RESUMO
Multidrug-resistant Neisseria gonorrhoeae has emerged as a threat to global health. The relationship between gepotidacin exposure and prevention of on-therapy amplification of drug-resistant N. gonorrhoeae was examined using a 7-day hollow-fiber in vitro infection model. The study design included both inactive (no-treatment and ciprofloxacin) and active (ceftriaxone) control regimens. Study drug concentration-time profiles were simulated in the in vitro system for a single oral 0.5 g ciprofloxacin dose, a single intramuscular 0.25 g ceftriaxone dose, and single or two (8 to 12 h apart) oral gepotidacin doses ranging from 0.75 to 12 g. The initial bacterial burden inoculated in the model was 106 CFU/ml. The gepotidacin, ciprofloxacin, and ceftriaxone broth MIC values for the challenge isolate (N. gonorrhoeae GSK #8) were 0.5, 2, and 0.002 mg/liter, respectively. Samples were collected for enumeration of total and drug-resistant bacterial populations and drug concentrations. The no-treatment control reached a bacterial density greater than 108 CFU/ml over 24 h and remained consistent over the 7-day study period. The bacterial density in the model system of the ciprofloxacin regimen matched that of the growth control throughout the study duration, while the ceftriaxone regimen sterilized the model system by the end of day 1. For gepotidacin, a full dose-response relationship was observed. While failure was observed for the 0.75-, 1.5-, and 3-g single-dose regimens, all gepotidacin single- or divided-dose regimens totaling at least 4.5 g prevented resistance amplification and sterilized the model system. These data are useful to provide gepotidacin dose selection support for treating patients with gonorrhea infections.
Assuntos
Gonorreia , Neisseria gonorrhoeae , Acenaftenos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Ceftriaxona/farmacologia , Ciprofloxacina/farmacologia , Ciprofloxacina/uso terapêutico , Farmacorresistência Bacteriana/genética , Gonorreia/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis , Humanos , Testes de Sensibilidade Microbiana , Neisseria gonorrhoeae/genéticaRESUMO
A phase 2 study of gepotidacin demonstrated the safety and efficacy of 3 gepotidacin doses (750 mg every 12 h [q12h], 1,000 mg q12h, and 1,000 mg every 8 h [q8h]) in hospitalized patients with suspected/confirmed Gram-positive acute bacterial skin and skin structure infections (ABSSSIs). Evaluating microbiology outcomes and responses were secondary endpoints. Pretreatment isolates recovered from infected lesions underwent susceptibility testing per Clinical and Laboratory Standards Institute guidelines. Staphylococcus aureus accounted for 78/102 (76%) of Gram-positive isolates; 54/78 (69%) were methicillin-resistant S. aureus (MRSA), and 24/78 (31%) were methicillin-susceptible S. aureus (MSSA). Posttherapy microbiological success (culture-confirmed eradication of the pretreatment pathogen or presumed eradication based on a clinical outcome of success) for S. aureus was 90% for the gepotidacin 750-mg q12h group, 89% for the 1,000-mg q12h, and 73% in the 1000-mg q8h group. For 78 S. aureus isolates obtained from pretreatment lesions, gepotidacin MIC50/MIC90 values were 0.25/0.5 µg/ml against both MRSA and MSSA. Isolates recovered from the few patients with posttreatment cultures showed no significant reduction in gepotidacin susceptibility (≥4-fold MIC increase) between pretreatment and posttreatment isolates. Two of the 78 S. aureus isolates from pretreatment lesions had elevated gepotidacin MICs and had mutations known to occur in quinolone-resistant S. aureus (GyrA S84L, ParC S80Y, and ParE D422E) or to confer elevated MICs to novel bacterial topoisomerase inhibitors (GyrA D83N, both isolates; ParC V67A, one isolate). This first report of microbiological outcomes and responses of gepotidacin in patients with ABSSSIs supports further evaluation of gepotidacin as a novel first-in-class antibacterial agent. (This study has been registered at ClinicalTrials.gov under identifier NCT02045797.).
