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In response to β-lactam chemotherapy, Staphylococcus aureus has acquired two resistance determinants: blaZ , coding for β-lactamase, which confers resistance to penicillins only, and mecA , coding for an extra cell wall cross-linking enzyme with reduced affinity for virtually all other β-lactams. The transcriptional control of both resistance determinants is regulated by homologous repressors (BlaI and MecI, respectively) and sensor inducers (BlaR1 and MecR1, respectively). There is a cross-talk between the two regulatory systems, and it has been demonstrated that bla regulators stabilize the mecA acquisition. In a recent study, we have unexpectedly observed that in most MRSA strains, there was no significant change in the resistance phenotype upon the overexpression in trans of a MecI repressor, whereas in those few strains negative for the bla locus, there was a massive decrease of resistance (D. C. Oliveira and H. de Lencastre, PLoS One 6:e23287, 2011). Here, we demonstrate that, contrary to what is currently accepted, the bla regulatory system efficiently disrupts the strong MecI-mediated repression on mecA , enabling the optimal expression of resistance. This effect appears to be due to the formation of MecI::BlaI heterodimers that might bind less efficiently to the mecA promoter and become nonfunctional due to the proteolytic inactivation of the BlaI monomer. In addition, we have also observed that the presence of bla regulators may enhance dramatically the expression of β-lactam resistance in MRSA strains with constitutive mecA expression, compensating for the fitness cost imposed by the large β-lactamase plasmid. These observations point to important unrecognized roles of the bla locus for the expression of the methicillin-resistant S. aureus (MRSA) phenotype.

HmrA is an antibiotic resistance factor of methicillin-resistant Staphylococcus aureus. Molecular analysis of this protein revealed that it is not a muramidase or β-lactamase but a non-specific double-zinc endopeptidase consisting of a catalytic domain and an inserted oligomerization domain, which probably undergo a relative interdomain hinge rotation upon substrate binding. The active-site cleft is located at the domain interface. Four HmrA protomers assemble to a large ∼170-kDa homotetrameric complex of 125 Å. All four active sites are fully accessible and ∼50-70 Å apart, far enough apart to act on a large meshwork substrate independently but simultaneously. In vivo studies with four S. aureus strains of variable resistance levels revealed that the extracellular addition of HmrA protects against loss of viability in the presence of oxacillin and that this protection depends on proteolytic activity. All of these results indicate that HmrA is a peptidase that participates in resistance mechanisms in vivo in the presence of β-lactams. Furthermore, our results have implications for most S. aureus strains of known genomic sequences and several other cocci and bacilli, which harbor close orthologs. This suggests that HmrA may be a new widespread antibiotic resistance factor in bacteria. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc., This study was supported in part by grants from European, Portuguese, Spanish, Swiss, and Catalan agencies (FP7-HEALTH-F3-2009-223101 “AntiPathoGN,” FP7-HEALTH-2010-261460 “GumsandJoints,” BIO2008-04080-E, BIO2009-10334, CSD2006-00015, PSE-010000-2009-8, 2009SGR1036, Fundacio´ “La Marato´ de TV3” 100372, POCTI/BIA-MIC/60320/2004, PTDC/BIA-MIC/64071/2006, SNF200020/125020/1).

This article contains supporting information online (a detailed description of the experimental procedures is provided in the SI Appendix. The latter also includes four supplementary tables, 10 supplementary figures, the Acknowledgments, and SI Appendix, SRD.) at http://www.pnas.org/content/suppl/2015/06/19/1506538112.DCSupplemental/pnas.1506538112.sapp.pdf, The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2 -macroglobulin (α2 M). Escherichia coli α2 M (ECAM) is a ∼180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric >snap trap.>., This study was supported in part by grants from public and private agencies from the European Community, Spain, and Catalonia (FP7-PEOPLE-2011-ITN-290246 “RAPID,” FP7-HEALTH-2012-306029-2 “TRIGGER;” BFU2011-25902, BFU2012-32862, BIO2013-49320-EXP, MDM-2014-0435; and 2014SGR9) and an FPU Ph.D. fellowship (AP2010-3799) from the Spanish Ministry for Education, Culture and Sport to I.G.-F. The Department of Structural Biology of IBMB is a “María de Maeztu” Unit of Excellence from the Ministry of Economy and Competitiveness. We acknowledge the help provided by ESRF and ALBA synchrotron local contacts. Funding for travelling and synchrotron data collection was provided in part by ESRF

