Electrophoretic mobility shift assay of relative MecI and BlaI affinities for the <i>mecA</i> promoter sequence.

<p>Binding reaction was performed with a labeled 200 bp DNA fragment encompassing the <i>mecA</i> promoter mixed with increasing amounts of purified MecI (panel A) and BlaI (panel B). Lanes are as follows: lane 1 – no protein (control); lane 2 – 0,001 µg; lane 3 – 0,01 µg; lane 4 – 0,05 µg; lane 5 – 0,1 µg; lane 6 – 0,25 µg; lane 7 – 0,5 µg; lane 8 – 1 µg; lane 9 – 0,1 ug of protein with a 125 molar excess of unlabelled DNA (specific competition).</p

Northern blot analysis of the <i>mecA</i> induction profile in three prototype MRSA strains.

<p>Induction of <i>mecA</i> transcription for three prototype strains upon exposure to oxacillin at 0′, 5′, 15′, 30′, and 60′. Strain relevant characteristics are as follows: strain PER34 - SCC<i>mec</i> type I, <i>mecI</i> negative, Δ<i>mecR1</i>; strain HU25 - SCC<i>mec</i> type III, <i>mecI</i>*, <i>mecR1</i>^WT; strain N315 - SCC<i>mec</i> type II, <i>mecI</i>^WT<i>mecR1</i>^WT. The three strains are β-lactamase positive.</p

Population analysis profiles of representative parental strains and recombinant strains.

<p>Panel A – strain COL and its recombinants strains COL+pGC2-<i>mecI</i>^WT, COL+pGC2-<i>mecI</i>*, and COL+pGC2-<i>mecImecR1</i> grown overnight with and without oxacillin before plating onto TSA plates. Panel B – strain PER34 (SCC<i>mec</i> type I) and PER34+pGC2-<i>mecI</i>^WT. Panel C – strain BK2464 (SCC<i>mec</i> type II) and BK2464+pGC2-<i>mecI</i>^WT. Panel D – strain HU25 (SCC<i>mec</i> type III) and HU25+pGC2-<i>mecI</i>^WT.</p

Characteristics of the extended collection of representative MRSA strains.

<p>Abbreviations: neg., negative; pos., positive; WT, wild-type; ND, not determined.</p><p>Notes:</p>a)<p>Clonal types as defined by MLST sequence type (ST) and SCC<i>mec</i> type.</p>b)<p>neg. – negative (due to IS<i>1272</i> or IS<i>431</i> insertions); WT – wild-type sequence (N315); * - mutated non-functional <i>mecI</i>.</p>c)<p>IS::Δ<i>mecR1</i> – <i>mecR1</i> with no C-terminal sensor domain (due to IS<i>1272</i> or IS431 insertions).</p>d)<p>The production of β-lactamase was tested for induced and no induced cultures (see text for details). All strains positive for β-lactamase were inducible. Strains negative for the nitrocefin assay were tested for the presence of <i>blaZ</i>, blaI, and <i>bla</i>R1 by PCR.</p>e)<p>MIC as determined by E-test strips.</p>f)<p>Parental strains transformed with a high copy number plasmid containing the wild-type <i>mecI</i> coding region (pGC2-<i>mecI</i>^WT).</p

Characteristics of the prototype MRSA strains used in this study.

<p>Abbreviations: neg., negative; pos., positive; WT, wild-type.</p><p>Notes:</p>a)<p>Clonal types as defined by MLST sequence type (ST) and SCC<i>mec</i> type.</p>b)<p>neg. – negative (due to IS<i>1272</i> insertion); WT – wild-type sequence (N315);</p><p>*- mutated non-functional <i>mecI</i> at Gln₆₈ (CAA→TAA);</p>c)<p>IS::Δ<i>mecR1</i> – <i>mecR1</i> with no C-terminal sensor domain (due to IS<i>1272</i> insertion); Δ<i>mecR1</i> – 160 bp deletion in the C-terminal inducer domain.</p>d)<p>The production of β-lactamase was assayed in induced and no induced cultures (see text for details). All strains positive for β-lactamase were inducible.</p>e)<p>MIC as determined by E-test strips.</p>f)<p>Parental strains transformed with a high copy number plasmid containing the wild-type <i>mecI</i> coding region (pGC2-<i>mecI</i>^WT).</p

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

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

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

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

<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