Assuntos
Acenaftenos/farmacologia , Antibacterianos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Mutação/genética , Pele/microbiologia , Dermatopatias Infecciosas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genéticaRESUMO
A paucity of novel acting antibacterials is in development to treat the rising threat of antimicrobial resistance, particularly in Gram-negative hospital pathogens, which has led to renewed efforts in antibiotic drug discovery. Fluoroquinolones are broad-spectrum antibacterials that target DNA gyrase by stabilizing DNA-cleavage complexes, but their clinical utility has been compromised by resistance. We have identified a class of antibacterial thiophenes that target DNA gyrase with a unique mechanism of action and have activity against a range of bacterial pathogens, including strains resistant to fluoroquinolones. Although fluoroquinolones stabilize double-stranded DNA breaks, the antibacterial thiophenes stabilize gyrase-mediated DNA-cleavage complexes in either one DNA strand or both DNA strands. X-ray crystallography of DNA gyrase-DNA complexes shows the compounds binding to a protein pocket between the winged helix domain and topoisomerase-primase domain, remote from the DNA. Mutations of conserved residues around this pocket affect activity of the thiophene inhibitors, consistent with allosteric inhibition of DNA gyrase. This druggable pocket provides potentially complementary opportunities for targeting bacterial topoisomerases for antibiotic development.
Assuntos
Antibacterianos , Clivagem do DNA , DNA Girase , Tiofenos , Antibacterianos/química , Antibacterianos/metabolismo , Cristalografia por Raios X , DNA Girase/química , DNA Girase/metabolismo , Descoberta de Drogas , Modelos Moleculares , Tiofenos/química , Tiofenos/metabolismoRESUMO
We evaluated microbiological correlates for the successful treatment of Neisseria gonorrhoeae isolates from a phase 2 study of gepotidacin, a novel triazaacenaphthylene antibacterial, for therapy of uncomplicated urogenital gonorrhea. Culture, susceptibility testing, genotypic characterization, and frequency of resistance (FoR) were performed for selected isolates. Microbiological success was defined as culture-confirmed eradication of N. gonorrhoeae Against 69 baseline urogenital isolates, gepotidacin MICs ranged from ≤0.06 to 1 µg/ml (MIC90 = 0.5 µg/ml). For gepotidacin, the ratio of the area under the free-drug concentration-time curve to the MIC (fAUC/MIC) was associated with therapeutic success. Success was 100% (61/61) at fAUC/MICs of ≥48 and decreased to 63% (5/8) for fAUC/MICs of ≤25. All 3 isolates from microbiological failures were ciprofloxacin resistant, had a baseline gepotidacin MIC of 1 µg/ml, and carried a preexisting ParC D86N mutation, a critical residue for gepotidacin binding. In a test-of-cure analysis, the resistance to gepotidacin emerged in 2 isolates (MICs increased ≥32-fold) with additional GyrA A92T mutations, also implicated in gepotidacin binding. Test-of-cure isolates had the same sequence type as the corresponding baseline isolates. For 5 selected baseline isolates, all carrying a ParC D86N mutation, the in vitro FoR to gepotidacin was low (10-9 to 10-10); the resistant mutants had the same A92T mutation as the 2 isolates in which resistance emerged. Five participants with isolates harboring the ParC D86N mutation were treatment successes. In summary, fAUC/MICs of ≥48 predicted 100% microbiological success, including 3 isolates with the ParC D86N mutation (fAUC/MICs ≥ 97). Pharmacokinetic/pharmacodynamic determinations may help to evaluate new therapies for gonorrhea; further study of gepotidacin is warranted. (This study has been registered at ClinicalTrials.gov under identifier NCT02294682.).
