RESUMEN
Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10(-7). In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole.
Asunto(s)
Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Nitroimidazoles/uso terapéutico , Oxazinas/uso terapéutico , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/farmacocinética , Células CACO-2 , Línea Celular Tumoral , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Farmacorresistencia Bacteriana/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Nitroimidazoles/farmacocinética , Oxazinas/farmacocinética , Oxazoles/uso terapéuticoRESUMEN
OBJECTIVE: To assess the activity of clofazimine (CFZ) against Mycobacterium tuberculosis persisters using an oxygen depletion model and a low-dose aerosol mouse model of chronic tuberculosis (TB). DESIGN: In in vitro experiments, CFZ showed much better activity than isoniazid under anaerobic conditions. In a low-dose aerosol mouse model of TB, we evaluated the efficacy of CFZ and moxifloxacin at different doses following treatment durations of 30, 60 and 90 days. RESULTS: CFZ showed significant bactericidal activity in the mouse model over the wide dose range of 2-200 mg/kg. CFZ activity was dose-dependent. The bacilli were eradicated in the CFZ 200 mg/kg group after treatment for 60 days, and in the CFZ 20 mg/kg group after 90 days of treatment. CONCLUSION: CFZ exhibits dose-dependent, sustained bactericidal activity against M. tuberculosis persisters, and thus warrants further study to demonstrate its potential to contribute significantly in a novel treatment-shortening regimen.
Asunto(s)
Antituberculosos/farmacología , Clofazimina/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Antituberculosos/sangre , Compuestos Aza/farmacología , Enfermedad Crónica , Clofazimina/sangre , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Farmacorresistencia Bacteriana , Fluoroquinolonas , Isoniazida/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Moxifloxacino , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/aislamiento & purificación , Quinolinas/farmacología , Rifampin/farmacología , Factores de Tiempo , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/microbiologíaRESUMEN
Human lactate dehydrogenase (LDH) is thought to contribute to the oxidation of glyoxylate to oxalate and thus to the pathogenesis of disorders of endogenous oxalate overproduction. Glyoxylate reductase (GRHPR) has a potentially protective role metabolising glyoxylate to the less reactive glycolate. In this paper, the kinetic parameters of recombinant human LDHA, LDHB and GR have been compared with respect to their affinity for glyoxylate and related substrates. The Km values and specificity constants (Kcat/K(M)) of purified recombinant human LDHA, LDHB and GRHPR were determined for the reduction of glyoxylate and hydroxypyruvate. K(M) values with glyoxylate were 29.3 mM for LDHA, 9.9 mM for LDHB and 1.0 mM for GRHPR. For the oxidation of glyoxylate, K(M) values were 0.18 mM and 0.26 mM for LDHA and LDHB respectively with NAD+ as cofactor. Overall, under the same reaction conditions, the specificity constants suggest there is a fine balance between the reduction and oxidation reactions of these substrates, suggesting that control is most likely dictated by the ambient concentrations of the respective intracellular cofactors. Neither LDHA nor LDHB utilised glycolate as substrate and NADPH was a poor cofactor with a relative activity less than 3% that of NADH. GRHPR had a higher affinity for NADPH than NADH (K(M) 0.011 mM vs. 2.42 mM). The potential roles of LDH isoforms and GRHPR in oxalate synthesis are discussed.
Asunto(s)
Oxidorreductasas de Alcohol/química , Glioxilatos/química , L-Lactato Deshidrogenasa/química , Oxidorreductasas de Alcohol/metabolismo , Catálisis , Glioxilatos/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Enfermedades Metabólicas/metabolismo , NAD/química , NADP/química , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Ethionamide (ETA) is an important component of second-line therapy for the treatment of multidrug-resistant tuberculosis. Synthesis of radiolabeled ETA and an examination of drug metabolites formed by whole cells of Mycobacterium tuberculosis (MTb) have allowed us to demonstrate that ETA is activated by S-oxidation before interacting with its cellular target. ETA is metabolized by MTb to a 4-pyridylmethanol product remarkably similar in structure to that formed by the activation of isoniazid by the catalase-peroxidase KatG. We have demonstrated that overproduction of Rv3855 (EtaR), a putative regulatory protein from MTb, confers ETA resistance whereas overproduction of an adjacent, clustered monooxygenase (Rv3854c, EtaA) confers ETA hypersensitivity. Production of EtaA appears to be negatively regulated by EtaR and correlates directly with [(14)C]ETA metabolism, suggesting that EtaA is the activating enzyme responsible for thioamide oxidation and subsequent toxicity. Coding sequence mutations in EtaA were found in 11 of 11 multidrug-resistant MTb patient isolates from Cape Town, South Africa. These isolates showed broad cross-resistance to thiocarbonyl containing drugs including ETA, thiacetazone, and thiocarlide.
