RESUMO
Toward minimizing bacterial colonization of surfaces, we present a one-step etching technique that renders aluminum alloys with micro- and nano-scale roughness. Such a multi-scale surface topography exhibited enhanced antibacterial effect against a wide range of pathogens. Multi-scale topography of commercially grade pure aluminum killed 97% of Escherichia coli and 28% of Staphylococcus aureus cells in comparison to 7% and 3%, respectively, on the smooth surfaces. Multi-scale topography on Al 5052 surface was shown to kill 94% of adhered E. coli cells. The microscale features on the etched Al 1200 alloy were not found to be significantly bactericidal, but shown to decrease the adherence of S. aureus cells by one-third. The fabrication method is easily scalable for industrial applications. Analysis of roughness parameters determined by atomic force microscopy revealed a set of significant parameters that can yield a highly bactericidal surface; thereby providing the design to make any surface bactericidal irrespective of the method of fabrication. The multi-scale roughness of Al 5052 alloy was also highly bactericidal to nosocomial isolates of E. coli, K. pneumoniae and P. aeruginosa. We envisage the potential application of engineered surfaces with multi-scale topography to minimize the spread of nosocomial infections.
RESUMO
BACKGROUND & OBJECTIVES: Outbreaks of infection due to Salmonella enterica servovar Typhi (S. Typhi) are a great threat to public health. A rapid molecular typing method to characterize strains implicated in an outbreak is critical in implementing appropriate control measures. This study was done to demonstrate the power of a PCR-based method to provide rapid insights into the genetic relatedness amongst the Salmonella isolates implicated in a suspected typhoid fever outbreak. METHODS: Forty two S. Typhi isolates originating from three geographically distinct areas, with one area suspected to have a single-source outbreak were included in the study. The genetic fingerprint of all isolates was generated using enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR). The antimicrobial susceptibility profiles were also evaluated. RESULTS: ERIC-PCR was found to be rapid and reproducible with a discriminatory index of 0.766. The dendrogram constructed based on ERIC-PCR fingerprinting revealed the existence of 12 distinct genotypes. The location suspected to have an outbreak displayed two genotypes amongst the 24 isolates. The other two locations (18 isolates) displayed genetic heterogeneity. The clonality of the outbreak isolates from the time-matched control isolates was established. The observed antimicrobial susceptibility profiles did not have any discriminatory power to subtype the isolates compared to the genetic fingerprints. INTERPRETATION & CONCLUSIONS: Our study demonstrated the discriminatory power and value of ERIC-PCR in the typing of S. Typhi isolates and providing valuable epidemiological insights.
Assuntos
Salmonella typhi/genética , Febre Tifoide/epidemiologia , Febre Tifoide/genética , Técnicas de Tipagem Bacteriana , Surtos de Doenças , Heterogeneidade Genética , Genótipo , Humanos , Índia/epidemiologia , Reação em Cadeia da Polimerase/métodos , Salmonella typhi/patogenicidade , Febre Tifoide/diagnóstico , Febre Tifoide/microbiologiaRESUMO
We report the discovery of benzothiazoles, a novel anti-mycobacterial series, identified from a whole cell based screening campaign. Benzothiazoles exert their bactericidal activity against Mycobacterium tuberculosis (Mtb) through potent inhibition of decaprenylphosphoryl-ß-d-ribose 2'-oxidase (DprE1), the key enzyme involved in arabinogalactan synthesis. Specific target linkage and mode of binding were established using co-crystallization and protein mass spectrometry studies. Most importantly, the current study provides insights on the utilization of systematic medicinal chemistry approaches to mitigate safety liabilities while improving potency during progression from an initial genotoxic hit, the benzothiazole N-oxides (BTOs) to the lead-like AMES negative, crowded benzothiazoles (cBTs). These findings offer opportunities for development of safe clinical candidates against tuberculosis. The design strategy adopted could find potential application in discovery of safe drugs in other therapy areas too.
