RESUMO
Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC is known to bind the Group A streptococcal cell wall, but the specific molecular target or the binding site within PlyC remain uncharacterized. Here we report for the first time, that the polyrhamnose backbone of the Group A streptococcal cell wall is the binding target of PlyC. We have also characterized the putative rhamnose binding groove of PlyC and found four key residues that were critical to either the folding or the cell wall binding action of PlyC. Based on our results, we suggest that the interaction between PlyC and the cell wall may not be a high-affinity interaction as previously proposed, but rather a high avidity one, allowing for PlyC's remarkable lytic activity. Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop the antibacterial agents with new modes of action. A detailed understanding of this potent endolysin may facilitate future developments of PlyC as a tool against the rise of antibiotic resistance.
Assuntos
Bacteriófagos/metabolismo , Endopeptidases/metabolismo , Peptidoglicano/metabolismo , Ramnose/metabolismo , Streptococcus pyogenes/virologia , Bacteriófagos/genética , Sítios de Ligação/fisiologia , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endopeptidases/genética , Simulação de Acoplamento Molecular , Ligação Proteica/fisiologia , Streptococcus pyogenes/metabolismoRESUMO
Clostridium difficile is an emerging nosocomial pathogen of increasing importance and virulence but our ability to study the molecular mechanisms underlying the pathogenesis of C. difficile-associated disease has been limited because of a lack of tools for its genetic manipulation. We have now developed a reproducible method for the targeted insertional inactivation of chromosomal C. difficile genes. The approach relies on the observation that an Escherichia coli-Clostridium perfringens shuttle vector is unstable in C. difficile and can be used as a form of conditional lethal vector to deliver gene constructs to the chromosome. We have used this methodology to insertionally inactivate two putative response regulator genes, rgaR and rgbR, which encode proteins with similarity to the toxin gene regulator, VirR, from C. perfringens. Transcriptomic analysis demonstrated that the C. difficile RgaR protein positively regulated four genes, including a putative agrBD operon. The RgaR protein was also purified and shown to bind specifically to sites that contained two consensus VirR boxes located just upstream of the putative promoters of these genes. The development of this methodology will significantly enhance our ability to use molecular approaches to develop a greater understanding of the ability of C. difficile to cause disease.
Assuntos
Cromossomos Bacterianos/genética , Clostridioides difficile/genética , Mutação/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clostridioides difficile/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Regulação Bacteriana da Expressão Gênica/genética , Teste de Complementação Genética , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Insercional/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Ligação Proteica , Homologia de Sequência de AminoácidosRESUMO
Clostridium difficile is a nosocomial pathogen that causes a range of chronic intestinal diseases, usually as a result of antimicrobial therapy. Macrolide-lincosamide-streptogramin B (MLS) resistance in C. difficile is encoded by the Erm B resistance determinant, which is thought to be located on a conjugative transposon, Tn5398. The 9630 bp Tn5398 element has been cloned and completely sequenced and its insertion site determined. Analysis of the resultant data reveals that Tn5398 is not a classical conjugative transposon but appears to be a mobilizable non-conjugative element. It does not carry any transposase or site-specific recombinase genes, nor any genes likely to be involved in conjugation. Furthermore, using PCR analysis it has been shown that isolates of C. difficile obtained from different geographical locations exhibit heterogeneity in the genetic arrangement of both Tn5398 and their Erm B determinants. These results indicate that genetic exchange and recombination between these determinants occurs in the clinical and natural environment.
Assuntos
Antibacterianos/farmacologia , Clostridioides difficile/efeitos dos fármacos , Elementos de DNA Transponíveis/genética , Farmacorresistência Bacteriana/genética , Eritromicina/farmacologia , Genoma Bacteriano , Macrolídeos , Sequência de Bases , Clostridioides difficile/genética , Conjugação Genética , Humanos , Lincosamidas , Metiltransferases/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Estreptogramina B/farmacologiaRESUMO
Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein from Clostridium perfringens with the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried the erm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in the erm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of the C. perfringens Erm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.
Assuntos
Clostridium perfringens/enzimologia , Metiltransferases/química , Metiltransferases/genética , Mutação Puntual , Sequência de Aminoácidos , Antibacterianos/farmacologia , Clostridium perfringens/química , Clostridium perfringens/genética , Farmacorresistência Bacteriana/genética , Eritromicina/farmacologia , Immunoblotting , Metiltransferases/metabolismo , Testes de Sensibilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Análise de Sequência de DNARESUMO
The Clostridium perfringens tetracycline resistance protein, TetA(P), is an inner-membrane protein that mediates the active efflux of tetracycline from the bacterial cell. This protein comprises 420 aa and is predicted to have 12 transmembrane domains (TMDs). Comparison of the TetA(P) amino acid sequence to that of several members of the major facilitator superfamily (MFS) identified a variant copy of the conserved Motif A. This region consists of the sequence E59xPxxxxxDxxxRK72 and is located within the putative loop joining TMDs 2 and 3 in the predicted structural model of the TetA(P) protein. To study the functional importance of the conserved residues, site-directed mutagenesis was used to construct 17 point mutations that were then analysed for their effect on tetracycline resistance and their ability to produce an immunoreactive TetA(P) protein. Changes to the conserved Phe-58 residue were tolerated, whereas three independent substitutions of Pro-61 abolished tetracycline resistance. Examination of the basic residues showed that Arg-71 is required for function, whereas tetracycline resistance was retained when Lys-72 was substituted with arginine. These results confirm that the region encoding this motif is important for tetracycline resistance and represents a distant version of the Motif A region found in other efflux proteins and members of the MFS family. In addition, it was shown that Glu-117 of the TetA(P) protein, which is predicted to be located in TMD4, is important for resistance although a derivative with an aspartate residue at this position is also functional.