Assuntos
Acenaftenos/farmacocinética , Antibacterianos/farmacocinética , DNA Topoisomerase IV/genética , Farmacorresistência Bacteriana/genética , Gonorreia/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Neisseria gonorrhoeae/efeitos dos fármacos , Acenaftenos/sangue , Acenaftenos/farmacologia , Administração Oral , Adulto , Antibacterianos/sangue , Antibacterianos/farmacologia , Área Sob a Curva , Técnicas de Tipagem Bacteriana , Hemocultura , Ciprofloxacina/uso terapêutico , DNA Topoisomerase IV/metabolismo , Esquema de Medicação , Feminino , Expressão Gênica , Gonorreia/sangue , Gonorreia/microbiologia , Gonorreia/patologia , Compostos Heterocíclicos com 3 Anéis/sangue , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Masculino , Testes de Sensibilidade Microbiana , Mutação , Neisseria gonorrhoeae/enzimologia , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/isolamento & purificação , Resultado do TratamentoRESUMO
GSK1322322 is a novel antibacterial agent under development, and it has known antibacterial activities against multidrug-resistant respiratory and skin pathogens through its inhibition of the bacterial peptide deformylase. Here, we used next-generation sequencing (NGS) of the bacterial 16S rRNA genes from stool samples collected from 61 healthy volunteers at the predosing and end-of-study time points to determine the effects of GSK1322322 on the gastrointestinal (GI) microbiota in a phase I, randomized, double-blind, and placebo-controlled study. GSK1322322 was administered either intravenously (i.v.) only or in an oral-i.v. combination in single- and repeat-dose-escalation infusions. Analysis of the 16S rRNA sequence data found no significant changes in the relative abundances of GI operational taxonomic units (OTUs) between the prestudy and end-of-study samples for either the placebo- or i.v.-only-treated subjects. However, oral-i.v. treatment resulted in significant decreases in some bacterial taxa, the Firmicutes and Bacteroidales, and increases in others, the Betaproteobacteria, Gammaproteobacteria, and Bifidobacteriaceae. Microbiome diversity plots clearly differentiated the end-of-study oral-i.v.-dosed samples from all others collected. The changes in genome function as inferred from species composition suggest an increase in bacterial transporter and xenobiotic metabolism pathways in these samples. A phylogenetic analysis of the peptide deformylase protein sequences collected from the published genomes of clinical isolates previously tested for GSK1322322 in vitro susceptibility and GI bacterial reference genomes suggests that antibiotic target homology is one of several factors that influences the response of GI microbiota to this antibiotic. Our study shows that dosing regimen and target class are important factors when considering the impact of antibiotic usage on GI microbiota. (This clinical trial was registered at the GlaxoSmithKline Clinical Study Register under study identifier PDF 113376.).
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Ácidos Hidroxâmicos/farmacologia , Microbiota/efeitos dos fármacos , Microbiota/genética , Betaproteobacteria/efeitos dos fármacos , Betaproteobacteria/genética , Bifidobacterium/efeitos dos fármacos , Bifidobacterium/genética , Método Duplo-Cego , Gammaproteobacteria/efeitos dos fármacos , Gammaproteobacteria/genética , Voluntários Saudáveis , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Ribossômico 16S/genéticaRESUMO
The continuous emergence of multidrug-resistant pathogenic bacteria is compromising the successful treatment of serious microbial infections. GSK1322322, a novel peptide deformylase (PDF) inhibitor, shows good in vitro antibacterial activity and has demonstrated safety and efficacy in human proof-of-concept clinical studies. In vitro studies were performed to determine the frequency of resistance (FoR) to this antimicrobial agent in major pathogens that cause respiratory tract and skin infections. Resistance to GSK1322322 occurred at high frequency through loss-of-function mutations in the formyl-methionyl transferase (FMT) protein in Staphylococcus aureus (4/4 strains) and Streptococcus pyogenes (4/4 strains) and via missense mutations in Streptococcus pneumoniae (6/21 strains), but the mutations were associated with severe in vitro and/or in vivo fitness costs. The overall FoR to GSK1322322 was very low in Haemophilus influenzae, with only one PDF mutant being identified in one of four strains. No target-based mutants were identified from S. pyogenes, and only one or no PDF mutants were isolated in three of the four S. aureus strains studied. In S. pneumoniae, PDF mutants were isolated from only six of 21 strains tested; an additional 10 strains did not yield colonies on GSK1322322-containing plates. Most of the PDF mutants characterized from those three organisms (35/37 mutants) carried mutations in residues at or in close proximity to one of three highly conserved motifs that are part of the active site of the PDF protein, with 30 of the 35 mutations occurring at position V71 (using the S. pneumoniae numbering system).
