Identification of a Staphylococcus aureus Efflux Pump Regulator Using a DNA-Protein Affinity Technique.

In this chapter, we describe the step-by-step identification of a putative regulator protein and demonstrate the function of this protein as a repressor of the expression of a specific efflux pump, causing resistance to quinolones in Staphylococcus aureus. We show that the knockout gene mutant has an increase in transcript levels of the target efflux pump when compared to that of the S. aureus parental strain RN6390. We provide a detailed protocol that includes the identification of the DNA-binding transcriptional regulatory protein from S. aureus cell extracts using DNA sequences linked to magnetic beads. In addition, we describe the real-time qRT-PCR assays and MIC testing to evaluate the effects of the regulator on S. aureus drug resistance phenotype.

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

The siderophores staphyloferrin A (SA) and staphyloferrin B (SB) of Staphylococcus aureus are essential for iron acquisition in the iron-restricted environment of the host, such as in subcutaneous abscesses. SA and SB are secreted by SfaA and SbnD transporters, respectively. To assess the further function of SfaA and SbnD in S. aureus fitness, we tested its effect on murine abscess models and intracellular replication in epithelial cells. Bacterial fitness in abscesses and in epithelial cells was studied, by comparing the parental strains RN6390 and MW2 and their ΔsfaA and ΔsbnD mutants using competition assays in a murine abscess model and invasion and replication assays with human lung adenocarcinoma cell line A549. In the murine abscess model using equal inocula of a ΔsfaA or ΔsbnD mutant and the wild-type RN6390 strain, the ΔsfaA mutant exhibited growth defects of 2.2-fold. Additionally, replication of the ΔsfaA mutant within A549 cells was decreased 3.0-fold. In complementation experiments, the ΔsfaA mutant carrying plasmid-borne sfaA restored the growth fitness in abscesses and epithelial cells. The ΔsbnD mutant, in contrast, showed no growth defect in either abscesses or epithelial cells. Our findings demonstrate that the efflux transporter of the siderophore SA contributes to the ability of S. aureus to replicate in abscesses and epithelial cells. Furthermore, fitness of S. aureus in these sites of replication is not compromised by the absence of transporter SbnD.

Regulation of expression of abcA and its response to environmental conditions.

The ATP-dependent transporter gene abcA in Staphylococcus aureus confers resistance to hydrophobic ß-lactams. In strain ISP794, abcA is regulated by the transcriptional regulators MgrA and NorG and shares a 420-nucleotide intercistronic region with the divergently transcribed pbp4 gene, which encodes the transpeptidase Pbp4. Exposure of exponentially growing cells to iron-limited media, oxidative stress, and acidic pH (5.5) for 0.5 to 2 h had no effect on abcA expression. In contrast, nutrient limitation produced a significant increase in abcA transcripts. We identified three additional regulators (SarA, SarZ, and Rot) that bind to the overlapping promoter region of abcA and pbp4 in strain MW2 and investigated their role in the regulation of abcA expression. Expression of abcA is decreased by 10.0-fold in vivo in a subcutaneous abscess model. In vitro, abcA expression depends on rot and sarZ regulators. Moenomycin A exposure of strain MW2 produced an increase in abcA transcripts. Relative to MW2, the MIC of moenomycin was decreased 8-fold for MW2ΔabcA and increased 10-fold for the MW2 abcA overexpresser, suggesting that moenomycin is a substrate of AbcA.

Native efflux pumps contribute resistance to antimicrobials of skin and the ability of Staphylococcus aureus to colonize skin.

