Roles of DHA2 Family Transporters in Drug Resistance and Iron Homeostasis in <i>Acinetobacter</i> spp.

Abstract

Background:Acinetobacterbaumannii is a major cause of nosocomial infections worldwide due to its fitness within clinical settings and recalcitrance to conventional therapies. The drug: H⁺ antiporter 2 (DHA2) family export systems encoded by A. baumannii were investigated for their roles in promoting the success ofthis organism as a human pathogen. Methods: Bioinformatic tools were used to identify the DHA2 family transporters encoded by Acinetobacter spp. and establish their phylogenetic relationships. The drug resistance phenotypes conferred by the transporters were tested using both heterologously expressed proteins in Escherichia coli and Acinetobacter deletion mutants. The transcriptional responses of DHA2 family transporter genes to their substrates were established by qRT-PCR. Results: Six highly conserved DHA2 family proteins were identified in A. baumannii. Drug resistance phenotypes were established for two DHA2 family transporters. The expression of a third DHA2 family protein is highly responsive to the availability of iron. The gene encoding this protein is located within a putative siderophore biosynthesis locus, suggesting a physiological role in iron uptake, possibly via the export of a siderophore. Conclusions: These results highlight functions for DHA2 family proteins in both drug resistance and the maintenance of stable cellular physiology, emphasizing their importance in A. baumannii infections.

Transcriptomic and biochemical analyses identify a family of chlorhexidine efflux proteins

“…Genes were amplified by PCR with Pfx supermix (Invitrogen) and cloned into the pTTQ18-RGSH6 vector as reported (7). Site-directed mutagenesis was conducted by using the QuikChange method as described (30), but using KOD hot start polymerase (Novagen).…”

“…The majority of chlorhexidine efflux systems described to date are multidrug efflux systems that confer resistance to a broad range of structurally diverse antimicrobial compounds, e.g., in A. baumannii the resistance/nodulation/division (RND) superfamily efflux systems AdeABC (Acinetobacter drug efflux) and AdeIJK (6), the major facilitator superfamily (MFS) AedF(Acinetobacter exporter of the DHA2 family) transporter (7), and the small multidrug resistance (SMR) family exporter AdeS (8) have been shown to confer chlorhexidine resistance. This study aimed to identify the core chlorhexidine resistance mechanisms operating in Gram-negative bacteria using a whole-genome transcriptomic analysis of A. baumannii as a representative Gram-negative pathogen and to functionally characterize proteins involved in the resistance response.…”

Fluorescence-Based Flow Sorting in Parallel with Transposon Insertion Site Sequencing Identifies Multidrug Efflux Systems in Acinetobacter baumannii

“…1; see also Data Set S1 in the supplemental material). Mutants carrying insertions in genes encoding several multidrug efflux pumps, particularly adeABC (12), adeIJK (10), and amvA (13, 14), and genes encoding the adeABC activator, adeRS (15), were overrepresented in the highly fluorescent populations (Fig. 1).…”

“…Selection of A. baumannii mutants carrying insertions in genes encoding the characterized efflux pumps AdeABC (12), AdeIJK (10), AdeFGH (20), AmvA (13, 14), CraA (21), AbeS (22), AbeM (23), and AceI (24, 25) and regulators AdeRS and BaeRS, which control expression of adeABC (15, 26); AdeN, which controls adeIJK (16); AdeL, which controls adeFGH (20); and AceR, which controls aceI (27). Bars represent the fold change in mutant abundance in cells selected for low ethidium fluorescence (blue), high ethidium fluorescence (red), or growth in 62.5 µg/ml (approximately 158 µM) ethidium bromide (hatched green; 1/4× MIC) compared to the starting mutant pool.…”

Mechanisms of Drug Efflux and Strategies to Overcome Them as a Way to Control Microbial Growth

“…Although half of the sequenced molecules are of unknown or putative function, others are known to be vital in multitude of physiological processes. And some subfamilies of exporters play a major role in multidrug resistance in bacteria and fungi (Hassan et al 2011;Prasad and Rawal 2014).…”