The Importance of a Critical Protonation State and the Fate of the Catalytic Steps in Class A β-Lactamases and Penicillin-binding Proteins

b-Lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to b-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active-site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins and crystal structures demonstrate that the side chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys73) in the TEM-1 b-lactamase from Escherichia coli. These techniques included perturbation of the pKa of Lys73 by the study of the g-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance and by computational treatment using the thermodynamic integration method. All three methods indicated that the pKa of Lys73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu166 and Lys73 of class A b-lactamases has actually raised the pKa of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot definitively rule out that Glu166 activates the active site water, which in turn promotes Ser70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu166 and unprotonated Lys73. As such, unprotonated Lys73 could promote serine for acylation, a process that should be shared among all active-site-serine b-lactamases and penicillin-binding proteins

Genetic Pathway in Acquisition and Loss of Vancomycin Resistance in a Methicillin Resistant Staphylococcus aureus (MRSA) Strain of Clonal Type USA300

An isolate of the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 with reduced susceptibility to vancomycin (SG-R) (i.e, vancomycin-intermediate S. aureus, VISA) and its susceptible “parental” strain (SG-S) were recovered from a patient at the end and at the beginning of an unsuccessful vancomycin therapy. The VISA phenotype was unstable in vitro generating a susceptible revertant strain (SG-rev). The availability of these 3 isogenic strains allowed us to explore genetic correlates of antibiotic resistance as it emerged in vivo. Compared to the susceptible isolate, both the VISA and revertant strains carried the same point mutations in yycH, vraG, yvqF and lspA genes and a substantial deletion within an intergenic region. The revertant strain carried a single additional frameshift mutation in vraS which is part of two component regulatory system VraSR. VISA isolate SG-R showed complex alterations in phenotype: decreased susceptibility to other antibiotics, slow autolysis, abnormal cell division and increased thickness of cell wall. There was also altered expression of 239 genes including down-regulation of major virulence determinants. All phenotypic properties and gene expression profile returned to parental levels in the revertant strain. Introduction of wild type yvqF on a multicopy plasmid into the VISA strain caused loss of resistance along with loss of all the associated phenotypic changes. Introduction of the wild type vraSR into the revertant strain caused recovery of VISA type resistance. The yvqF/vraSR operon seems to function as an on/off switch: mutation in yvqF in strain SG-R turns on the vraSR system, which leads to increase in vancomycin resistance and down-regulation of virulence determinants. Mutation in vraS in the revertant strain turns off this regulatory system accompanied by loss of resistance and normal expression of virulence genes. Down-regulation of virulence genes may provide VISA strains with a “stealth” strategy to evade detection by the host immune system

Comparative Study of the Susceptibilities of Major Epidemic Clones of Methicillin-Resistant Staphylococcus aureus to Oxacillin and to the New Broad-Spectrum Cephalosporin Ceftobiprole▿

Multidrug-resistant strains of Staphylococcus aureus continue to increase in frequency worldwide, both in hospitals and in the community, raising serious problems for the chemotherapy of staphylococcal disease. Ceftobiprole (BPR; BAL9141), the active constituent of the prodrug ceftobiprole medocaril (BAL5788), is a new cephalosporin which was already shown to have powerful activity against a number of bacterial pathogens, including S. aureus. In an effort to test possible limits to the antibacterial spectrum and efficacy of BPR, we examined the susceptibilities of the relatively few pandemic methicillin-resistant S. aureus (MRSA) clones that are responsible for the great majority of cases of staphylococcal disease worldwide. We also included in the tests the highly oxacillin-resistant subpopulations that are present with low frequencies in the cultures of these clones. Such subpopulations may represent a natural reservoir from which MRSA strains with decreased susceptibility to BPR may emerge in the future. We also tested the efficacy of BPR against MRSA strains with reduced susceptibility to vancomycin and against MRSA strains carrying the enterococcal vancomycin resistance gene complex. BPR was shown to be uniformly effective against all these resistant MRSA strains, and the mechanism of superb antimicrobial activity correlated with the strikingly increased affinity of the cephalosporin against penicillin-binding protein 2A, the protein product of the antibiotic resistance determinant mecA

The mechanism of heterogeneous beta-lactam resistance in MRSA: key role of the stringent stress response.

All methicillin resistant S. aureus (MRSA) strains carry an acquired genetic determinant--mecA or mecC--which encode for a low affinity penicillin binding protein -PBP2A or PBP2A'--that can continue the catalysis of peptidoglycan transpeptidation in the presence of high concentrations of beta-lactam antibiotics which would inhibit the native PBPs normally involved with the synthesis of staphylococcal cell wall peptidoglycan. In contrast to this common genetic and biochemical mechanism carried by all MRSA strains, the level of beta-lactam antibiotic resistance shows a very wide strain to strain variation, the mechanism of which has remained poorly understood. The overwhelming majority of MRSA strains produce a unique--heterogeneous--phenotype in which the great majority of the bacteria exhibit very poor resistance often close to the MIC value of susceptible S. aureus strains. However, cultures of such heterogeneously resistant MRSA strains also contain subpopulations of bacteria with extremely high beta-lactam MIC values and the resistance level and frequency of the highly resistant cells in such strain is a characteristic of the particular MRSA clone. In the study described in this communication, we used a variety of experimental models to understand the mechanism of heterogeneous beta-lactam resistance. Methicillin-susceptible S. aureus (MSSA) that received the mecA determinant in the laboratory either on a plasmid or in the form of a chromosomal SCCmec cassette, generated heterogeneously resistant cultures and the highly resistant subpopulations that emerged in these models had increased levels of PBP2A and were composed of bacteria in which the stringent stress response was induced. Each of the major heterogeneously resistant clones of MRSA clinical isolates could be converted to express high level and homogeneous resistance if the growth medium contained an inducer of the stringent stress response

Correction: The Mechanism of Heterogeneous Beta-Lactam Resistance in MRSA: Key Role of the Stringent Stress Response

Os estudos sobre as desigualdades sociais no Brasil concentram-se, em geral, nas dimensões objetivas do fenômeno, como sexo, cor, renda, ocupação e escolaridade. A amplitude do fosso entre ricos e pobres, graças às pesquisas já realizadas, é razoavelmente conhecida, mas pouco se sabe sobre a maneira como os brasileiros vêem essa disparidade. Em 2000, como parte do International Social Survey Programme tentou-se preencher essa lacuna com um survey aplicado a representantes das elites nacionais, entendendo-se por "elites" indivíduos pertencentes ao grupo dos 10% mais ricos do país. A pesquisa, de abrangência nacional - 2 mil entrevistas em 195 municípios -, buscou revelar diferenças entre as percepções da "elite" e do "povo" acerca da desigualdade. As questões levantadas pelo survey diziam respeito à imagem que os dois grupos têm da sociedade brasileira e da forma como ela está estruturada; as remunerações que seriam adequadas para trabalhadores de diferentes níveis de qualificação; os valores que deveriam prevalecer na distribuição da riqueza do país; os maiores problemas do Brasil; e as estratégias preferenciais de cada grupo para a redução da pobreza, com destaque para o papel do Estado. As respostas revelaram convergências e divergências surpreendentes entre povo e elite. Os dois grupos percebem a extensão das desigualdades sociais no país, mas tendem a defender estratégias distintas para reduzi-las, transferindo a solução das desigualdades ao Estado. Essas aproximações e diferenças de pontos de vista podem oferecer elementos importantes para se compreender os mecanismos de legitimação das desigualdades