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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.

No MeSH data available.


Impact of fumarate on motility of acrD::aph mutant cells compared to SL1344 cells. Areas covered by SL1344 and SL1344 acrD::aph mutant cells for swimming (a) and swarming (b) motility in LB alone and in the presence of increasing concentrations of fumarate are summarized. Data presented are from 13 replicates for LB alone and 4 replicates for fumarate, with mean values indicated. *, P < 0.05; **, P < 0.001.
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fig3: Impact of fumarate on motility of acrD::aph mutant cells compared to SL1344 cells. Areas covered by SL1344 and SL1344 acrD::aph mutant cells for swimming (a) and swarming (b) motility in LB alone and in the presence of increasing concentrations of fumarate are summarized. Data presented are from 13 replicates for LB alone and 4 replicates for fumarate, with mean values indicated. *, P < 0.05; **, P < 0.001.

Mentions: Compared to SL1344, the acrD mutant was impaired for swimming. SL1344 swam to cover an average area of 471.5 mm2, while the acrD mutant covered 266.7 mm2 (P = 0.0018) (Fig. 3a). Likewise, the acrD mutant was impaired for swarming compared with SL1344, which covered an average area of 6,090 mm2 while the acrD mutant covered 510 mm2 (P < 0.001) (Fig. 3b). The swimming assay produces results that are inherently variable, and so we minimized this impact by performing 13 replicates.


Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology
Impact of fumarate on motility of acrD::aph mutant cells compared to SL1344 cells. Areas covered by SL1344 and SL1344 acrD::aph mutant cells for swimming (a) and swarming (b) motility in LB alone and in the presence of increasing concentrations of fumarate are summarized. Data presented are from 13 replicates for LB alone and 4 replicates for fumarate, with mean values indicated. *, P < 0.05; **, P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: Impact of fumarate on motility of acrD::aph mutant cells compared to SL1344 cells. Areas covered by SL1344 and SL1344 acrD::aph mutant cells for swimming (a) and swarming (b) motility in LB alone and in the presence of increasing concentrations of fumarate are summarized. Data presented are from 13 replicates for LB alone and 4 replicates for fumarate, with mean values indicated. *, P < 0.05; **, P < 0.001.
Mentions: Compared to SL1344, the acrD mutant was impaired for swimming. SL1344 swam to cover an average area of 471.5 mm2, while the acrD mutant covered 266.7 mm2 (P = 0.0018) (Fig. 3a). Likewise, the acrD mutant was impaired for swarming compared with SL1344, which covered an average area of 6,090 mm2 while the acrD mutant covered 510 mm2 (P < 0.001) (Fig. 3b). The swimming assay produces results that are inherently variable, and so we minimized this impact by performing 13 replicates.

View Article: PubMed Central - PubMed

ABSTRACT

For over 20&nbsp;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.