MpeR regulates the mtr efflux locus in Neisseria gonorrhoeae and modulates antimicrobial resistance by an iron-responsive mechanism

Abstract

Previous studies have shown that the MpeR transcriptional regulator produced by Neisseria gonorrhoeae represses the expression of mtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation of mpeR has been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of the mtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that the mtrR gene, which encodes a direct repressor (MtrR) of mtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of the mtrCDE efflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression of mtrR was direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of the mtr efflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.

Fig 1

Regulation of mtrF by MpeR. (A) DNA sequence of a 161-bp fragment used in mtrF-lacZ expression analysis, with an asterisk marking the start of transcription. (B) The specific activities (nanomoles of o-nitrophenyl-β-d-galactopyranoside hydrolyzed per mg of protein) for measuring mtrF-lacZ expression in the FA19 mtrF-lacZ, JF5 mtrF-lacZ, and AD1 (JF5 mtrF-lacZ mpeR⁺ [Table 1]) strains are shown. Samples were harvested from gonococci to the mid-log phase of growth. The above-described experiment was done in triplicate and is a representative example of three independent experiments. Error bars represent one standard deviation. The difference in expression of mtrF-lacZ between FA19 and JF5 was significant (P = 0.001), as was the difference in expression of mtrF-lacZ between JF5 and AD1 (P = 0.001).

Fig 2

Maximal expression of mpeR. The specific activities (nanomoles of o-nitrophenyl-β-d-galactopyranoside hydrolyzed per mg of protein) for measuring mpeR-lacZ expression in strain AD2 (FA19 mpeR-lacZ [Table 1]) at different phases of growth under iron-replete conditions are shown. The above-described experiment was done in triplicate and is a representative example of three independent experiments. Error bars represent one standard deviation, and differences in expression of mpeR-lacZ at the early log, mid-log, late log, and stationary phases of growth were significant (P < 0.05).

Fig 3

Expression of mpeR under iron-replete and -depleted conditions. The growth of strain AD2 (FA19 mpeR-lacZ [Table 1]) under either iron-replete (+Fe) or iron-depleted (−Fe) conditions (top panel) and the expression of mpeR-lacZ (bottom panel) are shown. Samples from the growth curve were harvested from gonococci at the late log phase of growth. The experiment was done in triplicate and is a representative example of three independent experiments. Error bars represent one standard deviation. The difference in expression of mpeR-lacZ between cultures grown under iron-replete and iron-depleted conditions was significant (P = 0.007).

Fig 4

Regulation of mtrR by MpeR. mtrR expression in the FA19 mtrR-lacZ, JF5 mtrR-lacZ, and AD3 (JF5 mtrR-lacZ mpeR⁺ [Table 1]) strains as determined by translational lacZ fusions. Samples were harvested from gonococci at the late log phase of growth. The above-described experiment was done in triplicate and is a representative example of three independent experiments. Error bars represent one standard deviation. The difference in expression of mtrR-lacZ between FA19 and JF5 was significant (P ≤ 0.001), as was the difference in mtrR-lacZ expression between JF5 and AD3 (P ≤ 0.001).

Fig 5

Iron modulates levels of MtrR. Wild-type strain FA19 was grown under iron-replete and -depleted conditions. (A) Samples for each growth condition were harvested at the late log phase of growth, solubilized, and separated by SDS-PAGE; the electrophoretic mobility of each molecular mass marker is shown by the arrow. (B) Following transfer to nitrocellulose, the blot was probed with anti-MtrR antiserum and the difference in MtrR levels was determined as described in Materials and Methods.

Fig 6

Regulation of mtrCDE expression is modulated by mpeR expression and levels of free iron but not mtrR expression. (A) mtrCDE expression levels in the FA19 mtrC-lacZ, JF5 mtrC-lacZ, and AD4 (JF5 mtrC-lacZ mpeR⁺ [Table 1]) strains were measured. The difference in mtrCDE expression between strains FA19 and JF5 was significant (P = 0.002), as was that between strains JF5 and AD4 (P = 0.0001). (B) mtrCDE expression levels in the FA19 mtrC-lacZ and JF1 mtrC-lacZ (Table 1) strains grown under either iron-replete or iron-depleted conditions were determined. The difference in mtrCDE expression between FA19 +Fe and FA19 −Fe was significant (P = 0.0023), while that between JF1 +Fe and JF1 −Fe was not significant (P = 0.3132). The above-described experiments were done in triplicate and are representative examples of three independent experiments. Error bars represent one standard deviation.

Fig 7

MpeR binds specifically to the upstream region of mtrR. The 363-bp mtrR upstream region was radiolabeled, and 10 ng of this DNA was incubated with 15 μg of MBP-MpeR alone. In order to demonstrate that binding of MBP-MpeR to mtrR was specific, increasing concentrations (2×, 5×, and 10×) of cold specific competitor (mtrR) or increasing concentrations (2× and 10×) of cold nonspecific competitor (misR) were added. The arrow indicates the specific complex, while the asterisk indicates the nonspecific complex.

Fig 8

Model demonstrating the regulatory properties of MpeR that affect the high-level resistance mediated by the MtrCDE efflux system in an iron-dependent manner. (A) Under iron-replete conditions, Fur complexed with iron represses the expression of mpeR. (B) Under iron-depleted conditions, mpeR is derepressed and acts to repress the expression levels of both mtrF and mtrR in strain FA19. In strain FA1090, MpeR activates the expression of fetA (16). For all genes, the bent arrows identify promoter elements, while straight arrows indicate MpeR-activated genes and blocked lines designate MpeR-repressed genes.