P]PPi exchange assay. This adenylation reaction was competitively inhibited (Kiapp = 4 ± 0.7 nM) with the bisubstrate inhibitor 5’-O-[N-(2,3-dihydroxybenzoyl)sulfamoyl]adenosine (2,3-DHB-AMS). Despite this promising in vitro activity, growth inhibition (MIC99 = 25 μM) by 2,3-DHB-AMS in iron-limiting M9 medium was only observed with the co-addition of PAβN, a broad inhibitor of RND-type efflux systems. Cellular accumulation of ethidium bromide was also sensitive to PAβN indicating an active efflux system(s) in this strain. To confirm this prediction, we developed an LC-MS assay and showed that intracellular levels of 2,3-DHB-AMS are ~2.5-fold higher in cells treated with PAβN. We used qRT-PCR to observe that neither the vexAB and vexCD RND pumps, which are primarily responsible for antibiotic and bile efflux are induced by 2,3-DHB-AMS. Further, a previously characterized vexB vexD double mutant remained resistant to 2,3-DHB-AMS.
Conclusions: These observations predict that one or more of the other four RND systems annotated in the genome may be able to compensate for loss of the vex systems or are exclusively responsible for efflux of 2,3-DHB-AMS.
Brian Beck1, Courtney Aldrich2, Daniel Wilson2, J. Keller2, Laura Celia3 and  L. K. Celia, None., (1)ATCC, (2)University of Minnesota, (3)ATCC, Manassas, VA