Asunto(s)
Antituberculosos/metabolismo , Antituberculosos/farmacología , Resistencia a Múltiples Medicamentos , Etionamida/metabolismo , Etionamida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Farmacorresistencia Microbiana , Etionamida/síntesis química , Genes Bacterianos/genética , Genes Bacterianos/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Mutación/genética , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Oxidación-Reducción/efectos de los fármacos , Tioamidas/metabolismo , Tioamidas/farmacología , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
The Mycobacterium tuberculosis AhpC is similar to a family of bacterial and eukaryotic antioxidant proteins with alkylhydroperoxidase (Ahp) and thioredoxin-dependent peroxidase (TPx) activities. AhpC expression is associated with resistance to the front-line antitubercular drug isoniazid in the naturally resistant organisms E. coli and M. smegmatis. We identified several isoniazid-resistant M. tuberculosis isolates with ahpC promoter mutations resulting in AhpC overexpression. These strains were more resistant to cumene hydroperoxide than were wild-type strains. However, these strains were unchanged in their sensitivity to isoniazid, refuting a role for AhpC in detoxification of this drug. All the isoniazid-resistant, AhpC-overexpressing strains were also deficient in activity of the mycobacterial catalase-peroxidase KatG. KatG, the only known catalase in M. tuberculosis, is required for activation of isoniazid. We propose that compensatory ahpC promoter mutations are selected from KatG-deficient, isoniazid-resistant M. tuberculosis during infections, to mitigate the added burden imposed by organic peroxides on these strains.
Asunto(s)
Antituberculosos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Estrés Oxidativo , Peroxidasas/genética , Peroxidasas/metabolismo , Secuencia de Aminoácidos , Antioxidantes/metabolismo , Farmacorresistencia Microbiana , Isoniazida/farmacología , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Peroxidasas/química , Peroxirredoxinas , Regiones Promotoras Genéticas , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Although isoniazid (isonicotinic acid hydrazide, INH) is widely used for the treatment of tuberculosis, its molecular target has remained elusive. In response to INH treatment, saturated hexacosanoic acid (C26:0) accumulated on a 12-kilodalton acyl carrier protein (AcpM) that normally carried mycolic acid precursors as long as C50. A protein species purified from INH-treated Mycobacterium tuberculosis was shown to consist of a covalent complex of INH, AcpM, and a beta-ketoacyl acyl carrier protein synthase, KasA. Amino acid-altering mutations in the KasA protein were identified in INH-resistant patient isolates that lacked other mutations associated with resistance to this drug.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , Antituberculosos/farmacología , Isoniazida/farmacología , Mycobacterium tuberculosis/enzimología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/genética , Proteína Transportadora de Acilo/metabolismo , Secuencia de Aminoácidos , Farmacorresistencia Microbiana , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/metabolismo , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Peso Molecular , Mutación , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Ácidos Micólicos/metabolismo , Tuberculosis/microbiología , Regulación hacia ArribaRESUMEN
Isoniazid (INH), which acts by inhibiting mycolic acid biosynthesis, is very potent against the tuberculous mycobacteria. It is about 100-fold less effective against Mycobacterium avium. This difference has often been attributed to a decreased permeability of the cell wall. We measured the rate of conversion of radiolabelled INH to 4-pyridylmethanol by whole cells and cell-free extracts and estimated the permeability barrier imposed by the cell wall to INH influx in Mycobacterium tuberculosis and M. avium. There was no significant difference in the relative permeability to INH between these two species. However, the total conversion rate in M. tuberculosis was found to be four times greater. Examination of in vitro-generated mutants revealed that the major resistance mechanism for both species is loss of the catalase-peroxidase KatG. Analysis of lipid and protein biosynthetic profiles demonstrated that the molecular target of activated INH was identical for both species. M. avium, however, formed colonies at INH concentrations inhibitory for mycolic acid biosynthesis. These mycolate-deficient M. avium exhibited altered colony morphologies, modified cell wall ultrastructure and were 10-fold more sensitive to treatment with hydrophobic antibiotics, such as rifampin. These findings may significantly impact the design of new therapeutic regimens for the treatment of infections with atypical mycobacteria.