Assuntos
Amidoidrolases/antagonistas & inibidores , Antibacterianos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pyogenes/efeitos dos fármacos , Infecções por Haemophilus/tratamento farmacológico , Humanos , Testes de Sensibilidade Microbiana/métodos , Infecções Pneumocócicas/tratamento farmacológico , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
GSK2251052, a novel leucyl-tRNA synthetase (LeuRS) inhibitor, was in development for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. In a phase II study (study LRS114688) evaluating the efficacy of GSK2251052 in complicated urinary tract infections, resistance developed very rapidly in 3 of 14 subjects enrolled, with ≥32-fold increases in the GSK2251052 MIC of the infecting pathogen being detected. A fourth subject did not exhibit the development of resistance in the baseline pathogen but posttherapy did present with a different pathogen resistant to GSK2251052. Whole-genome DNA sequencing of Escherichia coli isolates collected longitudinally from two study LRS114688 subjects confirmed that GSK2251052 resistance was due to specific mutations, selected on the first day of therapy, in the LeuRS editing domain. Phylogenetic analysis strongly suggested that resistant Escherichia coli isolates resulted from clonal expansion of baseline susceptible strains. This resistance development likely resulted from the confluence of multiple factors, of which only some can be assessed preclinically. Our study shows the challenges of developing antibiotics and the importance of clinical studies to evaluate their effect on disease pathogenesis. (These studies have been registered at ClinicalTrials.gov under registration no. NCT01381549 for the study of complicated urinary tract infections and registration no. NCT01381562 for the study of complicated intra-abdominal infections.).
Assuntos
Compostos de Boro/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Leucina-tRNA Ligase/antagonistas & inibidores , Infecções Urinárias/tratamento farmacológico , Antibacterianos/farmacologia , Anti-Infecciosos Urinários/farmacologia , Compostos de Boro/uso terapêutico , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Genoma Bacteriano , Humanos , Mutação , Filogenia , Infecções Urinárias/microbiologiaRESUMO
There is a global emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. While the epidemiology of K. pneumoniae strains and occurrences of specific antibiotic resistance genes, such as plasmid-borne extended-spectrum ß-lactamases (ESBLs), have been extensively studied, only four complete genomes of K. pneumoniae are available. To better understand the multidrug resistance factors in K. pneumoniae, we determined by pyrosequencing the nearly complete genome DNA sequences of two strains with disparate antibiotic resistance profiles, broadly drug-susceptible strain JH1 and strain 1162281, which is resistant to multiple clinically used antibiotics, including extended-spectrum ß-lactams, fluoroquinolones, aminoglycosides, trimethoprim, and sulfamethoxazoles. Comparative genomic analysis of JH1, 1162281, and other published K. pneumoniae genomes revealed a core set of 3,631 conserved orthologous proteins, which were used for reconstruction of whole-genome phylogenetic trees. The close evolutionary relationship between JH1 and 1162281 relative to other K. pneumoniae strains suggests that a large component of the genetic and phenotypic diversity of clinical isolates is due to horizontal gene transfer. Using curated lists of over 400 antibiotic resistance genes, we identified all of the elements that differentiated the antibiotic profile of MDR strain 1162281 from that of susceptible strain JH1, such as the presence of additional efflux pumps, ESBLs, and multiple mechanisms of fluoroquinolone resistance. Our study adds new and significant DNA sequence data on K. pneumoniae strains and demonstrates the value of whole-genome sequencing in characterizing multidrug resistance in clinical isolates.
Assuntos
Genoma Bacteriano/genética , Genômica/métodos , Klebsiella pneumoniae/genética , Aminoglicosídeos/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Fluoroquinolonas/farmacologia , Klebsiella pneumoniae/efeitos dos fármacos , Sulfametoxazol/farmacologia , Trimetoprima/farmacologia , beta-Lactamas/farmacologiaRESUMO
This corrects the article DOI: 10.1038/ncomms16081.
