Staphylococcus aureus NorD, a Putative Efflux Pump Coregulated with the Opp1 Oligopeptide Permease, Contributes Selectively to Fitness In Vivo

Abstract

c Staphylococcus aureus readily infects humans, causing infections from mild superficial skin infections to lethal bacteremia and endocarditis. Transporters produced by S. aureus allow the pathogen to adapt to a variety of settings, including survival at sites of infection and in the presence of antibiotics. The native functions of many transporters are unknown, but their potential dual contribution to fitness and antimicrobial resistance highlights their importance in staphylococcal infections. Here, we show that S. aureus NorD, a newly recognized efflux pump of the major facilitator superfamily, contributes to fitness in a murine subcutaneous abscess model. In community-associated methicillin-resistant S. aureus (CA-MRSA) strain MW2, norD was selectively upregulated 36-fold at the infection site relative to growth in vitro, and the norD mutant demonstrated significant fitness impairment in abscesses, with fitness 20-to 40-fold lower than that of the parent MW2 strain. Plasmid-encoded NorD could complement the fitness defect of the MW2 norD mutant. Chromosomal norD expression is polycistronic with the upstream oligopeptide permease genes (opp1ABCDF), which encode an ABC oligopeptide transporter. Both norD and opp1 were upregulated in abscesses and iron-restricted culture medium and negatively regulated by Fur, but only NorD contributed to fitness in the murine abscess model. Staphylococcus aureus is a versatile pathogen that causes a variety of diseases from food poisoning, skin infections, and abscesses to lethal bacteremia and endocarditis (21). Its adaptability is facilitated by a range of virulence factors and by factors that contribute to its survival and fitness in multiple environments within the host (2). Among the contributors to S. aureus fitness are a range of transmembrane transporters that can mediate uptake of nutrients or export of metabolites and toxic substances, including in some cases export of antimicrobial agents causing antimicrobial resistance (5,9,15,16,26,28).We found previously that several staphylococcal efflux pumps were selectively overexpressed within the milieu of experimental subcutaneous abscesses and that these pumps not only contributed to antimicrobial resistance when overexpressed in vitro but also contributed to bacterial survival within the abscess in the absence of antimicrobial agents (12). Both NorB and Tet38, members of the major facilitator superfamily (MFS) of secondary transporters encoded on the S. aureus chromosome, contributed selectively to fitness in the abscess environment and conferred low-level resistance to fluoroquinolones and tetracycline, respectively.In order to identify other transporters that might also be important for bacterial survival in this environment, we evaluated the MFS transporter NorD. Like norB and tet38, norD expression was selectively upregulated in abscesses. Herein, we present data indicating that NorD contributes substantially to S. aureus fitness in an abscess infection model, and we identified a low-iron environment as a potential ...

Site-Specific Mutation of the Sensor Kinase GraS in Staphylococcus aureus Alters the Adaptive Response to Distinct Cationic Antimicrobial Peptides

“…All mutant strains were generated in an S. aureus MW2 background with in-frame deletion of target genes by allelic replacement, using the temperature-sensitive plasmid pMAD as described previously (15,30). S. aureus MW2 (USA400), a prototypical clinical methicillin-resistant S. aureus (MRSA) isolate, has been well characterized genotypically (e.g., available genome sequence information) and is virulent in vivo in animal models (31)(32)(33). Briefly, PCR was used to amplify an ϳ2-kb fragment comprising a 1-kb fragment upstream and another 1-kb fragment downstream of graS using genomic DNA as the template.…”

Efflux Transporter of Siderophore Staphyloferrin A in Staphylococcus aureus Contributes to Bacterial Fitness in Abscesses and Epithelial Cells

“…We previously demonstrated that the expression of some efflux pump genes in S. aureus was upregulated in murine subcutaneous abscesses and that these genes contribute to S. aureus survival in abscesses and on skin (1,2). Thus, we infected mice with strain RN6390 and assessed the relative expression of sfaA and sbnD in total bacterial RNA recovered from an abscess versus that of in vitro cultures using real-time quantitative reverse transcription- (Fig.…”

“…norA, like sfaA and sbnD, encodes an MFS-type efflux pump and is regulated by Fur (9, 12, 17). Fur also indirectly regulates the expression of norD encoding MFS-type efflux pump NorD (2). norD also contributes MW2 (B).…”

“…A number of efflux transporters conferring antimicrobial resistance are present in S. aureus. Of these transporters, we previously demonstrated that the expression levels of norB (resistance to quinolones), norD, and tet38 (resistance to tetracycline) genes were increased in murine subcutaneous abscesses and that these genes contribute to S. aureus survival in abscesses and on skin (1,2). Furthermore, we found that antimicrobial fatty acids are natural substrates of Tet38 (3).…”

“…Furthermore, we found that antimicrobial fatty acids are natural substrates of Tet38 (3). NorB, NorD, and Tet38 are members of the major facilitator superfamily (MFS)-type efflux pump encoded on the S. aureus chromosome and are energized by proton motive force (2,4).…”