BACKGROUND: Staphylococcus aureus colonizes skin in the presence of antimicrobial fatty acids and polyamines. The chromosomally encoded Tet38 efflux transporter confers resistance to tetracycline and fitness in abscesses, but its natural substrates and those of the Nor quinolone efflux pumps are unknown. METHODS: Susceptibility of tet38 and other pump mutants to and pump gene induction by fatty acids and polyamines were compared. Transport of fatty acids by Tet38 was determined in membrane vesicles. Survival on skin was tested in an adapted mouse skin infection model. RESULTS: The tet38 expression caused a 5- to 8-fold increase in resistance to palmitoleic and undecanoic acids but not polyamines. Subinhibitory concentrations of these fatty acids induced 4-fold increases in tet38 transcripts and competitively inhibited transport of Hoechst 33 342 dye in Tet38 membrane vesicles. Colonization of skin in BALB/c mice was decreased 5-fold in a Δtet38 mutant, which was complemented by plasmid-encoded tet38. Although polyamine minimum inhibitory concentrations (MICs) decreased 4-fold in a norC::cat mutant and increased 8-fold with norC overexpression, spermidine did not induce expression of norC and other pump genes, and norC::cat exhibited wild-type colonization. CONCLUSION: Antibacterial fatty acids may be natural substrates of Tet38, which contributes to resistance and the ability of S. aureus to colonize skin.

Association of norB overexpression and fluoroquinolone resistance in clinical isolates of Staphylococcus aureus from Korea.

OBJECTIVES: Although the prevalence of fluoroquinolone resistance among methicillin-resistant Staphylococcus aureus (MRSA) is known to be higher than in methicillin-susceptible S. aureus (MSSA), the reasons have never been identified. METHODS: We randomly selected 115 isolates of S. aureus collected from 10 different hospitals in Korea between June 2009 and May 2011. To investigate the difference in fluoroquinolone resistance mechanisms between MRSA and MSSA, we evaluated gyrA and parC mutations and the relative expression of the multidrug efflux pump genes norA, norB and norC. RESULTS: All 62 ciprofloxacin-resistant S. aureus had either gyrA or parC mutations. The S84L mutation of gyrA (59/62, 95.2%) and the S80F mutation of parC (61/62, 98.4%) were the most common. Fifty-eight (93.6%) strains had both the S84L mutation of gyrA and the S80F mutation of parC. Among the 115 isolates, norB overexpression was the most common, occurring in 49 (42.6%) strains. There were only two (1.7%) strains with norA overexpression and none with norC overexpression. Strains overexpressing norB were more common among ciprofloxacin-resistant S. aureus (33/62, 53.2%) than ciprofloxacin-susceptible S. aureus (16/53, 30.2%) (P = 0.013). When we analysed 62 ciprofloxacin-resistant S. aureus strains, those overexpressing norB were more common in ciprofloxacin-resistant MRSA (28/46, 60.9%) than in ciprofloxacin-resistant MSSA (5/16, 31.3%) (P = 0.041). CONCLUSIONS: Increased expression of norB can be a factor that contributes to ciprofloxacin resistance in MRSA strains.

Reduced aeration affects the expression of the NorB efflux pump of Staphylococcus aureus by posttranslational modification of MgrA.

We previously showed that at acid pH, the transcription of norB, encoding the NorB efflux pump, increases due to a reduction in the phosphorylation level of MgrA, which in turn leads to a reduction in bacterial killing by moxifloxacin, a substrate of the NorB efflux pump. In this study, we demonstrated that reduced oxygen levels did not affect the transcript levels of mgrA but modified the dimerization of the MgrA protein, which remained mostly in its monomeric form. Under reduced aeration, we also observed a 21.7-fold increase in the norB transcript levels after 60 min of growth that contributed to a 4-fold increase in the MICs of moxifloxacin and sparfloxacin for Staphylococcus aureus RN6390. The relative proportions of MgrA in monomeric and dimeric forms were altered by treatment with H(2)O(2), but incubation of purified MgrA with extracts of cells grown under reduced but not normal aeration prevented MgrA from being converted to its dimeric DNA-binding form. This modification was associated with cleavage of a fragment of the dimerization domain of MgrA without change in MgrA phosphorylation and an increase in transcript levels of genes encoding serine proteases in cells incubated at reduced aeration. Taken together, these data suggest that modification of MgrA by proteases underlies the reversal of its repression of norB and increased resistance to NorB substrates in response to reduced-aeration conditions, illustrating a third mechanism of posttranslational modification, in addition to oxidation and phosphorylation, that modulates the regulatory activities of MgrA.

Implication of the NorB efflux pump in the adaptation of Staphylococcus aureus to growth at acid pH and in resistance to moxifloxacin.