Assuntos
Oxirredutases do Álcool/metabolismo , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Benzotiazóis/química , Benzotiazóis/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Oxirredutases do Álcool/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Humanos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
The MIC of the novel antituberculosis (anti-TB) drug AZD5847 was determined against 146 clinical isolates from diverse geographical regions, including eastern Europe, North America, Africa, and Asia, using the automated Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system. These isolates originated from specimen sources such as sputum, bronchial alveolar lavage fluid, pleural fluid, abscess material, lung biopsies, and feces. The overall MIC90 was 1.0 mg/liter (range, 0.125 to 4 mg/liter). The MICs of AZD5847 for isolates of Mycobacterium tuberculosis were similar among drug-sensitive strains, multidrug-resistant (MDR) strains, and extensively drug resistant (XDR) strains. The good in vitro activity of AZD5847 against M. tuberculosis and the lack of cross-resistance make this agent a promising anti-TB drug candidate.
Assuntos
Antituberculosos/uso terapêutico , Tuberculose Extensivamente Resistente a Medicamentos/tratamento farmacológico , Mycobacterium tuberculosis/efeitos dos fármacos , Oxazolidinonas/uso terapêutico , Tuberculose Pulmonar/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla , Tuberculose Extensivamente Resistente a Medicamentos/microbiologia , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Tuberculose Pulmonar/microbiologiaRESUMO
Oxazolidinones represent a new class of antituberculosis drugs that exert their function by inhibiting protein synthesis. Here, we compared the activities of three oxazolidinones, linezolid, PNU-100480, and AZD5847, against latent tuberculosis using a simple model employing the streptomycin-starved Mycobacterium tuberculosis strain 18b. The in vitro drug susceptibility results showed that the three oxazolidinones had a bacteriostatic effect against actively growing bacilli but potent bactericidal activity against nonreplicating cells. In the murine model of latent infection with M. tuberculosis 18b, the efficacy of the three compounds varied greatly. Indeed, AZD5847 or its prodrug exhibited no activity or only modest activity, respectively, after 2 months of treatment, whereas both linezolid and PNU-100480 were effective against latent bacilli in mice and showed promising outcomes in combination therapy with rifampin. Moreover, the potency of PNU-100480 was significantly greater than that of linezolid, making it an attractive drug candidate in the development of new combination therapies for latent tuberculosis.
Assuntos
Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Latente/tratamento farmacológico , Mycobacterium tuberculosis/efeitos dos fármacos , Oxazolidinonas/farmacologia , Oxazolidinonas/uso terapêutico , Acetamidas/farmacologia , Acetamidas/uso terapêutico , Animais , Células Cultivadas , Contagem de Colônia Microbiana , Feminino , Hipóxia , Tuberculose Latente/microbiologia , Linezolida , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade MicrobianaRESUMO
Moxifloxacin has shown excellent activity against drug-sensitive as well as drug-resistant tuberculosis (TB), thus confirming DNA gyrase as a clinically validated target for discovering novel anti-TB agents. We have identified novel inhibitors in the pyrrolamide class which kill Mycobacterium tuberculosis through inhibition of ATPase activity catalyzed by the GyrB domain of DNA gyrase. A homology model of the M. tuberculosis H37Rv GyrB domain was used for deciphering the structure-activity relationship and binding interactions of inhibitors with mycobacterial GyrB enzyme. Proposed binding interactions were later confirmed through cocrystal structure studies with the Mycobacterium smegmatis GyrB ATPase domain. The most potent compound in this series inhibited supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of <5 nM, an MIC of 0.03 µg/ml against M. tuberculosis H37Rv, and an MIC90 of <0.25 µg/ml against 99 drug-resistant clinical isolates of M. tuberculosis. The frequency of isolating spontaneous resistant mutants was â¼10(-6) to 10(-8), and the point mutation mapped to the M. tuberculosis GyrB domain (Ser208 Ala), thus confirming its mode of action. The best compound tested for in vivo efficacy in the mouse model showed a 1.1-log reduction in lung CFU in the acute model and a 0.7-log reduction in the chronic model. This class of GyrB inhibitors could be developed as novel anti-TB agents.
Assuntos
Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/patogenicidade , Tuberculose/tratamento farmacológico , Animais , Linhagem Celular , Humanos , Camundongos , Mycobacterium tuberculosis/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis.
