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Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology

View Article: PubMed Central - PubMed

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.

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The gene expression changes of SL1344 acrD::aph compared to wild-type SL1344. Genes with altered expression in the acrD mutant were categorized according to function. The horizontal axis indicates the number of genes in each category, with negative values indicating genes that were downregulated and positive values indicating genes that were upregulated.
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fig1: The gene expression changes of SL1344 acrD::aph compared to wild-type SL1344. Genes with altered expression in the acrD mutant were categorized according to function. The horizontal axis indicates the number of genes in each category, with negative values indicating genes that were downregulated and positive values indicating genes that were upregulated.

Mentions: The transcriptome of the acrD mutant revealed significantly altered expression of 403 genes compared to the parental SL1344 strain (for a list of genes with significantly altered expression, see Table S1 in the supplemental material; full microarray data are available at http://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-2975/samples/). Specifically, in the acrD mutant expression of genes encoding proteins involved in anaerobic growth, ATP synthesis, amino acid metabolism, sugar transport, glycolysis/gluconeogenesis, ribosomal subunit biosynthesis, RNA polymerase, and oxidative phosphorylation were generally increased (Fig. 1). Expression of genes associated with pathogenicity, sigma factors, stress response, the tricarboxylic acid (TCA) cycle, and purine metabolism were generally decreased (Fig. 1). Surprisingly, very few of the transcriptomic changes could be directly linked to drug export.


Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology
The gene expression changes of SL1344 acrD::aph compared to wild-type SL1344. Genes with altered expression in the acrD mutant were categorized according to function. The horizontal axis indicates the number of genes in each category, with negative values indicating genes that were downregulated and positive values indicating genes that were upregulated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120143&req=5

fig1: The gene expression changes of SL1344 acrD::aph compared to wild-type SL1344. Genes with altered expression in the acrD mutant were categorized according to function. The horizontal axis indicates the number of genes in each category, with negative values indicating genes that were downregulated and positive values indicating genes that were upregulated.
Mentions: The transcriptome of the acrD mutant revealed significantly altered expression of 403 genes compared to the parental SL1344 strain (for a list of genes with significantly altered expression, see Table S1 in the supplemental material; full microarray data are available at http://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-2975/samples/). Specifically, in the acrD mutant expression of genes encoding proteins involved in anaerobic growth, ATP synthesis, amino acid metabolism, sugar transport, glycolysis/gluconeogenesis, ribosomal subunit biosynthesis, RNA polymerase, and oxidative phosphorylation were generally increased (Fig. 1). Expression of genes associated with pathogenicity, sigma factors, stress response, the tricarboxylic acid (TCA) cycle, and purine metabolism were generally decreased (Fig. 1). Surprisingly, very few of the transcriptomic changes could be directly linked to drug export.

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Related in: MedlinePlus