Staphylococcus aureus is an important pathogen that adapts and survives in low-pH environments. One component of this adaptation involves the regulation of genes encoding bacterial transporters that could affect response to antibiotics under these conditions. We previously demonstrated that the transcriptional regulator MgrA in its phosphorylated form (MgrA-P) represses the expression of norB, encoding the NorB multidrug resistance efflux pump. In this study, we focused on changes in the expression of mgrA at the transcriptional and posttranslational levels, following a shift from pH 7.0 to pH 4.5. We then correlated those changes with modifications in transcript levels of norB and to resistance to moxifloxacin, a substrate of NorB. At pH 4.5, S. aureus MgrA increased 2-fold and MgrA-P decreased 4-fold, associated with an 8-fold increase in norB transcripts and a 6-fold reduction in bacterial killing by moxifloxacin, and the phenomenon was dependent on intact mgrA. Taken together, these new data showed that phosphoregulation of MgrA at low pH reverses its repression of norB expression, conferring resistance to moxifloxacin.

Phosphorylation of MgrA and its effect on expression of the NorA and NorB efflux pumps of Staphylococcus aureus.

MgrA is a global regulator in Staphylococcus aureus that controls the expression of diverse genes encoding virulence factors and multidrug resistance (MDR) efflux transporters. We identified pknB, which encodes the (Ser/Thr) kinase PknB, in the S. aureus genome. PknB was able to autophosphorylate as well as phosphorylate purified MgrA. We demonstrated that rsbU, which encodes a Ser/Thr phosphatase and is involved in the activation of the SigB regulon, was able to dephosphorylate MgrA-P but not PknB-P. Serines 110 and 113 of MgrA were found to be phosphorylated, and Ala substitutions at these positions resulted in reductions in the level of phosphorylation of MgrA. DNA gel shift binding assays using norA and norB promoters showed that MgrA-P was able to bind the norB promoter but not the norA promoter, a pattern which was the reverse of that for unphosphorylated MgrA. The double mutant MgrA(S110A-S113A) bound to the norA promoter but not the norB promoter. The double mutant led to a 2-fold decrease in norA transcripts and a 2-fold decrease in the MICs of norfloxacin and ciprofloxacin in strain RN6390. Thus, phosphorylation of MgrA results in loss of binding to the norA promoter, but with a gain of the ability to bind the norB promoter. Loss of the ability to phosphorylate MgrA by Ala substitution resulted in increased repression of norA expression and in reductions in susceptibilities to NorA substrates.

Posttranslational modification influences the effects of MgrA on norA expression in Staphylococcus aureus.

MgrA is a global regulator in Staphylococcus aureus. Differences in the effects of MgrA on norA expression have been reported for different strains, which varied in rsbU, a gene that affects the expression of sigB, which encodes an alternative sigma factor involved in stress responses. We hypothesized that MgrA was modified by sigB-dependent factors that affected its ability to control the expression of the norA efflux pump. Heterologously expressed MgrA purified from Escherichia coli was incubated with crude extracts (CE) from strains RN6390 (rsbU) and SH1000 (rsbU(+)) and tested for binding to the norA promoter. Purified MgrA exhibited greater binding to norA promoter DNA after being incubated with SH1000 CE than MgrA incubated with the RN6390 CE. Phosphorylation of MgrA occurring in cell extracts caused it to lose the ability to bind norA promoter DNA. Overexpression of pknB, encoding a candidate serine/threonine kinase, produced increased phospho-MgrA and led to a fivefold increase in the transcript level of norA for both RN6390 and SH1000, as well as a fourfold increase in the MICs of norfloxacin and ciprofloxacin for these two strains. The levels of expression of pknB in RN6390 and SH1000, however, indicated that additional factors related to rsbU or sigB contribute to the differential regulatory effects of MgrA on norA expression.

The transcriptional regulators NorG and MgrA modulate resistance to both quinolones and beta-lactams in Staphylococcus aureus.

