The global consequence of disruption of the AcrAB-TolC efflux pump in Salmonella enterica includes reduced expression of SPI-1 and other attributes required to infect the host

Abstract

The mechanisms by which RND pumps contribute to pathogenicity are currently not understood. Using the AcrAB-TolC system as a paradigm multidrug-resistant efflux pump and Salmonella enterica serovar Typhimurium as a model pathogen, we have demonstrated that AcrA, AcrB, and TolC are each required for efficient adhesion to and invasion of epithelial cells and macrophages by Salmonella in vitro. In addition, AcrB and TolC are necessary for Salmonella to colonize poultry. Mutants lacking acrA, acrB, or tolC showed differential expression of major operons and proteins involved in pathogenesis. These included chemotaxis and motility genes, including cheWY and flgLMK and 14 Salmonella pathogenicity island (SPI)-1-encoded type III secretion system genes, including sopE, and associated effector proteins. Reverse transcription-PCR confirmed these data for identical mutants in two other S. Typhimurium backgrounds. Western blotting showed reduced production of SipA, SipB, and SipC. The absence of AcrB or TolC also caused widespread repression of chemotaxis and motility genes in these mutants, and for acrB::aph, this was associated with decreased motility. For mutants lacking a functional acrA or acrB gene, the nap and nir operons were repressed, and both mutants grew poorly in anaerobic conditions. All phenotypes were restored to that of the wild type by trans-complementation with the wild-type allele of the respective inactivated gene. These data explain how mutants lacking a component of AcrAB-TolC are attenuated and that this phenotype is a result of decreased expression of numerous genes encoding proteins involved in pathogenicity. The link between antibiotic resistance and pathogenicity establishes the AcrAB-TolC system as fundamental to the biology of Salmonella.

FIG. 1.

Validation of transcriptome data. Expression of sipA, sipC, cheM, flgM, nirD, and ramA, as measured by RT-PCR. Open bars indicate SL1344 and mutants thereof; gray bars indicate 14028S and mutants thereof; and black bars indicate L3 and mutants thereof. WT, wild type.

FIG. 2.

Complementation of expression of genes altered in mutants. Expression of ramA (open boxes), cheM (gray boxes), nirD (boxes with horizontal lines), sipC (boxes with vertical lines), flgM (boxes with diagonal lines), sipA (black boxes) was measured in SL1344 and L773 (L110 pKSW30acrB), L774 (L109 pKSW30tolC), and L989 (L884 pKSW30acrA). Expression was measured by comparative RT-PCR, and results are shown as change relative to SL1344.

FIG. 3.

Comparison of transcriptomes of the acrA, acrB, and tolC mutants. Venn diagram showing overlap of genes with significantly altered (increased or decreased) expression in L884 (acrA::aph), L110 (acrB::aph), and L109 (tolC::aph) compared with SL1344. Regulators with altered expression in each group are indicated. Bold type indicates genes with increased expression; lightface type those with decreased expression.

FIG. 4.

Production of SPI-1 proteins. Western blot of SipB and SipC of protein supernatants prepared from L884 (acrA::aph), L110 (acrB::aph), and L109 (tolC::aph) grown in minimal media.

FIG. 5.

Motility of mutants. Average zones of motility (± standard deviation) of SL1344, L884 (acrA::aph), L110 (acrB::aph), and L109 (tolC::aph) in three concentrations of semisolid minimal medium agar. Asterisks indicate statistically significant (P < 0.05) differences.

FIG. 6.

Growth kinetics of mutants. Growth (± standard deviation) of L884 (acrA::aph), L110 (acrB::aph), and L109 (tolC::aph) in anaerobic conditions. Asterisks indicate statistically significant (P < 0.05) differences.