RESUMO
BACKGROUND: Proteins from the LytR-CpsA-Psr family are found in almost all Gram-positive bacteria. Although LCP proteins have been studied in other pathogens, their functions in enterococci remain uncharacterized. The Psr protein from Enterococcus hirae, here renamed LcpA, previously associated with the regulation of the expression of the low-affinity PBP5 and ß-lactam resistance, has been characterized. RESULTS: LcpA protein of E. hirae ATCC 9790 has been produced and purified with and without its transmembrane helix. LcpA appears, through different methods, to be localized in the membrane, in agreement with in silico predictions. The interaction of LcpA with E. hirae cell wall indicates that LcpA binds enterococcal peptidoglycan, regardless of the presence of secondary cell wall polymers. Immunolocalization experiments showed that LcpA and PBP5 are localized at the division site of E. hirae. CONCLUSIONS: LcpA belongs to the LytR-CpsA-Psr family. Its topology, localization and binding to peptidoglycan support, together with previous observations on defective mutants, that LcpA plays a role related to the cell wall metabolism, probably acting as a phosphotransferase catalyzing the attachment of cell wall polymers to the peptidoglycan.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Streptococcus faecium ATCC 9790/metabolismo , Peptidoglicano/metabolismo , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Membrana Celular/metabolismo , Parede Celular/metabolismo , Clonagem Molecular , DNA Bacteriano , Streptococcus faecium ATCC 9790/citologia , Streptococcus faecium ATCC 9790/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Fosfotransferases/metabolismo , Mapas de Interação de Proteínas , Proteínas Recombinantes , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Resistência beta-LactâmicaRESUMO
The opportunistic human pathogen Enterococcus faecium overproduces the low-affinity PBP5. In clinical strains, mutations in PBP5 further reduce its acylation rate by ß-lactams. Previous studies have reported that ceftaroline had poor inhibitory activity against ß-lactam-resistant E. faecium strains. In this study, we show that ceftaroline exhibits killing activity against our laboratory-derived ampicillin-resistant E. faecium mutant that overproduces a wild-type PBP5 and that ceftaroline inactivates PBP5 much faster than benzylpenicillin and faster than ceftobiprole.
Assuntos
Antibacterianos/farmacologia , Cefalosporinas/farmacologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/genética , Resistência a Ampicilina/genética , Proteínas de Bactérias/genética , Resistência beta-Lactâmica/genética , CeftarolinaRESUMO
Ceftobiprole is a new cephalosporin that exhibits a high level of affinity for methicillin-resistant Staphylococcus aureus PBP 2a. It was reported that ceftobiprole did not interact with a mutated form of the low-affinity protein Enterococcus faecium PBP 5 (PBP 5fm) that, when overexpressed, confers a beta-lactam resistance phenotype to the bacterium. Our results show that ceftobiprole binds to unmutated PBP 5fm to form a stable acyl-enzyme and that ceftobiprole is able to efficiently kill a penicillin-resistant Enterococcus faecium strain that produces this protein.
Assuntos
Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Cefalosporinas/metabolismo , Cefalosporinas/farmacologia , Enterococcus faecium/efeitos dos fármacos , Enterococcus faecium/metabolismo , Proteínas de Bactérias/genética , Enterococcus faecium/genética , Testes de Sensibilidade Microbiana , Penicilina G/farmacologia , Ligação Proteica/genéticaRESUMO
The contribution of penicillin-binding protein 5 (PBP5) and the PBP5 synthesis repressor (Psr) to the beta-lactam resistance, growth, and cell autolysis of wild-type strain ATCC 9790 and resistant strain R40 of Enterococcus hirae was investigated by disruption or substitution of the corresponding pbp5 and psr genes by Campbell-type recombination. The resulting modifications were confirmed by hybridization and PCR. The low susceptibility of E. hirae to beta-lactams was confirmed to be largely dependent on the presence of PBP5. However, against all expectations, inactivation of psr in ATCC 9790 or complementation of R40 cells with psr did not modify the susceptibility to benzylpenicillin or the growth and cell autolysis rates. These results indicated that the psr gene does not seem to be involved in the regulation of PBP5 synthesis and consequently in beta-lactam resistance or in the regulation of cell autolysis in E. hirae.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriólise , Enterococcus/fisiologia , Proteínas Repressoras/metabolismo , Resistência beta-Lactâmica , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Sequência de Bases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Enterococcus/efeitos dos fármacos , Enterococcus/genética , Enterococcus/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Hexosiltransferases/genética , Hexosiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/genética , Muramilpentapeptídeo Carboxipeptidase/metabolismo , Mutação , Proteínas de Ligação às Penicilinas , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Recombinação Genética , Proteínas Repressoras/genética , Análise de Sequência de DNARESUMO
A penicillin-resistant mutant, JH2-2r (MIC 75 microg ml(-1)), was isolated from Enterococcus faecalis JH2-2 (MIC 5 microg ml(-1)) by successive passages on plates containing increasing concentrations of benzylpenicillin. A comparison of the penicillin-binding protein (PBP) profiles in the two strains revealed a more intensely labelled PBP4 in JH2-2r. Because the sequences of the JH2-2 and JH2-2r pbp4 genes were strictly identical, even in their promoter regions, this intensive labelling could only be associated with an overproduction of the low-affinity PBP4. No psr gene analogous to that proposed to act as a regulator of PBP5 synthesis in Enterococcus hirae and Enterococcus faecium could be identified in the vicinity of pbp4 in E. faecalis JH2-2 and JH2-2r. However, a psr-like gene distant from pbp4 was identified. The cloning and sequencing of that psr-like gene from both E. faecalis strains indicated that they were identical. It is therefore postulated that the PBP4 overproduction in E. faecalis JH2-2r results from the modification of an as yet unidentified factor.
Assuntos
Proteínas de Bactérias , Proteínas de Transporte/biossíntese , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/metabolismo , Hexosiltransferases , Muramilpentapeptídeo Carboxipeptidase/biossíntese , Resistência às Penicilinas/fisiologia , Peptidil Transferases , Sequência de Bases , Proteínas de Transporte/genética , Clonagem Molecular , DNA Bacteriano/genética , Enterococcus faecalis/genética , Enterococcus faecalis/patogenicidade , Escherichia coli/genética , Expressão Gênica , Genes Bacterianos , Humanos , Dados de Sequência Molecular , Muramilpentapeptídeo Carboxipeptidase/genética , Resistência às Penicilinas/genética , Proteínas de Ligação às Penicilinas , Plasmídeos/genéticaRESUMO
A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a DD- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.