RESUMO
The identification and prioritization of chemically tractable therapeutic targets is a significant challenge in the discovery of new medicines. We have developed a novel method that rapidly screens multiple proteins in parallel using DNA-encoded library technology (ELT). Initial efforts were focused on the efficient discovery of antibacterial leads against 119 targets from Acinetobacter baumannii and Staphylococcus aureus. The success of this effort led to the hypothesis that the relative number of ELT binders alone could be used to assess the ligandability of large sets of proteins. This concept was further explored by screening 42 targets from Mycobacterium tuberculosis. Active chemical series for six targets from our initial effort as well as three chemotypes for DHFR from M. tuberculosis are reported. The findings demonstrate that parallel ELT selections can be used to assess ligandability and highlight opportunities for successful lead and tool discovery.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Descoberta de Drogas/métodos , Biblioteca Gênica , Mycobacterium tuberculosis/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , Staphylococcus aureus/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Avaliação Pré-Clínica de Medicamentos , Terapia de Alvo Molecular , Mycobacterium tuberculosis/metabolismo , Staphylococcus aureus/metabolismoRESUMO
Undecaprenyl pyrophosphate synthase (UppS) is an essential enzyme in bacterial cell wall synthesis. Here we report the discovery of Staphylococcus aureus UppS inhibitors from an Encoded Library Technology screen and demonstrate binding to the hydrophobic substrate site through cocrystallography studies. The use of bacterial strains with regulated uppS expression and inhibitor resistant mutant studies confirmed that the whole cell activity was the result of UppS inhibition, validating UppS as a druggable antibacterial target.
Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antibacterianos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Alquil e Aril Transferases/metabolismo , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Pirazóis/síntese química , Pirazóis/química , Staphylococcus aureus/enzimologia , Relação Estrutura-AtividadeRESUMO
The gastrointestinal tract microbiome has been suggested as a potential therapeutic target for metabolic diseases such as obesity and Type 2 diabetes mellitus (T2DM). However, the relationship between changes in microbial communities and metabolic disease-phenotypes are still poorly understood. In this study, we used antibiotics with markedly different antibacterial spectra to modulate the gut microbiome in a diet-induced obesity mouse model and then measured relevant biochemical, hormonal and phenotypic biomarkers of obesity and T2DM. Mice fed a high-fat diet were treated with either ceftazidime (a primarily anti-Gram negative bacteria antibiotic) or vancomycin (mainly anti-Gram positive bacteria activity) in an escalating three-dose regimen. We also dosed animals with a well-known prebiotic weight-loss supplement, 10% oligofructose saccharide (10% OFS). Vancomycin treated mice showed little weight change and no improvement in glycemic control while ceftazidime and 10% OFS treatments induced significant weight loss. However, only ceftazidime showed significant, dose dependent improvement in key metabolic variables including glucose, insulin, protein tyrosine tyrosine (PYY) and glucagon-like peptide-1 (GLP-1). Subsequently, we confirmed the positive hyperglycemic control effects of ceftazidime in the Zucker diabetic fatty (ZDF) rat model. Metagenomic DNA sequencing of bacterial 16S rRNA gene regions V1-V3 showed that the microbiomes of ceftazidime dosed mice and rats were enriched for the phylum Firmicutes while 10% OFS treated mice had a greater abundance of Bacteroidetes. We show that specific changes in microbial community composition are associated with obesity and glycemic control phenotypes. More broadly, our study suggests that in vivo modulation of the microbiome warrants further investigation as a potential therapeutic strategy for metabolic diseases.
Assuntos
Antibacterianos/farmacologia , Diabetes Mellitus Tipo 2/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Obesidade/microbiologia , Animais , Ceftazidima/farmacologia , Dieta/efeitos adversos , Modelos Animais de Doenças , Masculino , Camundongos , Obesidade/etiologia , Fenótipo , Prebióticos , RatosRESUMO
New antibacterials are needed to tackle antibiotic-resistant bacteria. Type IIA topoisomerases (topo2As), the targets of fluoroquinolones, regulate DNA topology by creating transient double-strand DNA breaks. Here we report the first co-crystal structures of the antibacterial QPT-1 and the anticancer drug etoposide with Staphylococcus aureus DNA gyrase, showing binding at the same sites in the cleaved DNA as the fluoroquinolone moxifloxacin. Unlike moxifloxacin, QPT-1 and etoposide interact with conserved GyrB TOPRIM residues rationalizing why QPT-1 can overcome fluoroquinolone resistance. Our data show etoposide's antibacterial activity is due to DNA gyrase inhibition and suggests other anticancer agents act similarly. Analysis of multiple DNA gyrase co-crystal structures, including asymmetric cleavage complexes, led to a 'pair of swing-doors' hypothesis in which the movement of one DNA segment regulates cleavage and religation of the second DNA duplex. This mechanism can explain QPT-1's bacterial specificity. Structure-based strategies for developing topo2A antibacterials are suggested.
