title: Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology.
abstract: 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.
  
  
  
  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.
authors.affiliation: Antimicrobials Research Group, Institute of Microbiology and Infection, College of Medical and Dental Sciences, The University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors.forename: Michelle M C
authors.initials: MM
authors.lastname: Buckner
authors.affiliation: Antimicrobials Research Group, Institute of Microbiology and Infection, College of Medical and Dental Sciences, The University of Birmingham, Edgbaston, Birmingham, United Kingdom.
authors.forename: Jessica M A
authors.identifier: 0000-0001-6904-4253
authors.initials: JM
authors.lastname: Blair
authors.affiliation: School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom.
authors.forename: Roberto M
authors.initials: RM
authors.lastname: La Ragione
authors.affiliation: School of Biosciences and Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, United Kingdom.
authors.forename: Jane
authors.initials: J
authors.lastname: Newcombe
authors.affiliation: Department of Cell Biology and Molecular Genetics, Institute for Physical Science and Technology, Department of Bioengineering, Maryland Pathogen Research Institute, University of Maryland, College Park, Maryland, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Dwyer
authors.affiliation: Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, Scotland.
authors.forename: Alasdair
authors.initials: A
authors.lastname: Ivens
authors.affiliation: Antimicrobials Research Group, Institute of Microbiology and Infection, College of Medical and Dental Sciences, The University of Birmingham, Edgbaston, Birmingham, United Kingdom l.j.v.piddock@bham.ac.uk.
authors.forename: Laura J V
authors.identifier: 0000-0003-1460-473X
authors.initials: LJ
authors.lastname: Piddock
mesh_terms: D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001693:Biological Transport, Active; D024881:Drug Resistance, Bacterial; D017353:Gene Deletion; D020869:Gene Expression Profiling; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*
chemical_list: C485379:AcrD protein, Salmonella enterica; D000900:Anti-Bacterial Agents; D026901:Membrane Transport Proteins