Characterization of the RND family of multidrug efflux pumps: in silico to in vivo confirmation of four functionally distinct subgroups

Abstract

SummaryWe have developed a generalized profile that identifies members of the root‐nodulation‐cell‐division (RND) family of efflux pumps and classifies them into four functional subfamilies. According to Z‐score values, efflux pumps can be grouped by their metabolic function, thus making it possible to distinguish pumps involved in antibiotic resistance (group 1) from those involved in metal resistance (group 3). In silico data regarding efflux pumps in group 1 were validated after identification of RND efflux pumps in a number of environmental microbes that were isolated as resistant to ethidium bromide. Analysis of the Pseudomonas putida KT2440 genome identified efflux pumps in all groups. A collection of mutants in efflux pumps and a screening platform consisting of 50 drugs were created to assign a function to the efflux pumps. We validated in silico data regarding efflux pumps in groups 1 and 3 using 9 different mutants. Four mutants belonging to group 2 were found to be more sensitive than the wild‐type to oxidative stress‐inducing agents such as bipyridyl and methyl viologen. The two remaining mutants belonging to group 4 were found to be more sensitive than the parental to tetracycline and one of them was particularly sensitive to rubidium and chromate. By effectively combining in vivo data with generalized profiles and gene annotation data, this approach allowed the assignment, according to metabolic function, of both known and uncharacterized RND efflux pumps into subgroups, thereby providing important new insight into the functions of proteins within this family.

Mechanisms of Resistance to Chloramphenicol in Pseudomonas putida KT2440

“…In this study, we have isolated a mutant in the ttgB gene that exhibited increased sensitivity to chloramphenicol, confirming Godoy and colleagues' observations (23). The TtgABC efflux pump is a multidrug efflux system that expels chloramphenicol from the cell, as it is known to do for tetracycline, ethidium bromide, ampicillin, and other antibiotics (14,18,23,58).…”

“…Genome annotation of several natural chloramphenicol-resistant P. putida strains failed to show any specific chloramphenicol-modifying enzymes (36,41,45,67); however, Godoy and colleagues (23) showed that P. putida mutants in the multidrug TtgABC pump exhibited compromised growth in the presence of chloramphenicol (21). Further studies in P. putida DOT-T1E confirmed the role of the TtgABC efflux pump in tolerance to chloramphenicol and other toxic compounds while unveiling the molecular mechanisms involved in the regulation of the expression of this pump in this strain (14,18,58).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…Their regulators (e.g., MexR, EsrC, ArmZ, and MexT) can respond to various stresses caused by, for example, membrane-damaging or ribosome-disrupting agents, reactive oxygen species, and/or nitrosative stress and subsequently cause increased activities of these pumps against the stresses (441,454,461,467,798,799,827,835,911). Consistently, P. putida RND pump mutants are categorized into 4 functionally distinct subgroups, and one of them is more susceptible to oxidation-inducing agents (912). In a situation mimicking chronic cystic fibrosis infections with antibiotic therapy, oxidative stress and antibiotic exposure result in the hyperexpression of AmrAB-OprM of Burkholderia vietnamiensis (913).…”

Correlation between AcrB Trimer Association Affinity and Efflux Activity

“…9−11 RND family efflux pumps play a major role in multidrug resistance in Gram-negative bacteria. AcrB is an obligate trimer that exists and functions exclusively in the trimeric form (Figure 1A).…”