The Anti-Repressor MecR2 Promotes the Proteolysis of the <em>mecA</em> Repressor and Enables Optimal Expression of β-lactam Resistance in MRSA

<div><p>Methicillin-resistant <em>Staphylococcus aureus</em> (MRSA) is an important human pathogen, which is cross-resistant to virtually all β-lactam antibiotics. MRSA strains are defined by the presence of <em>mecA</em> gene. The transcription of <em>mecA</em> can be regulated by a sensor-inducer (MecR1) and a repressor (MecI), involving a unique series of proteolytic steps. The induction of <em>mecA</em> by MecR1 has been described as very inefficient and, as such, it is believed that optimal expression of β-lactam resistance by MRSA requires a non-functional MecR1-MecI system. However, in a recent study, no correlation was found between the presence of functional MecR1-MecI and the level of β-lactam resistance in a representative collection of epidemic MRSA strains. Here, we demonstrate that the <em>mecA</em> regulatory locus consists, in fact, of an unusual three-component arrangement containing, in addition to <em>mecR1-mecI</em>, the up to now unrecognized <em>mecR2</em> gene coding for an anti-repressor. The MecR2 function is essential for the full induction of <em>mecA</em> expression, compensating for the inefficient induction of <em>mecA</em> by MecR1 and enabling optimal expression of β-lactam resistance in MRSA strains with functional <em>mecR1-mecI</em> regulatory genes. Our data shows that MecR2 interacts directly with MecI, destabilizing its binding to the <em>mecA</em> promoter, which results in the repressor inactivation by proteolytic cleavage, presumably mediated by native cytoplasmatic proteases. These observations point to a revision of the current model for the transcriptional control of <em>mecA</em> and open new avenues for the design of alternative therapeutic strategies for the treatment of MRSA infections. Moreover, these findings also provide important insights into the complex evolutionary pathways of antibiotic resistance and molecular mechanisms of transcriptional regulation in bacteria.</p> </div

<i>mecR2</i> interferes with the <i>mecI</i>-mediated repression of β-lactam resistance.

<p>(A) Co-overexpression of <i>mecI</i> and <i>mecR2</i> region (COL+<i>mecI-mecR2</i>) reverted the effect of <i>mecI</i> overexpression (COL+<i>mecI</i>) on the oxacillin-resistance phenotype in strain COL, as evaluated with diffusion disks containing 1 mg of oxacillin. (B) Northern blotting analysis of <i>mecA</i> transcription shows that in the presence of <i>mecR2</i> locus the repressor effect of <i>mecI</i> is reverted.</p

MecR2 interacts directly with MecI, interfering with the binding of MecI to the <i>mecA</i> promoter and fostering the proteolysis of MecI.

<p>(A) <i>In vivo</i> analysis of the MecR2::MecI interaction using the bacterial two-hybrid strategy. This strategy is based on the restoration of the adenylate cyclase (CyaA) activity of <i>E. coli</i>, which activates a specific reporter gene, <i>lacZ</i>. Interactions between protein fusions were evaluated in liquid cultures through the hydrolysis of the chromogenic X-gal substrate by the activated β-galoctasidase. The MecR2::MecI interaction was evaluated using the eight possible combinations: fusions either with T25 or T18 fragments of CyaA at either the N′ or C′ terminals. Tube 1, T18-MecR2::MecI-T25; tube 2, T18-MecR2::T25-MecI; tube 3, MecR2-T18::MecI-T25; tube 4, MecR2-T18::T25-MecI; tube 5, MecR2-T25::T18-MecI; tube 6, T25-MecR2::T18-MecI; tube 7, MecR2-T25::MecI-T18; tube 8, T25-MecR2::MecI-T18; tube 9, positive control provided by the manufacturer (Zip-T25::Zip-T18); tube 10, negative control (T25::T18); tube 11, “in-house” positive control testing the MecI-MecI interaction T18-MecI::T25-MecI. (B) Electrophoretic mobility shift assay (EMSA) of the binding of purified MecI to a labeled 212 bp DNA fragment encompassing the <i>mecA</i> promoter in the presence of purified MecR2. MecI concentration was constant in all binding reactions (0.05 µg). Lane 1, negative control, labeled DNA only; lane 2, 8-fold excess of MecI; lane 3, 4-fold excess of MecI; lane 4, binding control, MecI only; lane 5, 2-fold excess of MecI; lane 6, equimolar amounts of MecI and MecR2; lane 7, 2-fold excess of MecR2; lane 8, control for specific binding, MecI with a 125 molar excess of unlabelled DNA. (C) Western blotting analysis of MecI cleavage in total protein extracts (60–80 mg/lane). Lane 1, prototype strain N315; lane 2, <i>mecR2</i> null-mutant (N315::Δ<i>mecR</i>2); lane 3, strain HT0350 co-overexpressing MecI and MecR2 (HT0350+<i>mecImecR2</i>); lane 4, strain HT0350 overexpressing MecI (HT0350+<i>mecI</i>). Cultures of N315 and N315::Δ<i>mecR2</i> cultures were induced with a sub-MIC concentration of oxacillin (0.05 mg/L).</p

Effect of <i>mecR2</i> on the induction of <i>mecA</i> transcription.

