Reduced Aeration Affects the Expression of the NorB Efflux Pump of Staphylococcus aureus by Posttranslational Modification of MgrA

Abstract

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.

Effects of Chlorophyll-Derived Efflux Pump Inhibitor Pheophorbideaand Pyropheophorbideaon Growth and Macrolide Antibiotic Resistance of Indicator and Anaerobic Swine Manure Bacteria

“…Subsequent spectrophotometric determination of growth parameters of S. aureus confirmed that pyp in combination with erythromycin was most effective, that is, synergistic already at the lower concentration of 0.5 g mL −1 , in highly resistant cultures under anaerobic conditions ( Figures 4 and 5(c)) and revealed that pyp was intrinsically inhibitory to anaerobic, naïve cultures ( Figures 4 and 5(b)). As could be expected [8,94], the growth rate in the absence and at a low concentration of erythromycin (0.0625 g mL −1 ) (Figure 4(b)) was greatest in the aerobic, naïve cultures and lowest for the highly resistant strain-the latter effect being due to a "fitness cost" as entailed by most antimicrobial resistance mechanisms [88]. The intrinsic growth inhibition of anaerobic, naïve cultures of S. aureus indicates that pyp, provided it equals php in its function as an EPI [51,52], acts against a metabolically regulated MDR pump with broader, compound nonspecific functions unrelated to antimicrobials.…”

“…Thirdly, efflux-mediated antimicrobial resistance is controlled by the metabolic condition of bacteria and can be altered by a switch from an aerobic to an anaerobic metabolism through the influence of metabolically integrated global regulators, different endogenous cellular metabolites, such as ROS from aerobic respiration or anaerobic fermentation end products which often times are the natural pump substrates, and an altered transmembrane electrochemical proton gradient as energy source for secondary active transporters [27,28,32,80]. Hence, it can be presumed that the higher level of induced resistance in S. aureus under anaerobic conditions was contingent on a switch from glycolysis, the pentose phosphate pathway and ROS generating tricarboxylic acid cycle under aerobiosis to anaerobic glucose fermentation and ATPase-mediated proton efflux, generating a greater motive force for erythromycin-proton antiporters [28,80,94], such as LmrS, MdeA, Mef(A), and conceivably NorA [27,32,[95][96][97].…”

“…ROS have antimicrobial activity and contribute to dysregulation primarily of MDR efflux pump expression by oxidative inactivation of global regulator proteins [29,32]. For example, in S. aureus, reduced aeration causes an increase of norB expression and hence increased resistance to antimicrobial NorB substrates via an effect on MgrA, an oxidative stresssensitive global regulator of 350 genes including norA and norB [92][93][94]. Thirdly, efflux-mediated antimicrobial resistance is controlled by the metabolic condition of bacteria and can be altered by a switch from an aerobic to an anaerobic metabolism through the influence of metabolically integrated global regulators, different endogenous cellular metabolites, such as ROS from aerobic respiration or anaerobic fermentation end products which often times are the natural pump substrates, and an altered transmembrane electrochemical proton gradient as energy source for secondary active transporters [27,28,32,80].…”

Regulation of Expression of abcA and Its Response to Environmental Conditions

“…We have recently shown that reduced aera- tion induces a 4-fold decrease of abcA expression (39). Exposure of exponentially growing cells to iron limitation, oxidative stress (H 2 O 2 ), and acidic pH (5.5), all of which may exist in abscess environments for 0.5 to 2 h, had no effect on abcA or pbp4 expression (data not shown).…”

“…Because other efflux pumps, such as NorA (38) and NorB (39), are expressed differently in response to environmental conditions that could affect the activity of the antimicrobials that are their substrates, we evaluated a range of conditions that have been shown to affect expression of other pumps for their effects on abcA expression. Expression data for response to different conditions included abscess and exposure to antimicrobials (cell envelope stress).…”