Asunto(s)
Proteínas Bacterianas , Farmacorresistencia Microbiana/fisiología , Isoniazida/farmacología , Mycobacterium avium/fisiología , División Celular/genética , Diseño de Fármacos , Cinética , Microscopía Electrónica , Datos de Secuencia Molecular , Mutagénesis/genética , Mycobacterium avium/ultraestructura , Mycobacterium tuberculosis/fisiología , Ácidos Micólicos/química , Permeabilidad , Peroxidasas/deficiencia , Peroxidasas/genética , Fenotipo , Rifampin/farmacologíaRESUMEN
Isoniazid (INH) is a widely used front-line antituberculous agent with bacteriocidal activity at concentrations as low as 150 nM against Mycobacterium tuberculosis. INH is a prodrug and requires activation by an endogenous mycobacterial enzyme, the catalase-peroxidase KatG, before exerting toxic effects on cellular targets. Resistance to INH develops primarily through failure to activate the prodrug due to point mutations in the katG gene. In addition to mutations in katG, mutations in several other loci, such as the alkylhydroperoxidase AhpC and the enoylreductase InhA, may contribute to INH resistance. Although these markers can be used to accurately predict clinical INH resistance in a large number of cases, the molecular mechanisms involved remain largely speculative and incomplete.
RESUMEN
The Mycobacterium tuberculosis H37Rv efpA gene encodes a putative efflux protein, EfpA, of 55,670 Da. The deduced EfpA protein was similar in secondary structure to Pur8, MmrA, TcmA, LfrA, EmrB, and other members of the QacA transporter family (QacA TF) which mediate antibiotic and chemical resistance in bacteria and yeast. The predicted EfpA sequence possessed all transporter motifs characteristic of the QacA TF, including those associated with proton-antiport function and the motif considered to be specific to exporters. The 1,590-bp efpA open reading frame was G+C rich (65%), whereas the 40-bp region immediately upstream had an A+T bias (35% G+C). Reverse transcriptase-PCR assays indicated that efpA was expressed in vitro and in situ. Putative promoter sequences were partially overlapped by the A+T-rich region and by a region capable of forming alternative secondary structures indicative of transcriptional regulation in analogous systems. PCR single-stranded conformational polymorphism analysis demonstrated that these upstream flanking sequences and the 231-bp, 5' coding region are highly conserved among both drug-sensitive and multiply-drug-resistant isolates of M. tuberculosis. The efpA gene was present in the slow-growing human pathogens M. tuberculosis, Mycobacterium leprae, and Mycobacterium bovis and in the opportunistic human pathogens Mycobacterium avium and Mycobacterium intracellular. However, efpA was not present in 17 other opportunistically pathogenic or nonpathogenic mycobacterial species.
Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Transporte de Membrana , Mycobacterium tuberculosis/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Transporte Biológico/genética , Proteínas Portadoras/metabolismo , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Mycobacterium bovis/genética , Mycobacterium leprae/genética , Reacción en Cadena de la PolimerasaRESUMEN
An examination of the pattern of lipid biosynthetic responses to isoniazid (INH) treatment of Mycobacterium tuberculosis and Mycobacterium smegmatis suggests that the mode of action of activated INH differs between these 2 organisms. Transformation of M. smegmatis with inhA on a plasmid construct conferred high-level resistance to INH, while the same construct failed to confer resistance upon M. tuberculosis. The inhA region from 2 clinical isolates whose resistance has been attributed to changes in the upstream promoter region has been cloned and was not sufficient to impart INH resistance to the level of the parent strain on sensitive M. tuberculosis. These putative mutant promoter elements appear to elevate expression levels of gene fusion reporter constructs, suggesting some noncausal connection between the observed mutations and the lipid metabolism of drug-resistant organisms. These results suggest that InhA is not the major target for activated INH in M. tuberculosis.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/genética , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas , Biotransformación , Farmacorresistencia Microbiana , Isoniazida/metabolismo , Lípidos/biosíntesis , Mutación , Regiones Promotoras GenéticasRESUMEN
The intrinsic resistance of many mycobacterial species to chemotherapy is largely attributable to their impermeable cell wall. The composition of the cell wall of a particular species appears to be influenced by the environmental niche that the species occupies. The complex regulatory and biosynthetic pathways involved in cell wall biosynthesis and construction offer useful chemotherapeutic targets against mycobacteria.
Asunto(s)
Pared Celular/química , Mycobacterium/fisiología , Adaptación Fisiológica , Pared Celular/efectos de los fármacos , Pared Celular/fisiología , Etambutol/farmacología , Isoniazida/farmacología , Membrana Dobles de Lípidos/química , Lipopolisacáridos/metabolismo , Modelos Biológicos , Mycobacterium/efectos de los fármacos , Mycobacterium/ultraestructura , Permeabilidad , Fosfatidilinositoles/fisiologíaRESUMEN
Mutations that eliminate KatG catalase-peroxidase activity prevent activation of isoniazid and are a major mechanism of resistance to this principal drug for the treatment of Mycobacterium tuberculosis infections. However, the loss of KatG activity in clinical isolates seemed paradoxical because KatG is considered an important factor for the survival of the organism. Expression of either KatG or the recently identified alkyl hydroperoxidase AhpC was sufficient to protect bacilli against the toxic effects of organic peroxides. To survive during infection, isoniazid-resistant KatG mutants have apparently compensated for the loss of KatG catalase-peroxidase activity by a second mutation, resulting in hyperexpression of AhpC.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas , Isoniazida/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Oxidorreductasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Farmacorresistencia Microbiana/genética , Sinergismo Farmacológico , Inducción Enzimática , Regulación Bacteriana de la Expresión Génica , Peróxido de Hidrógeno/farmacología , Datos de Secuencia Molecular , Mutación , Mycobacterium bovis/efectos de los fármacos , Mycobacterium bovis/genética , Peroxidasas/biosíntesis , Peroxidasas/genética , Peroxidasas/metabolismo , Peroxirredoxinas , Regiones Promotoras GenéticasRESUMEN
Phagemid vectors were constructed to allow fusions of alkaline phosphatase to proteins encoded by G+C-rich DNA, by engineering a BstBI site (TT/CGAA) in front of a phoA gene that lacks an encoded signal peptide. Three vectors (pJDT1, pJDT2 and pJDT3), each with phoA in a different reading frame with respect to the BstBI site, were produced; a lacP region is present in each plasmid upstream of the BstBI site. The presence of the BstBI site allows the random cloning of G+C-rich DNA digested with a number of restriction enzymes that generate cohesive ends.
Asunto(s)
Fosfatasa Alcalina/biosíntesis , Vectores Genéticos , Proteínas Recombinantes de Fusión/biosíntesis , Fosfatasa Alcalina/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Bacteriano/genética , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genéticaRESUMEN
We constructed mutant strains of Francisella tularensis biotype novicida by insertional mutagenesis with a kanamycin resistance (KmR) cassette. One mutant, KEM7, was defective for survival in macrophages in comparison with the wild-type (WT) strain and a random insertion strain, KEM21. While all three strains exhibited intracellular growth, the number of viable KEM7 present after 24-48 h of infection was approximately 10 times less than that of WT or KEM21. This observation was apparently due to a reduced number of viable KEM7 associated with the macrophages one hour after phagocytosis. KEM7 was approximately 3 times more susceptible than WT or KEM21 to killing by the products of the xanthine-xanthine oxidase reaction or by hydrogen peroxide. KEM7 was also found to be susceptible to killing by serum, whereas WT and KEM21 were resistant. Upon intravenous inoculation of C57BL/6 mice, the number of KEM7 in the livers and spleens 48 h post-infection was found to be 1,000- to 10,000-times less than that of either KEM21 or WT. DNA sequence analysis at the KmR insertion site suggested that the F. tularensis homologue of minD had been interrupted. Western immunoblot analysis confirmed the presence of a MinD homologue in F. tularensis WT and KEM21, and demonstrated its absence in KEM7.
Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Escherichia coli , Francisella tularensis/genética , Mutagénesis Insercional , Secuencia de Aminoácidos , Animales , Francisella tularensis/patogenicidad , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oxidación-Reducción , Alineación de Secuencia , VirulenciaRESUMEN
Francisella novicida is a facultative intracellular pathogen that can survive and grow in macrophages by preventing phagolysosomal fusion. In this study in vitro cassette mutagenesis was used to generate a library of insertion mutants of F.novicida. Two related mutants, KM14 and KM14S, initially identified as defective for growth in macrophages, were found to be sensitive to serum. These mutants were also found to grow approximately 1000-fold less well in the livers and spleens of infected mice. We cloned a genetic locus that was presumably mutagenized in these mutants and found that it included genes that had high similarity in their deduced amino acid sequence to those of msbA and orfE of Escherichia coli. The former is a member of the superfamily of ABC transporter proteins. We named the corresponding genes in F. novicida, valAB. Integration of a cloned valAB locus into the chromosome of KM14S partially restored the serum resistance phenotype found in wild-type F. novicida.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Francisella/genética , Genes Bacterianos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Medios de Cultivo , ADN Bacteriano/genética , Escherichia coli/genética , Femenino , Francisella/crecimiento & desarrollo , Francisella/patogenicidad , Prueba de Complementación Genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Fenotipo , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Virulencia/genéticaRESUMEN
DNA cloned from Chlamydia trachomatis is able to direct the formation of the genus-specific lipopolysaccharide epitope of chlamydiae in enteric Gram-negative bacteria. We now demonstrate that a single C. trachomatis gene (gseA) is sufficient to impart this trait to Escherichia coli. The deduced amino acid sequence of gseA shows 23% identity (66% similarity) to kdtA, an E. coli gene that codes for a bifunctional enzyme catalyzing the addition of two 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A precursors (Clementz, T., and Raetz, C. R. H. (1991) J. Biol. Chem. 266, 9687-9696). Extracts of E. coli expressing gseA transfer at least one additional Kdo unit from CMP-Kdo to precursors already bearing the two Kdo residues attached by the kdtA gene product. Introduction of gseA into an E. coli mutant with a thermolabile kdtA gene product endows cell extracts with the ability to transfer not only the third but also the first two Kdos to lipid A precursors, demonstrating that the C. trachomatis enzyme is at least trifunctional. Given the similarities of these two Kdo transferases and the essentiality of Kdo in Gram-negative bacteria, lipopolysaccharide biosynthesis may be a target for development of novel drugs effective against chlamydiae.
Asunto(s)
Chlamydia trachomatis/enzimología , Epítopos/genética , Expresión Génica , Transferasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía en Capa Delgada , Clonación Molecular , Escherichia coli/genética , Datos de Secuencia Molecular , Plásmidos , Biosíntesis de Proteínas , Recombinación Genética , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Transferasas/inmunologíaRESUMEN
A gene locus that is functionally analogous to the recA gene of Escherichia coli was molecularly cloned from Francisella novicida. The cloned gene was found to suppress the sensitivity of an E. coli strain to DNA-damaging agents and to support genetic recombination in E. coli. After transposon mutagenesis, the recA-like gene locus was returned to F. novicida and a UV-sensitive F. novicida strain was isolated. In contrast to the wild-type strain, this UV-sensitive strain could not be transformed with chromosomal DNA.