RND pumps across the genus Acinetobacter: AdeIJK is the universal efflux pump

Abstract

Acinetobacter are generally soil-dwelling organisms that can also cause serious human infections. A. baumannii is one of the most common causative agents of Acinetobacter infections and is often multidrug resistant. However, an additional 25 species within the genus have also been associated with infection. A. baumannii encodes six resistance nodulation division (RND) efflux pumps, the most clinically relevant class of efflux pumps for antibiotic export, but the distribution and types of RND efflux pumps across the genus is currently unknown. Sixty-four species making up the genus Acinetobacter were searched for RND systems within their genomes. We also developed a novel method using conserved RND residues to predict the total number of RND proteins including currently undescribed RND pump proteins. The total number of RND proteins differed both within a species and across the genus. Species associated with infection tended to encode more pumps. AdeIJK/AdeXYZ was found in all searched species of Acinetobacter, and through genomic, structural and phenotypic work we show that these genes are actually homologues of the same system. This interpretation is further supported by structural analysis of the potential drug-binding determinants of the associated RND-transporters, which reveal their close similarity to each other, and distinctiveness from other RND-pumps in Acinetobacter , such as AdeB. Therefore, we conclude that AdeIJK is the fundamental RND system for species in the genus Acinetobacter . AdeIJK can export a broad range of antibiotics and provides crucial functions within the cell, for example lipid modulation of the cell membrane, and therefore it is likely that all Acinetobacter require AdeIJK for survival and homeostasis. In contrast, additional RND systems, such as AdeABC and AdeFGH, were only found in a subset of Acinetobacter that are associated with infection. By understanding the roles and mechanisms of RND efflux systems in Acinetobacter , treatments for infections can avoid efflux-mediated resistance and improve patient outcomes.

Types and Mechanisms of Efflux Pump Systems and the Potential of Efflux Pump Inhibitors in the Restoration of Antimicrobial Susceptibility, with a Special Reference to Acinetobacter baumannii

“…RND transporters are trimers that contain interdependent protomers. These protomers cycle through loose, tight, and open conformations and have proximal and distal binding pockets that have a variety of substrates they can bind [ 24 ]. In the RND efflux pump family, the proximal binding pocket (PBP) is conserved, except for residues 660–688 (using the numbering from AdeJ in A. baumannii ).…”

“…In the RND efflux pump family, the proximal binding pocket (PBP) is conserved, except for residues 660–688 (using the numbering from AdeJ in A. baumannii ). The region that is not conserved forms the bottom of the PBP and covers the flexible loop (F-loop) [ 24 ]. The MFP plays a role in the stabilization of the OMP by bringing the inner and outer membranes closer together [ 23 ].…”

“…This pump needs to be further characterized to understand its regulation [ 8 ]. AdeXYZ shares 93% nucleotide identity and 99% amino acid identity with AdeIJK, but they have been shown to be separate subfamilies of the RND efflux pumps [ 24 , 85 ]. The amino acid sequences of AdeJ and AdeY are identical, except for a single insertion after position 602 (position based off AdeJ found in A. baumannii ) in AdeY.…”

“…Three of these residues (A46, Q91, and T128) are located at the back of the DBP and are likely responsible for the differences in substrate binding. Residue AdeJ F611 plays a critical role in ligand binding in the DBP and is also conserved in AdeY and AcrB (F610) [ 24 ]. Across the DBPs of AdeJ, AdeY, adeb, and AcrB, F136, F178, Y327, V613, F618, and F629 (position based off AdeJ found in A. baumannii) are universally conserved.…”

“…AcrB shares highly conserved regions with AdeB and is so structurally similar to AdeB that their binding pockets can be directly compared. Residue F610 in AcrB plays a critical role in ligand binding in the DBP and is also conserved in AdeY and AcrB (F611) [ 24 ]. R717 at the entrance of the periplasmic cleft plays a role in substrate specificity [ 78 ].…”