Assuntos
Antituberculosos/farmacologia , Inibidores Enzimáticos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Western Blotting , Técnicas de Silenciamento de Genes , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Fenótipo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Conformação Proteica , Quinolonas/farmacologia , Relação Estrutura-Atividade , Triazóis/farmacologiaRESUMO
Scaffold hopping from the thiazolopyridine ureas led to thiazolopyridone ureas with potent antitubercular activity acting through inhibition of DNA GyrB ATPase activity. Structural diversity was introduced, by extension of substituents from the thiazolopyridone N-4 position, to access hydrophobic interactions in the ribose pocket of the ATP binding region of GyrB. Further optimization of hydrogen bond interactions with arginines in site-2 of GyrB active site pocket led to potent inhibition of the enzyme (IC50 2 nM) along with potent cellular activity (MIC=0.1 µM) against Mycobacterium tuberculosis (Mtb). Efficacy was demonstrated in an acute mouse model of tuberculosis on oral administration.
Assuntos
Mycobacterium tuberculosis/efeitos dos fármacos , Piridonas/síntese química , Tiazóis/síntese química , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/farmacologia , Ureia/síntese química , Ureia/farmacologia , Administração Oral , Animais , Antituberculosos/síntese química , Antituberculosos/química , Antituberculosos/farmacologia , Modelos Animais de Doenças , Concentração Inibidora 50 , Camundongos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Piridonas/química , Piridonas/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Inibidores da Topoisomerase II/química , Ureia/químicaRESUMO
Imidazo[1,2-a]pyridine-8-carboxamides as a novel antimycobacterial lead were generated by whole cell screening of a focused library against Mycobacterium tuberculosis. Herein, we describe the synthesis and structure activity relationship evaluation of this class of inhibitors and the optimization of physicochemical properties. These are selective inhibitors of Mycobacterium tuberculosis with no activity on either gram positive or gram negative pathogens.
Assuntos
Antituberculosos/química , Antituberculosos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Amidas/química , Amidas/farmacologia , Humanos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Relação Estrutura-Atividade , Tuberculose/tratamento farmacológicoRESUMO
Insulin therapy is indispensable for achieving glycemic control in all patients with type 1 diabetes mellitus and many patients with type 2 diabetes mellitus. Insulin injections are associated with negative connotations in patients owing to administration discomfort and adverse effects such as hypoglycemia and weight gain. Insulin administered orally can overcome these limitations by providing a convenient and effective mode of delivery with a potentially lower risk of hypoglycemia. Oral insulin mimics the physiologic process of insulin secretion, absorption into the portal circulation, and subsequent peripheral delivery, unlike the subcutaneous route that results in peripheral hyperinsulinemia. Insulin tregopil (IN-105), a new generation human recombinant insulin, methoxy (polyethylene glycol) hexanoyl human recombinant insulin, is developed by Biocon as an ultra-fast onset short-acting oral insulin analog. This recombinant oral insulin is a single short-chain amphiphilic oligomer modified with the covalent attachment of methoxy-triethylene-glycol-propionyl moiety at Lys-ß29-amino group of the B-chain via an amide linkage. Sodium caprate, an excipient in the insulin tregopil formulation, is a permeation enhancer that increases its absorption through the gastrointestinal tract. Also, meal composition has been shown to non-significantly affect its absorption. Several global randomized, controlled clinical trials have been conducted in type 1 and type 2 diabetes patients towards the clinical development of insulin tregopil. The formulation shows post-prandial glucose control that is more effective than placebo throughout the meal period; however, compared with an active comparator insulin aspart, the post-prandial control is more effective mainly in the early post-meal period. It shows a good safety profile with a lower incidence of clinically significant hypoglycemia. This review covers the overall clinical development of insulin tregopil establishing it as an ultra-fast onset, short-acting oral insulin analog for optimizing post-prandial glucose.
Assuntos
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Hipoglicemia , Humanos , Glicemia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemia/tratamento farmacológico , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Insulina/análogos & derivados , Insulina/uso terapêutico , Insulina Aspart/uso terapêutico , Proteínas Recombinantes/uso terapêuticoRESUMO
With the advent of HIV and the widespread emergence of drug-resistant strains of Mycobacterium tuberculosis, newer control strategies in the form of a better vaccine could decrease the global incidence of tuberculosis. A desirable trait in an effective live attenuated vaccine strain is an ability to persist within the host in a limited fashion in order to produce important protective antigens in vivo. Attenuated M. tuberculosis vaccine candidates have been constructed by deleting genes required for growth in mice. These candidate vaccines did not elicit adequate protective immunity in animal models, due to their inability to persist sufficiently long within the host tissues. Here we report that an auxotrophic mutant of M. tuberculosis defective in the de novo biosynthesis of pantothenic acid (vitamin B5) is highly attenuated in immunocompromised SCID mice and in immunocompetent BALB/c mice. SCID mice infected with the pantothenate auxotroph survived significantly longer (250 days) than mice infected with either bacille Calmette-Guerin (BCG) vaccine or virulent M. tuberculosis (77 and 35 days, respectively). Subcutaneous immunization with this auxotroph conferred protection in C57BL/6J mice against an aerosol challenge with virulent M. tuberculosis, which was comparable with that afforded by BCG vaccination. Our findings highlight the importance of de novo pantothenate biosynthesis in limiting the intracellular survival and pathogenesis of M. tuberculosis without reducing its immunogenicity in vaccinated mice.