Assuntos
Antibacterianos/química , Antineoplásicos/química , DNA Girase/química , Etoposídeo/química , Fluoroquinolonas/química , Staphylococcus aureus/enzimologia , Inibidores da Topoisomerase II/química , Antibacterianos/farmacologia , Antineoplásicos/farmacologia , DNA Girase/genética , DNA Girase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Farmacorresistência Bacteriana , Etoposídeo/farmacologia , Fluoroquinolonas/farmacologia , Modelos Moleculares , Estrutura Molecular , Moxifloxacina , Staphylococcus aureus/química , Staphylococcus aureus/efeitos dos fármacos , Inibidores da Topoisomerase II/farmacologiaRESUMO
Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.
Assuntos
DNA Topoisomerases Tipo II/genética , Farmacorresistência Bacteriana/genética , Evolução Molecular , Transferência Genética Horizontal , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Anti-Infecciosos/farmacologia , Sequência de Bases , DNA Girase/genética , Fluoroquinolonas/farmacologia , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
The screening of new antibiotics against several bacterial strains often reveals unexpected occurrences of natural drug resistance. Two examples of this involve specific inhibitors of Staphylococcus aureus isoleucyl-transfer-RNA synthetase 1 (IleRS1) and, more recently, Streptococcus pneumoniae methionyl-tRNA synthetase 1 (MetRS1). In both cases, resistance is due to the presence of a second gene that encodes another synthetase (IleRS2 or MetRS2). Here, we show that both S. pneumoniae MetRS2 and S. aureus IleRS2 have closely related homologues in the Gram-positive bacterium Bacillus anthracis, the causative agent of anthrax. Furthermore, similar to drug-resistant pathogens, strains of B. anthracis and its closest relative, B. cereus, also have wild-type ileS1 and metS1 genes. Clostridium perfringens, the causative agent of gangrene, also has two metS genes, whereas Oceanobacillus iheyensis isolated from deep-sea sediments has a single ileS2-type gene. This study shows the importance of understanding complex evolutionary networks of ancient horizontal gene transfer for the development of novel antibiotics.
Assuntos
Bacillus anthracis/genética , Farmacorresistência Bacteriana/genética , Transferência Genética Horizontal/genética , Bactérias Gram-Positivas/enzimologia , Bactérias Gram-Positivas/genética , Isoleucina-tRNA Ligase/genética , Metionina tRNA Ligase/genética , Sequência de Aminoácidos , Antraz/microbiologia , Bacillus anthracis/enzimologia , Biologia Computacional , Evolução Molecular , Isoleucina-tRNA Ligase/química , Metionina tRNA Ligase/química , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de AminoácidosRESUMO
Streptococcus pneumoniae is an important human pathogen capable of causing serious infections. NADH oxidase, a factor necessary for infection, was previously identified as part of a signature-tagged mutagenesis screen of a S. pneumoniae clinical isolate, 0100993. The mutant, with a plasmid insertion disrupting the nox gene, was attenuated for virulence in a murine respiratory tract infection model. A complete refined nox deletion mutant was generated by allelic-replacement mutagenesis and found to be attenuated for virulence 10(5)-fold in the murine respiratory tract infection model and at least 10(4)-fold in a Mongolian gerbil otitis media infection model, confirming the importance of the NADH oxidase for both types of S. pneumoniae infection. NADH oxidase converts O(2) to H(2)O. If O(2) is not fully reduced, it can form superoxide anion (O2(-)) and hydrogen peroxide (H(2)O(2)), both of which can be toxic to cells. Bacterial cell extracts from the allelic-replacement mutant were found to lack NADH oxidase activity and the mutant was unable to grow exponentially under conditions of vigorous aeration. In contrast, the mutant displayed normal growth characteristics under conditions of limited aeration. The S. pneumoniae nox gene was cloned and expressed in E. coli. The purified His-tagged NADH oxidase was shown to oxidize NADH with a K:(m) of 32 microM, but was unable to oxidize NADPH. Oxidation of NADH was independent of exogenous FAD or FMN.
