The novel MFS efflux pump SxtP, regulated by the LysR-type transcriptional activator SxtR, is involved in the susceptibility to sulfamethoxazole/trimethoprim (SXT) and the pathogenesis of Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a notorious opportunistic pathogen responsible for healthcare-associated infections worldwide. Efflux pumps play crucial roles in mediating antimicrobial resistance, motility, and virulence. In this study, we present the identification and characterization of the new A. baumannii efflux pump SxtP belonging to the MFS superfamily (major facilitator superfamily), along with its associated activator LysR-type transcriptional regulator (LTTR) SxtR, demonstrating their roles in sulfamethoxazole/trimethoprim (also known as co-trimoxazole or SXT) resistance, surface-associated motility and virulence.

Keywords: Acinetobacter; LysR-type transcriptional regulator; MFS efflux pump; co-trimoxazole.

Fig 1

(A) Intergenic region showing the 138 bp between the sxtP and sxtR genes in A. baumannii strain ATCC 17978. Surrounding genes are also shown. The TTA−N₇−TAA DNA motif is shown in bold and underlined. (B) EMSA of a DIG-labeled DNA fragment containing the intergenic sequence present between the genes coding SxtP and SxtR proteins. The experiment was carried out in the presence (+) or absence (−) of 2 µM of the SxtR protein, with at least a tenfold molar excess of nonlabeled and nonspecific DNA in all lanes and nonlabeled and specific DNA (third lane). All EMSAs were performed at least three times obtaining reproducible results. A representative image is shown.

Fig 2

(A) Surface-associated motility assays of wild-type A. baumannii strain ATCC 17978 (WT), and the derivative mutants lacking the genes encoding either the SxtP or the SxtR proteins. All surface-associated motility assays were performed at least three times obtaining reproducible results. A representative image is shown. (B) G. mellonella killing assay of the specified strains. Larvae (n = 10 per group) were inoculated with either ~10⁶ CFU of the indicated strain or PBS as a negative control (not shown). *P < 0.001 compared to the A. baumannii parental ATCC 17978 strain (WT). All G. mellonella killing experiments were performed at least three times obtaining reproducible results. A representative graph of larval survival is shown.