Assuntos
Mycobacterium tuberculosis/imunologia , Ácido Pantotênico/metabolismo , Vacinas contra a Tuberculose , Tuberculose/prevenção & controle , Animais , Genes Bacterianos , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos SCID , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Taxa de Sobrevida , Tuberculose/imunologia , Tuberculose/patologia , Vacinas AtenuadasRESUMO
We report a novel benzimidazole (BI) based DprE1 inhibitor that resulted from scaffold morphing of a 1,4-azaindole series. The clinical progression of the 1,4-azaindole series from our previous work validates the potential of exploring newer chemical entities with antimycobacterial activity driven via a noncovalent inhibition of the decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). The representative compounds from the new scaffold reported in this study exhibited an improved solubility and higher free plasma fraction, while retaining potent DprE1 inhibition and antimycobacterial activity. A representative compound from the benzimidazole series demonstrated good efficacy in a murine model of tuberculosis. Furthermore, molecular modeling of the BI scaffold suggests plausible modes of binding in the active site of DprE1 enzyme from Mycobacterium tuberculosis that can be used for further exploration of the series.
RESUMO
OBJECTIVES: The aim of this study was to determine the in vitro activity of lipiarmycin against drug-resistant strains of Mycobacterium tuberculosis (MTB) and to establish the resistance mechanism of MTB against lipiarmycin using genetic approaches. METHODS: MIC values were measured against a panel of drug-resistant strains of MTB using the broth microdilution method. Spontaneous lipiarmycin-resistant mutants of MTB were tested for cross-resistance to standard anti-TB drugs, and their rpoB and rpoC genes were sequenced to identify mutations. RESULTS: Lipiarmycin exhibited excellent inhibitory activity against multidrug-resistant strains of MTB with MIC values of <0.1 mg/L. Sequence analysis of the rpoB and rpoC genes from spontaneous lipiarmycin-resistant mutants of MTB revealed that missense mutations in these genes caused resistance to lipiarmycin. Although both lipiarmycin and rifampicin are known to inhibit the bacterial RNA polymerase, the sites of mutation in the rpoB gene were found to be different in MTB strains resistant to these inhibitors. Whereas all six rifampicin-resistant MTB strains tested had mutation in the 81 bp hotspot region of the rpoB gene spanning codons 507-533, 16 of 18 lipiarmycin-resistant strains exhibited mutation between codons 977 and 1150. The remaining two lipiarmycin-resistant strains had mutation in the rpoC gene. CONCLUSIONS: Lipiarmycin has excellent bactericidal activity against MTB and lacks cross-resistance to standard anti-TB drugs. Furthermore, rifampicin-resistant strains remained fully susceptible to lipiarmycin, and none of the lipiarmycin-resistant MTB strains became resistant to rifampicin, highlighting the lack of cross-resistance.