<p>(A) Northern blot and (B) qRT-PCR analysis of the <i>mecA</i> induction profile in parental strain N315, <i>mecR2</i> null-mutant (N315::Δ<i>mecR2</i>) and complemented mutant (N315::Δ<i>mecR2</i>+<i>spac</i>::<i>mecR2</i>, IPTG 100 µM). Cultures were induced with a sub-MIC concentration of oxacillin (0.05 mg/L) and samples were taken at 0′, 5′, 10′ 30′ and 60′.</p

Model for the <i>mecA</i> induction by MecR1-MecI-MecR2.

<p>In the presence of a β-lactam antibiotic, MecR1 is activated and rapidly induces the expression of <i>mecA</i> and <i>mecR1-mecI-mecR2</i>. The anti-repressor activity of MecR2 is essential to sustain the <i>mecA</i> induction since it promotes the inactivation of MecI by proteolytic cleavage. In the absence of β-lactams, MecR1 is not activated and a steady state is established with stable MecI-dimers bound to the <i>mecA</i> promoter and residual copies of MecR1 at the cell membrane.</p

The <i>mecR2</i> function is not dependent of <i>mecR1</i> neither of the β-lactamase locus.

<p>(A) Prototype strain HT0350 is negative for <i>mecR1-mecI</i> and for the β-lactamase locus. Co-overexpression of <i>mecI</i> and <i>mecR2</i> in strain HT0350 (HT0350+<i>mecI-mecR2</i>), reverts the effect of <i>mecI</i> overexpression (HT0350+<i>mecI</i>). (B) The strategy used to delete <i>mecR2</i> in prototype strain N315 generated an intermediate mutant that has lost the β-lactamase plasmid. The chromosomal <i>mecR2</i> deletion was then transduced back to the parental strain generating a β-lactamase positive <i>mecR2</i> null mutant. In both variants, the deletion of <i>mecR2</i> caused a sharp decrease of the resistance level to oxacillin. (C) Prototype strain HU25 is positive for <i>mecR2</i> and the β-lactamase locus and has a truncated non-functional MecI and, as such, the <i>mecA</i> expression is under the exclusive control of <i>bla</i> regulatory genes. Deletion of <i>mecR2</i> in strain HU25 (HU25::Δ<i>mecR2</i>) has no effect on the phenotypic expression of oxacillin resistance.</p

<i>mecR2</i> transcription analysis.

<p>qRT-PCR analysis of the <i>mecR2</i> induction profile in parental strain N315 and its complemented <i>mecR2</i> mutant (N315::Δ<i>mecR2</i>+<i>spac</i>::<i>mecR2</i>, IPTG 100 µM). Cultures were induced with a sub-MIC concentration of oxacillin (0.05 mg/L).</p

Reconstruction of the <i>mecA</i> regulatory locus in prototype strain COL.

<p>Reconstruction of the <i>mecR1-mecI</i> locus in the chromosome of strain COL (COL::RI) causes a decrease of the resistance level to oxacillin, which can be reverted by the reconstruction of the full <i>mecA</i> regulatory locus, <i>mecR1-mecI-mecR2</i> (COL::RI-R2). Control experiments with <i>mecI</i> overexpressed in trans (COL::RI+<i>mecI</i> and COL::RI-R2+<i>mecI</i>) demonstrate that the functions of <i>mecR1</i> and <i>mecR2</i> are not affected by high levels of MecI. For comparative purposes the overexpression of <i>mecI</i> in parental strain COL (COL+<i>mecI</i>) is also shown. The oxacillin-resistance levels were evaluated by diffusion disks containing 1 mg of oxacillin (left) or by population analysis profiles (PAP's) (right).</p

Role of <i>mecR2</i> on the optimal expression of β-lactam resistance.

<p>(A) Deletion of <i>mecR2</i> from the chromosome of strain N315 (N315::Δ<i>mecR2</i>) causes a decrease on the resistance level to oxacillin, which can be reverted upon complementation with <i>mecR2</i> expressed from an inducible promoter (N315::Δ<i>mecR2</i>+<i>spac</i>::<i>mecR2</i>) in the presence of the inducer (IPTG 100 µM). (B) The poor expression of oxacillin resistance by recombinant strain COL::RI, can also be reverted upon complementation with <i>mecR2</i> expressed from an inducible promoter (COL::RI+<i>spac</i>::<i>mecR2</i>) in the presence of the inducer (IPTG 100 µM). The oxacillin-resistance levels were evaluated by diffusion disks containing 1 mg of oxacillin (left) and by population analysis profiles (PAP's) (right).</p

<i>mecR2</i> is essential for the optimal expression of β-lactam resistance in strains with functional <i>mecI-mecR1</i> regulatory locus.

<p>(A) Deletion of <i>mecR2</i> from the chromosome of prototype epidemic strains USA100, USA200 and USA600 harboring SCC<i>mec</i> type II causes a decrease on the resistance level to oxacillin, which can be reverted upon complementation with <i>mecR2</i> expressed from an inducible promoter (<i>spac</i>::<i>mecR2</i>) in the presence of the inducer (IPTG 100 µM). (B) Northern blot analysis of the <i>mecA</i> induction profile in parental strains USA100, USA200 and USA600 and respective <i>mecR2</i> null-mutants. Cultures were induced with a sub-MIC concentration of oxacillin (0.05 mg/L) and samples were taken at 0′, 10′ and 60′. For comparative purposes the profile of parental strain N315 and <i>mecR2</i> null-mutant were also repeated. Note that film was exposed for 4 h whereas in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002816#ppat-1002816-g005" target="_blank">Figure 5A</a> it was exposed for 48 h.</p