Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in
Salmonella
Biology
Authors: Buckner Michelle M. C. , Blair Jessica M. A. , La Ragione Roberto M. , Newcombe Jane , Dwyer Daniel J. , Ivens Alasdair , Piddock Laura J. V. 
Published: November 2016

For over 20 years, bacterial multidrug resistance (MDR) efflux pumps have been studied because of their impact on resistance to antimicrobials. However, critical questions remain, including why produce efflux pumps under non-antimicrobial treatment conditions, and why have multiple pumps if their only purpose is antimicrobial efflux?
Salmonella
spp. possess five efflux pump families, including the resistance-nodulation-division (RND) efflux pumps. Notably, the RND efflux pump AcrD has a unique substrate profile, distinct from other
Salmonella
efflux pumps. Here we show that inactivation of
acrD
results in a profoundly altered transcriptome and modulation of pathways integral to
Salmonella
biology. The most significant transcriptome changes were central metabolism related, with additional changes observed in pathogenicity, environmental sensing, and stress response pathway expression. The extent of tricarboxylic acid cycle and fumarate metabolism expression changes led us to hypothesize that
acrD
inactivation may result in motility defects due to perturbation of metabolite concentrations, such as fumarate, for which a role in motility has been established. Despite minimal detectable changes in flagellar gene expression, we found that an
acrD
mutant
Salmonella enterica
serovar Typhimurium isolate was significantly impaired for swarming motility, which was restored by addition of fumarate. The
acrD
mutant outcompeted the wild type in fitness experiments. The results of these diverse experiments provide strong evidence that the AcrD efflux pump is not simply a redundant system providing response resilience, but also has distinct physiological functions. Together, these data indicate that the AcrD efflux pump has a significant and previously underappreciated impact on bacterial biology, despite only minor perturbations of antibiotic resistance profiles.

IMPORTANCE
Efflux pumps in Gram-negative bacteria are studied because of their important contributions to antimicrobial resistance. However, the role of these pumps in bacterial biology has remained surprisingly elusive. Here, we provide evidence that loss of the AcrD efflux pump significantly impacts the physiology of
Salmonella enterica
serovar Typhimurium. Inactivation of
acrD
led to changes in the expression of 403 genes involved in fundamental processes, including basic metabolism, virulence, and stress responses. Pathways such as these allow
Salmonella
to grow, survive in the environment, and cause disease. Indeed, our data show that the
acrD
mutant is more fit than wild-type
Salmonella
under standard lab conditions. We hypothesized that inactivation of
acrD
would alter levels of bacterial metabolites, impacting traits such as swarming motility. We demonstrated this by exogenous addition of the metabolite fumarate, which partially restored the
acrD
mutant’s swarming defect. This work extends our understanding of the role of bacterial efflux pumps.