Assuntos
Aminoglicosídeos/farmacologia , Antituberculosos/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Tuberculose Resistente a Múltiplos Medicamentos/microbiologia , Adaptação Biológica , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Proteínas de Bactérias/genética , Análise Mutacional de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Fidaxomicina , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Mycobacterium tuberculosis/isolamento & purificação , Rifampina/farmacologia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
PURPOSE: The purpose of this study was to evaluate the discriminatory power of two DNA-based technique and a protein-based technique for the typing of nosocomial isolates of Klebsiella pneumoniae. A second objective was to determine the antimicrobial susceptibility pattern and characterise the presence of genes encoding extended-spectrum beta-lactamases (ESBLs) and carbapenemases. MATERIALS AND METHODS: Forty-six K. pneumoniae isolates from patients with bloodstream infections at a tertiary care hospital in India between December 2014 and December 2015 were studied. All isolates were typed using enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction (ERIC-PCR), randomly amplified polymorphic DNA (RAPD) analysis and matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry. Antimicrobial susceptibility profiles and ESBLs were detected using the BD Phoenix system. The types of ESBL and carbapenemase genes present were detected using PCR. RESULTS: Isolates were subtyped into 31, 30 and 33 distinct genotypes by ERIC-PCR, RAPD and MALDI-TOF, respectively. Several isolates displaying identical DNA fingerprints were binned into different branches based on their proteomic fingerprint. Antimicrobial susceptibility tests revealed that 33/46 strains were multidrug resistant (MDR); a majority of the strains (83%) were sensitive to colistin. PCR-based analysis demonstrated 19 strains to harbour two or more ESBL and carbapenemase genes. CONCLUSION: ERIC-PCR was the most reproducible method for typing K. pneumoniae isolates and could not be substituted by MALDI-TOF for clonality analysis. A high degree of genetic diversity and the presence of MDR genes highlight the challenges in treating K. pneumoniae-associated infections.
Assuntos
Infecção Hospitalar/microbiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/classificação , Tipagem Molecular/métodos , Proteínas de Bactérias/análise , DNA Bacteriano/genética , Humanos , Índia , Klebsiella pneumoniae/química , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/isolamento & purificação , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Centros de Atenção TerciáriaRESUMO
The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.
Assuntos
Trifosfato de Adenosina/metabolismo , Antituberculosos/uso terapêutico , Mycobacterium tuberculosis/efeitos dos fármacos , Piridinas/uso terapêutico , Quinina/análogos & derivados , Tuberculose/tratamento farmacológico , Animais , Antituberculosos/química , Antituberculosos/farmacocinética , Antituberculosos/farmacologia , Éteres/química , Éteres/farmacocinética , Éteres/farmacologia , Éteres/uso terapêutico , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacologia , Quinina/química , Quinina/farmacocinética , Quinina/farmacologia , Quinina/uso terapêutico , Tuberculose/metabolismoRESUMO
Adoptively transferred dendritic cells presenting antigens derived from different pathogens have been shown to elicit specific T cell responses and to induce protective antibacterial immunity. We describe here the induction of high levels of protective immunity in mice using dendritic cells infected with auxotrophic mutants of Mycobacterium tuberculosis. We provide evidence that protection is superior to BCG and that it is associated with increased priming of CD4+ and CD8+ T cells specific for mycobacterial antigens. This method for generating high levels of anti-bacterial protective immunity could be helpful in the design of novel vaccines against tuberculosis and other intracellular pathogens.
Assuntos
Transferência Adotiva , Células Dendríticas/imunologia , Mycobacterium tuberculosis/imunologia , Vacinas contra a Tuberculose/imunologia , Tuberculose Pulmonar/prevenção & controle , Animais , Células Cultivadas , Células Dendríticas/microbiologia , Células Dendríticas/transplante , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Mycobacterium tuberculosis/genética , Tuberculose Pulmonar/imunologiaRESUMO
Discovery of mupirocin, an antibiotic that targets isoleucyl-tRNA synthetase, established aminoacyl-tRNA synthetase as an attractive target for the discovery of novel antibacterial agents. Despite a high degree of similarity between the bacterial and human aminoacyl-tRNA synthetases, the selectivity observed with mupirocin triggered the possibility of targeting other aminoacyl-tRNA synthetases as potential drug targets. These enzymes catalyse the condensation of a specific amino acid to its cognate tRNA in an energy-dependent reaction. Therefore, each organism is expected to encode at least twenty aminoacyl-tRNA synthetases, one for each amino acid. However, a bioinformatics search for genes encoding aminoacyl-tRNA synthetases from Mycobacterium smegmatis returned multiple genes for glutamyl (GluRS), cysteinyl (CysRS), prolyl (ProRS) and lysyl (LysRS) tRNA synthetases. The pathogenic mycobacteria, namely, Mycobacterium tuberculosis and Mycobacterium leprae, were also found to possess two genes each for CysRS and LysRS. A similar search indicated the presence of additional genes for LysRS in gram negative bacteria as well. Herein, we describe sequence and structural analysis of the additional aminoacyl-tRNA synthetase genes found in M. smegmatis. Characterization of conditional expression strains of Cysteinyl and Lysyl-tRNA synthetases generated in M. smegmatis revealed that the canonical aminoacyl-tRNA synthetase are essential, while the additional ones are not essential for the growth of M. smegmatis.
Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Lisina-tRNA Ligase/química , Lisina-tRNA Ligase/metabolismo , Mutação/genética , Mycobacterium smegmatis/enzimologia , Aminoacil-tRNA Sintetases/genética , Humanos , Lisina-tRNA Ligase/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , RNA de Transferência/metabolismoRESUMO
Nitroarenes are less preferred in drug discovery due to their potential to be mutagenic. However, several nitroarenes were shown to be promising antitubercular agents with specific modes of action, namely, nitroimidazoles and benzothiazinones. The nitro group in these compounds is activated through different mechanisms, both enzymatic and non-enzymatic, in mycobacteria prior to binding to the target of interest. From a whole-cell screening program, we identified a novel lead nitrobenzothiazole (BT) series that acts by inhibition of decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) of Mycobacterium tuberculosis (Mtb). The lead was found to be mutagenic to start with. Our efforts to mitigate mutagenicity resulted in the identification of 6-methyl-7-nitro-5-(trifluoromethyl)-1,3-benzothiazoles (cBTs), a novel class of antitubercular agents that are non-mutagenic and exhibit an improved safety profile. The methyl group ortho to the nitro group decreases the electron affinity of the series, and is hence responsible for the non-mutagenic nature of these compounds. Additionally, the co-crystal structure of cBT in complex with Mtb DprE1 established the mode of binding. This investigation led to a new non-mutagenic antitubercular agent and demonstrates that the mutagenic nature of nitroarenes can be solved by modulation of stereoelectronic properties.
Assuntos
Antituberculosos/farmacologia , Benzotiazóis/farmacologia , Mutagênicos/química , Mycobacterium tuberculosis/efeitos dos fármacos , Nitrocompostos/farmacologia , Antituberculosos/efeitos adversos , Antituberculosos/química , Benzotiazóis/efeitos adversos , Benzotiazóis/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Nitrocompostos/efeitos adversos , Nitrocompostos/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The recent advances in genetic tools to manipulate Mycobacterium tuberculosis have led to the construction of defined mutants and to the study of their role in the virulence and pathogenesis of tuberculosis. The safety and vaccine potential of a few of these M. tuberculosis mutants as candidate vaccines against tuberculosis are discussed.
Assuntos
Mycobacterium tuberculosis/genética , Vacinas contra a Tuberculose , Animais , Mutação , Mycobacterium tuberculosis/imunologia , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/imunologia , Vacinas contra a Tuberculose/toxicidade , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/toxicidadeRESUMO
Conditional expression strains serve as a valuable tool to study the essentiality and to establish the vulnerability of a target under investigation in a drug discovery program. While essentiality implies an absolute requirement of a target function, vulnerability provides valuable information on the extent to which a target function needs to be depleted to achieve bacterial growth inhibition followed by cell death. The critical feature of an ideal conditional expression system is its ability to tightly regulate gene expression to achieve the full spectrum spanning from a high level of expression in order to support growth and near zero level of expression to mimic conditions of gene knockout. A number of bacterial conditional expression systems have been reported for use in mycobacteria. The utility of an isopropylthiogalactoside (IPTG) inducible system in mycobacteria has been reported for protein overexpression and anti-sense gene expression from a replicating multi-copy plasmid. Herein, we report the development of a versatile set of non-replicating IPTG inducible vectors for mycobacteria which can be used for generation of conditional expression strains through homologous recombination. The role of a single lac operator versus a double lac operator to regulate gene expression was evaluated by monitoring the expression levels of ß-galactosidase in Mycobacterium smegmatis. These studies indicated a significant level of leaky expression from the vector with a single lac operator but none from the vector with double lac operator. The significance of the double lac operator vector for target validation was established by monitoring the growth kinetics of an inhA, a rpoB and a ftsZ conditional expression strain grown in the presence of different concentrations of IPTG. The utility of this inducible system in identifying target specific inhibitors was established by screening a focussed library of small molecules using an inhA and a rpoB conditional expression strain.