Recently, we have demonstrated that the cognate regulatory locus of the mecA gene in methicillin-resistant Staphylococcus aureus (MRSA) is in fact a three-component system containing the novel mecR2 gene coding for an antirepressor. MecR2 interacts with the repressor MecI, disturbing its binding to the mecA promoter and fostering its proteolysis. Here, we engineered a point mutation in the putative cleavage site of MecI and demonstrated that MecI proteolysis is strictly required for the optimal expression of β-lactam resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen, which is cross-resistant to virtually all β-lactam antibiotics. MRSA strains are defined by the presence of mecA gene. The transcription of mecA can be regulated by a sensor-inducer (MecR1) and a repressor (MecI), involving a unique series of proteolytic steps. The induction of mecA 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 mecA regulatory locus consists, in fact, of an unusual three-component arrangement containing, in addition to mecR1-mecI, the up to now unrecognized mecR2 gene coding for an anti-repressor. The MecR2 function is essential for the full induction of mecA expression, compensating for the inefficient induction of mecA by MecR1 and enabling optimal expression of β-lactam resistance in MRSA strains with functional mecR1-mecI regulatory genes. Our data shows that MecR2 interacts directly with MecI, destabilizing its binding to the mecA 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 mecA 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., Author Summary Methicillin-resistance Staphylococcus aureus (MRSA) is an important human pathogen, causing a wide range of infections. MRSA strains are resistant to virtually all β-lactam antibiotics and often are also resistant to many other classes of antibiotics, leaving physicians with few therapeutic options. MRSA is defined by the presence of the mecA gene. The induction of mecA transcription in response to β-lactams involves a unique series of proteolytic steps and some critical details of this signal transduction mechanism are still illusive. For instance, it is not fully explained why the induction of mecA by its cognate regulatory genes mecR1-mecI appears to be very inefficient and it is not clear if the observed MecI repressor proteolysis is mediated directly by the activated MecR1 sensor-inducer. In this study, we demonstrate that the mecA regulatory locus is not a two-component system but instead it is a three-component system containing the previously unrecognized anti-repressor mecR2 gene. MecR2 disturbs the binding of the repressor MecI to the mecA promoter, which leads to its proteolytic inactivation independently from MecR1. Moreover, our data shows that in the presence of functional mecR1-mecI genes, mecR2 is essential for a robust induction of mecA transcription and, as consequence, for the optimal expression of β-lactam resistance.

Dissertação para obtenção do Grau de Doutor em Biologia Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen, causing a wide range of infections. MRSA has not only developed an intrinsic resistance to all β-lactams, but has also acquired resistance to virtually all classes of antimicrobial agents. The characteristic MRSA phenotype is conferred by the presence of mecA gene which is regulated by a sensor-inducer (MecR1) and a repressor (MecI). However, mecA induction by its cognate sensor/inducer is very inefficient and, therefore, it is believed that optimal expression of β-lactam resistance in MRSA requires a non-functional MecR1-MecI system. Surprisingly, 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, suggesting the existence of other mecA regulators. In these studies, we show that the mecA regulatory locus is not a two-component system but, actually, it is a three-component system containing besides mecR1-mecI, the previously unidentified antirepressor mecR2. The crystal structure of MecR2 reveals a three-domain architecture, with an N-terminal DNA-binding-like domain, an intermediate scaffold domain, and a C-terminal dimerization domain, important to the functional dimeric oligomerization state. MecR2 disturbs the binding of the repressor MecI to the mecA promoter, which leads to its proteolytic inactivation independently from MecR1, presumably by non-specific cytoplasmatic proteases. Our data also demonstrates that in the presence of functional mecR1-mecI genes, mecR2 is essential for a robust induction of mecA transcription and, consequently, for the optimal expression of resistance phenotype in MRSA. These observations point to a revision of the current model for the transcriptional control of the mecA gene. Fundação para a Ciência e a Tecnologia - Doctoral Project (SFRH/BD/38316/2007)