MgrA is a known regulator of the expression of several multidrug transporters in Staphylococcus aureus. We identified another regulator of multiple efflux pumps, NorG, by its ability, like that of MgrA, to bind specifically to the promoter of the gene encoding the NorA efflux pump. NorG is a member of the family of the GntR-like transcriptional regulators, and it binds specifically to the putative promoters of the genes encoding multidrug efflux pumps NorA, NorB, NorC, and AbcA. Overexpression of norG produces a threefold increase in norB transcripts associated with a fourfold increase in the level of resistance to quinolones. In contrast, disruption of norG produces no change in the level of transcripts of norA, norB, and norC but causes an increase of at least threefold in the transcript level of abcA, associated with a fourfold increase in resistance to methicillin, cefotaxime, penicillin G, and nafcillin. Overexpression of cloned abcA caused an 8- to 128-fold increase in the level of resistance to all four beta-lactam antibiotics. Furthermore, MgrA and NorG have opposite effects on norB and abcA expression. MgrA acts as an indirect repressor for norB and a direct activator for abcA, whereas NorG acts as a direct activator for norB and a direct repressor for abcA.

NorC, a new efflux pump regulated by MgrA of Staphylococcus aureus.

NorC, a new efflux pump, like NorB, contributes to quinolone resistance that includes resistance to moxifloxacin and sparfloxacin in Staphylococcus aureus. norC expression, like that of norB and tet38, is negatively regulated by MgrA, and overexpression of both norC and norB contributes to the quinolone resistance phenotype of an mgrA mutant.

DX-619, a novel des-fluoro(6) quinolone manifesting low frequency of selection of resistant Staphylococcus aureus mutants: quinolone resistance beyond modification of type II topoisomerases.

DX-619, a novel des-fluoro(6) quinolone, was 16- to 32-fold, twofold, and four- to eightfold more potent than ciprofloxacin, gemifloxacin, and garenoxacin, respectively, against wild-type Staphylococcus aureus. DX-619 manifested equal fourfold increases in MIC against a common parC mutant and a common gyrA mutant and selected for mutants at up to two- to fourfold its MIC, consistent with dual-targeting properties. Of the four independent single-step mutants selected, two had new single mutations in parC (V87F and R17H), and two shared a new gyrA mutation (A26V), one with an additional deletion mutation in parE (delta215-7). By allelic exchange, the ParC but not the GyrA or ParE mutation was shown to be fully responsible for the resistance phenotypes, suggesting an as yet undefined mechanism of resistance operating in conjunction with type II topoisomerase mutations contributed to resistance to DX-619. Studies with purified topoisomerase IV and gyrase from S. aureus also showed that DX-619 had similar activity against topoisomerase IV and gyrase (50% stimulation of cleavage complexes concentration, 1.25 and 0.62 to 1.25 mug/ml, respectively). Susceptibility studies with DX-619 and an array of efflux pump substrates with and without reserpine, an inhibitor of efflux pumps, suggested that resistance in DX-619-selected mutants is affected by mechanisms other than mutations in topoisomerases or known reserpine-inhibitable pumps in S. aureus and thus are likely novel.

Characterization of NorR protein, a multifunctional regulator of norA expression in Staphylococcus aureus.

We characterized a Staphylococcus aureus norA gene expression regulator, NorR, initially identified from its binding to the norA promoter. The norR gene was 444 bp in length, located approximately 7 kb upstream from the norA gene, and encoded a predicted 17.6-kDa protein. Overexpression of norR in wild-type S. aureus strain ISP794 led to a fourfold decrease in sensitivity to quinolones and ethidium bromide and an increase in the level of norA transcripts, suggesting that NorR acts as a positive regulator of norA expression. Overexpression of norR in sarA and agr mutants did not alter quinolone sensitivity or levels of norA transcription, indicating that the presence of these two global regulatory systems is necessary for NorR to affect the expression of norA. Insertion and disruption of norR in ISP794 increased resistance to quinolones by 4- to 16-fold but had no effect on norA transcription, suggesting that NorR acts as a repressor for another unidentified efflux pump or pumps. These mutants also exhibited an exaggerated clumping phenotype in liquid media, which was complemented fully by a plasmid-encoded norR gene. Collectively, these results indicate that NorR is a multifunctional regulator, affecting cell surface properties as well as the expression of NorA and likely other multidrug resistance efflux pumps.