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Clavulanate inactivation of Staphylococcus aureus beta-lactamase.

ABSTRACT: The interaction of clavulanic acid with beta-lactamase from Staphylococcus aureus was investigated, particularly with a view to determining whether conformational effects are involved. The inactivation at neutral pH is essentially stoichiometric, leading to an inactive species with an enamine chromophore. Two forms of the enamine were observed, the first-formed having a positive ellipticity with a maximum near 290 nm. This species slowly converted into the stable form of the inactivated enzyme that had a negative ellipticity with a minimum at 275 nm. This change in sign of the ellipticity of the enamine is consistent with the previously proposed cis-trans isomerization of the enamine [Cartwright & Coulson (1979) Nature (London) 278, 360-361). Both the far-u.v.c.d. and the intrinsic viscosity of the inactivated enzyme indicated that negligible change in conformation of the enzyme accompanied inactivation. The rates of inactivation and enamine formation were compared at low temperatures, where the initial rates were slow enough to be monitored. The rate of loss of 95% of the catalytic activity was almost 100-fold faster than the rate of formation of the first-formed enamine species. The remaining 5% activity was lost with a rate comparable with that for formation of the initial enamine. The simplest explanation of these results is that a relatively stable acyl-enzyme intermediate builds up initially and more slowly partitions between turnover (hydrolysis) and enamine formation. The initially formed enamine is in the cis conformation but slowly isomerizes to the more stable trans form.

SUBMITTER: Rizwi I

PROVIDER: S-EPMC1138342 | biostudies-other | 1989 Feb

REPOSITORIES: biostudies-other

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Project description:Staphylococcus aureus is an opportunistic pathogen with a clinical spectrum ranging from asymptomatic skin colonization to invasive infections. While traditional antibiotic therapies can be effective against S. aureus, the increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. Photodynamic inactivation (PDI) is an innovative and promising alternative to antibiotics. While progress has been made in our understanding of the bacterial response to PDI, major gaps remain in our knowledge of PDI tolerance, the global cellular response, and adaptive genomic mutations acquired as a result of PDI. To address these gaps, S. aureus HG003 and isogenic mutants with mutations in agr, mutS, mutL, and mutY exposed to single or multiple doses of PDI were assessed for survival and tolerance and examined by global transcriptome and genome analyses to identify regulatory and genetic adaptations that contribute to tolerance. Pathways in inorganic ion transport, oxidative response, DNA replication recombination and repair, and cell wall and membrane biogenesis were identified in a global cellular response to PDI. Tolerance to PDI was associated with superoxide dismutase and the S. aureus global methylhydroquinone (MHQ)-quinone transcriptome network. Genome analysis of PDI-tolerant HG003 identified a nonsynonymous mutation in the quinone binding domain of the transcriptional repressor QsrR, which mediates quinone sensing and oxidant response. Acquisition of a heritable QsrR mutation through repeated PDI treatment demonstrates selective adaption of S. aureus to PDI. PDI tolerance of a qsrR gene deletion in HG003 confirmed that QsrR regulates the S. aureus response to PDI.IMPORTANCE Staphylococcus aureus can cause disease at most body sites, with illness ranging from asymptomatic infection to death. The increasing prevalence of antibiotic-resistant strains results in treatment failures and high mortality rates. S. aureus acquires resistance to antibiotics through multiple mechanisms, often by genetic variation that alters antimicrobial targets. Photodynamic inactivation (PDI), which employs a combination of a nontoxic dye and low-intensity visible light, is a promising alternative to antibiotics that effectively eradicates S. aureus in human infections when antibiotics are no longer effective. In this study, we demonstrate that repeated exposure to PDI results in resistance of S. aureus to further PDI treatment and identify the underlying bacterial mechanisms that contribute to resistance. This work supports further analysis of these mechanisms and refinement of this novel technology as an adjunctive treatment for S. aureus infections.

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Clavulanate inactivation of Staphylococcus aureus beta-lactamase.

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