Assuntos
Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/genética , NADH NADPH Oxirredutases/metabolismo , Infecções Pneumocócicas/fisiopatologia , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/patogenicidade , Alelos , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Mutação , Otite Média/microbiologia , Otite Média/fisiopatologia , Filogenia , Infecções Pneumocócicas/microbiologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/fisiopatologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crescimento & desenvolvimento , VirulênciaRESUMO
As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, > or =8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors.
Assuntos
Inibidores Enzimáticos/farmacologia , Metionina tRNA Ligase/antagonistas & inibidores , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Sequência de Aminoácidos , Sequência de Bases , DNA Bacteriano/química , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Metionina tRNA Ligase/genética , Metionina tRNA Ligase/metabolismo , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutagênese , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Streptococcus pneumoniae/genéticaRESUMO
The promoter of the Streptococcus pneumoniae putative fuculose kinase gene (fcsK), the first gene of a novel fucose utilization operon, is induced by fucose and repressed by glucose or sucrose. When the streptococcal polypeptide deformylase (PDF) gene (def1, encoding PDF) was placed under the control of P(fcsK), fucose-dependent growth of the S. pneumoniae (P(fcsK)::def1) strain was observed, confirming the essential nature of PDF in this organism. The mode of antibacterial action of actinonin, a known PDF inhibitor, was also confirmed with this strain. The endogenous fuculose kinase promoter is a tightly regulated, titratable promoter which will be useful for target validation and for confirmation of the mode of action of novel antibacterial drugs in S. pneumoniae.
Assuntos
Amidoidrolases , Antibacterianos/farmacologia , Fucose/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Essenciais , Regiões Promotoras Genéticas/genética , Streptococcus pneumoniae/efeitos dos fármacos , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Inibidores Enzimáticos/farmacologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Dados de Sequência Molecular , Óperon , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/crescimento & desenvolvimento , Streptococcus pneumoniae/metabolismoRESUMO
Homologues of Escherichia coli bacA, encoding extremely hydrophobic proteins, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Allelic replacement mutagenesis demonstrated that the gene is not essential for in vitro growth in either organism, and the mutants showed no significant changes in growth rate or morphology. The Staph. aureus bacA mutant showed slightly reduced virulence in a mouse model of infection and an eightfold increase in bacitracin susceptibility. However, a Strep. pneumoniae bacA mutant was highly attenuated in a mouse model of infection, and demonstrated an increase in susceptibility to bacitracin of up to 160000-fold. These observations are consistent with the previously proposed role of BacA protein as undecaprenol kinase.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Staphylococcus aureus/genética , Streptococcus pneumoniae/genética , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Bacitracina/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Modelos Animais de Doenças , Feminino , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos CBA , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Infecções Pneumocócicas/microbiologia , Homologia de Sequência de Aminoácidos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/patogenicidade , VirulênciaRESUMO
Attempted allelic replacement of 144 Streptococcus pneumoniae open reading frames of previously uncharacterized function led to the identification of 36 genes essential for growth under laboratory conditions. Of these, 14 genes (obg, spoIIIJ2, trmU, yacA, yacM, ydiC, ydiE, yjbN, yneS, yphC, ysxC, ytaG, yloI and yxeH4) were also essential in Staphylococcus aureus and Haemophilus influenzae or Escherichia coli, 2 genes (yrrK and ydiB) were only essential in H. influenzae as well as S. pneumoniae and 8 genes were necessary for growth of S.pneumoniae and S. aureus and did not have a homolog in H. influenzae(murD2, ykqC, ylqF, yqeH, ytgP, yybQ) or were not essential in that organism (yqeL, yhcT). The proteins encoded by these genes could represent good targets for novel antibiotics covering different therapeutic profiles. The putative functions of some of these essential proteins, inferred by bioinformatic analysis, are presented. Four mutants, with deletions of loci not essential for in vitro growth, were found to be severely attenuated in a murine respiratory tract infection model, suggesting that not all targets for antibacterial therapeutics are revealed by simple in vitro essentiality testing. The results of our experiments together with those collated from previously reported studies including Bacillus subtilis, E. coli and Mycoplasma sp. demonstrate that gene conservation amongst bacteria does not necessarily indicate that essentiality in one organism can be extrapolated to others. Moreover, this study demonstrates that different experimental procedures can produce apparently contradictory results.