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

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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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SPI-1 expression of the acrD::aph strain. Strains containing the promoter of prgH (SPI-1) fused to gfp were analyzed by flow cytometry and the number of fluorescent and nonfluorescent cells were enumerated. (a) Mean results ± standard deviations. *, P < 0.05. (b) A representative example of the flow cytometry results for SL1344 and SL1344 acrD::aph cells.
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fig2: SPI-1 expression of the acrD::aph strain. Strains containing the promoter of prgH (SPI-1) fused to gfp were analyzed by flow cytometry and the number of fluorescent and nonfluorescent cells were enumerated. (a) Mean results ± standard deviations. *, P < 0.05. (b) A representative example of the flow cytometry results for SL1344 and SL1344 acrD::aph cells.

Mentions: Genes from SPI-1 have been shown to be expressed by only a portion of the total population (31–33), so techniques such as microarrays and reverse transcription-PCR (RT-PCR), which measure gene expression across a whole population, only provide partial information about gene expression of individual bacteria in a population. The expression of the SPI-1 gene prgH, which encodes the essential basal component of the needle complex of the SPI-1 type III secretion system, has been used previously to measure expression of SPI-1 in single cells (31, 34). Therefore, strains containing the promoter of prgH fused to the green fluorescent protein gene gfp were used to compare the percentage of cells in the population that expressed SPI-1 in the acrD mutant versus in the wild type, as described previously (31, 34). This showed that when acrD was inactivated, fewer cells expressed SPI-1 than did wild-type Salmonella cells (Fig. 2). These findings correlate with the decreased detection of SPI-1 transcripts in the acrD mutant microarray analysis (Table S1).


Beyond Antimicrobial Resistance: Evidence for a Distinct Role of the AcrD Efflux Pump in Salmonella Biology
SPI-1 expression of the acrD::aph strain. Strains containing the promoter of prgH (SPI-1) fused to gfp were analyzed by flow cytometry and the number of fluorescent and nonfluorescent cells were enumerated. (a) Mean results ± standard deviations. *, P < 0.05. (b) A representative example of the flow cytometry results for SL1344 and SL1344 acrD::aph cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: SPI-1 expression of the acrD::aph strain. Strains containing the promoter of prgH (SPI-1) fused to gfp were analyzed by flow cytometry and the number of fluorescent and nonfluorescent cells were enumerated. (a) Mean results ± standard deviations. *, P < 0.05. (b) A representative example of the flow cytometry results for SL1344 and SL1344 acrD::aph cells.
Mentions: Genes from SPI-1 have been shown to be expressed by only a portion of the total population (31–33), so techniques such as microarrays and reverse transcription-PCR (RT-PCR), which measure gene expression across a whole population, only provide partial information about gene expression of individual bacteria in a population. The expression of the SPI-1 gene prgH, which encodes the essential basal component of the needle complex of the SPI-1 type III secretion system, has been used previously to measure expression of SPI-1 in single cells (31, 34). Therefore, strains containing the promoter of prgH fused to the green fluorescent protein gene gfp were used to compare the percentage of cells in the population that expressed SPI-1 in the acrD mutant versus in the wild type, as described previously (31, 34). This showed that when acrD was inactivated, fewer cells expressed SPI-1 than did wild-type Salmonella cells (Fig. 2). These findings correlate with the decreased detection of SPI-1 transcripts in the acrD mutant microarray analysis (Table S1).

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.


Related in: MedlinePlus