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Discrete steps in sensing of beta-lactam antibiotics by the BlaR1 protein of the methicillin-resistant Staphylococcus aureus bacterium.

ABSTRACT: Chemical sensing by cell-surface receptors to effect signal transduction is a ubiquitous biological event. Despite extensive structural biochemical study, detailed knowledge of how signal transduction occurs is largely lacking. We report herein a kinetic and structural study, obtained by stopped-flow IR spectroscopy, of the activation of the BlaR1 receptor of the Staphylococcus aureus bacterium by beta-lactam antibiotics. The cell-surface BlaR1 receptor alerts the bacterium to the presence of beta-lactam antibiotics, resulting in expression of the gene for a beta-lactamase enzyme. This enzyme hydrolytically destroys the remaining beta-lactam antibiotics. IR spectroscopic interrogation of the beta-lactam-BlaR1 receptor reaction has allowed the simultaneous measurement of the chemical events of receptor recognition of the beta-lactam and the characterization of the conformational changes in the BlaR1 receptor that result. The key chemical events in beta-lactam recognition are serine acylation and subsequent irreversible decarboxylation of the BlaR1 active site lysine carbamate. Both events are observed by stopped-flow IR kinetics and (13)C isotope-edited IR spectroscopy. The secondary structural changes in the BlaR1 receptor conformation that occur as a consequence of this acylation/decarboxylation are predicted to correlate to the signal transduction event accomplished by this receptor.

SUBMITTER: Thumanu K

PROVIDER: S-EPMC1502283 | biostudies-literature | 2006 Jul

REPOSITORIES: biostudies-literature

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Discrete steps in sensing of beta-lactam antibiotics by the BlaR1 protein of the methicillin-resistant Staphylococcus aureus bacterium.

Thumanu Kanjana K Cha Jooyoung J Fisher Jed F JF Perrins Richard R Mobashery Shahriar S Wharton Christopher C

Proceedings of the National Academy of Sciences of the United States of America 20060630 28

Chemical sensing by cell-surface receptors to effect signal transduction is a ubiquitous biological event. Despite extensive structural biochemical study, detailed knowledge of how signal transduction occurs is largely lacking. We report herein a kinetic and structural study, obtained by stopped-flow IR spectroscopy, of the activation of the BlaR1 receptor of the Staphylococcus aureus bacterium by beta-lactam antibiotics. The cell-surface BlaR1 receptor alerts the bacterium to the presence of be ...[more]

PMID: 16815972

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Project description:Bacterial infections remain the leading killer worldwide which is worsened by the continuous emergence of antibiotic resistance. In particular, methicillin-sensitive (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) are prevalent and the latter can be difficult to treat. The traditional strategy of novel therapeutic drug development inevitably leads to emergence of resistant strains, rendering the new drugs ineffective. Therefore, rejuvenating the therapeutic potentials of existing antibiotics offers an attractive novel strategy. Plectasin, a defensin antimicrobial peptide, potentiates the activities of other antibiotics such as ?-lactams, aminoglycosides and glycopeptides against MSSA and MRSA. We performed in vitro and in vivo investigations to test against genetically diverse clinical isolates of MSSA (n = 101) and MRSA (n = 115). Minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The effects of combining plectasin with ?-lactams, aminoglycosides and glycopeptides were examined using the chequerboard method and time kill curves. A murine neutropenic thigh model and a murine peritoneal infection model were used to test the effect of combination in vivo. Determined by factional inhibitory concentration index (FICI), plectasin in combination with aminoglycosides (gentamicin, neomycin or amikacin) displayed synergistic effects in 76-78% of MSSA and MRSA. A similar synergistic response was observed when plectasin was combined with ?-lactams (penicillin, amoxicillin or flucloxacillin) in 87-89% of MSSA and MRSA. Interestingly, no such interaction was observed when plectasin was paired with vancomycin. Time kill analysis also demonstrated significant synergistic activities when plectasin was combined with amoxicillin, gentamicin or neomycin. In the murine models, plectasin at doses as low as 8 mg/kg augmented the activities of amoxicillin and gentamicin in successful treatment of MSSA and MRSA infections. We demonstrated that plectasin strongly rejuvenates the therapeutic potencies of existing antibiotics in vitro and in vivo. This is a novel strategy that can have major clinical implications in our fight against bacterial infections.

Project description:Methicillin-resistant Staphylococcus aureus (MRSA) resists nearly all β-lactam antibiotics that have a bactericidal activity. However, whether the empirically used β-lactams enhance MRSA pathogenicity in vivo remains unclear. In this study, we showed that a cluster of lipoprotein-like genes (lpl, sa2275 to sa2273 [sa2275-sa2273]) was upregulated in MRSA in response to subinhibitory concentrations of β-lactam induction. The increasing expression of lpl by β-lactams was directly controlled by the global regulator SarA. The β-lactam-induced Lpls stimulated the production of interleukin-6 and tumor necrosis factor alpha in RAW 264.7 macrophages. The lpl deletion mutants (N315Δlpl and USA300Δlpl) decreased the proinflammatory cytokine levels in vitro and in vivo Purified lipidated SA2275-his proteins could trigger a Toll-like-receptor-2 (TLR2)-dependent immune response in primary mouse bone marrow-derived macrophages and C57BL/6 mice. The bacterial loads of N315Δlpl in the mouse kidney were lower than those of the wild-type N315. The β-lactam-treated MRSA exacerbated cutaneous infections in both BALB/c and C57BL/6 mice, presenting increased lesion size; destroyed skin structure; and easily promoted abscess formation compared with those of the untreated MRSA. However, the size of abscesses caused by the β-lactam-treated N315 was negligibly different from those caused by the untreated N315Δlpl in C57BL/6 TLR2-/- mice. Our findings suggest that β-lactams must be used carefully because they might aggravate the outcome of MRSA infection compared to inaction in treatment.IMPORTANCE β-Lactam antibiotics are widely applied to treat infectious diseases. However, certain poor disease outcomes caused by β-lactams remain poorly understood. In this study, we have identified a cluster of lipoprotein-like genes (lpl, sa2275-sa2273) that is upregulated in the major clinically prevalent MRSA clones in response to subinhibitory concentrations of β-lactam induction. The major highlight of this work is that β-lactams stimulate the expression of SarA, which directly binds to the lpl cluster promoter region and upregulates lpl expression in MRSA. Deletion of lpl significantly decreases proinflammatory cytokine levels in vitro and in vivo The β-lactam-induced Lpls enhance host inflammatory responses by triggering the Toll-like-receptor-2-mediated expressions of interleukin-6 and tumor necrosis factor alpha. The β-lactam-induced Lpls are important virulence factors that enhance MRSA pathogenicity. These data elucidate that subinhibitory concentrations of β-lactams can exacerbate the outcomes of MRSA infection through induction of lpl controlled by the global regulator SarA.

Dataset Information

Discrete steps in sensing of beta-lactam antibiotics by the BlaR1 protein of the methicillin-resistant Staphylococcus aureus bacterium.

Publications

Discrete steps in sensing of beta-lactam antibiotics by the BlaR1 protein of the methicillin-resistant Staphylococcus aureus bacterium.

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