RESUMO
The ability of Mycobacterium tuberculosis to respond and adapt to various stresses such as oxygen/nitrogen radicals and low pH inside macrophages is critical for the persistence of this human pathogen inside its host. We have previously shown that an AraC/XylS-type transcriptional regulator, VirS, which is induced in low pH, is involved in remodeling the architecture of the bacterial cell envelope. However, how VirS influences gene expression to coordinate these pH responses remains unclear. Here, using a genetic biosensor of cytoplasmic pH, we demonstrate that VirS is required for the intracellular pH maintenance in response to acidic stress and inside acidified macrophages. Furthermore, we observed that VirS plays an important role in blocking phagosomal-lysosomal fusions. Transcriptomics experiments revealed that VirS affects the expression of genes encoding metabolic enzymes, cell-wall envelope proteins, efflux pumps, ion transporters, detoxification enzymes, and transcriptional regulators expressed under low-pH stress. Employing electrophoretic mobility-shift assays, DNA footprinting, and in silico analysis, we identified a DNA sequence to which VirS binds and key residues in VirS required for its interaction with DNA. A significant role of VirS in M. tuberculosis survival in adverse conditions suggested it as a potential anti-mycobacterial drug target. To that end, we identified VirS inhibitors in a virtual screen; the top hit compounds inhibited its DNA-binding activity and also M. tuberculosis growth in vitro and inside macrophages. Our findings establish that VirS mediates M. tuberculosis responses to acidic stress and identify VirS-inhibiting compounds that may form the basis for developing more effective anti-mycobacterial agents.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Tuberculose/tratamento farmacológico , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Bacteriano/genética , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Fagocitose , Conformação Proteica , Homologia de Sequência , Tuberculose/metabolismo , Tuberculose/microbiologiaRESUMO
The difference in the defect structures produced by different ion masses in a tungsten lattice is investigated using 80 MeV Au7+ ions and 10 MeV B3+ ions. The details of the defects produced by ions in recrystallized tungsten foil samples are studied using transmission electron microscopy. Dislocations of type b = 1/2[111] and [001] were observed in the analysis. While highly energetic gold ion produced small clusters of defects with very few dislocation lines, boron has produced large and sparse clusters with numerous dislocation lines. The difference in the defect structures could be due to the difference in separation between primary knock-on atoms produced by gold and boron ions.
RESUMO
The extent of pathogenicity of the mycobacterial infections depends on virulence factors that mediate survival inside macrophages. Virulence factors are generally believed to be specific for pathogenic species and mutated/non-functional in nonpathogenic strains. Mycobacterial TlyA can modulate the phagolysosome maturation pathway, immediately after entry into macrophages. Over-expression of open reading frame (ORF) ML1358 (tlyA) in tissues of leprosy patients by partial DNA chip and real time PCR analysis during active infection attracted our interest to explore the properties of this gene at molecular and serological levels, to understand its role in the host. Molecular properties were studied by cloning and expression of the corresponding gene in pASK-iba 43(þ) expression vector in E. coli and bioinformatics tools while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA were applied to investigate the serological significance of rTlyA protein in different clinical states of leprosy. We observed that TlyA has a close relation among mycobacteria with specific protein domains in slow growing intracellular adapted pathogenic species. The presence of trans-membrane domains indicates its association to the cell membrane. The study revealed its highly significant sero-reactivity (P value , 0·001) in borderline lepromatous (BL) patients, and those with reversal reaction (RR) and erythema nodosum leprosum (ENL). Its role in active infection, association with the cell membrane, presence in pathogenic species and high sero-reactivity, suggested the tlyA gene as a strong disease progression marker.
Assuntos
Proteínas de Bactérias/sangue , Proteínas Hemolisinas/sangue , Hanseníase/sangue , Hanseníase/microbiologia , Mycobacterium leprae/metabolismo , Proteínas de Bactérias/genética , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática , Proteínas Hemolisinas/genética , Humanos , Hanseníase/diagnóstico , Mycobacterium leprae/genéticaRESUMO
Unsatisfactory performance of the existing BCG vaccines, especially against the adult pulmonary disease, has urged the need for an effective vaccine against tuberculosis (TB). In this study, we employed differential proteomics to obtain a list of antigens as potential vaccine candidates. Bacterial epitopes being presented at early stages on MHC class I and class II molecules of macrophages infected with Mycobacterium tuberculosis (M. tb) were identified using iTRAQ labelling and reverse phase LC-MS/MS. The putative vaccine candidates, thus identified, were tested as plasmid DNA vaccines in mice to ascertain their protective efficacy against the aerosolized M. tb challenge, based on their ability to reduce the bacterial load in the lungs of infected mice. Here, we observed that 4 out of the 17 selected antigens imparted significant protection against the challenge of M. tb. The four shortlisted antigens were further assessed in a more stringent guinea pig model, where too, they demonstrated.significant protection. It concludes that combining a proteomics approach with the in vivo assessment of vaccine candidates in animal models can be valuable in identifying new potential candidates to expand the antigenic repertoire for novel vaccines against TB.
Assuntos
Antígenos de Bactérias/imunologia , Macrófagos/imunologia , Mycobacterium tuberculosis/imunologia , Proteômica/métodos , Vacinas contra a Tuberculose/imunologia , Tuberculose/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Bactérias/genética , Antígenos de Bactérias/metabolismo , Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Feminino , Cobaias , Interações Hospedeiro-Parasita/imunologia , Humanos , Imunização/métodos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Mycobacterium tuberculosis/fisiologia , Células NIH 3T3 , Espectrometria de Massas em Tandem , Resultado do Tratamento , Tuberculose/parasitologia , Tuberculose/prevenção & controle , Vacinas contra a Tuberculose/administração & dosagem , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a leading infectious disease taking one human life every 15s globally. Mycobacterium undergoes reductive evolution; the ancestors have bigger genome size and rich in metabolic pathways. Mycobacterium indicus pranii (MIP) is placed much above Mycobacterium tuberculosis (M.tb) in evolutionary scale and is a non-pathogenic, saprophytic mycobacterium. Our in silico comparative proteomic analyses of virulence factors of M.tb and their homologs in 12 different Mycobacterial species, including MIP, point toward gene cooption as an important mechanism in evolution of mycobacteria. We propose that adaptive changes in niche factors of non-pathogenic mycobacterium, together with novel gene acquisitions, are key players in the evolution of pathogenicity. Antigenic analyses between M.tb and MIP highlighted the importance of PE/PPE family in host immunomodulation, further supporting the likely potential of MIP as an effective vaccine against TB.
Assuntos
Proteínas de Bactérias/análise , Evolução Biológica , Mycobacterium/química , Proteoma/análise , Proteínas de Bactérias/genética , Biologia Computacional/métodos , Humanos , Mycobacterium/genética , Proteoma/genética , Fatores de Virulência/genéticaRESUMO
Understanding the evolutionary and genomic mechanisms responsible for turning the soil-derived saprophytic mycobacteria into lethal intracellular pathogens is a critical step towards the development of strategies for the control of mycobacterial diseases. In this context, Mycobacterium indicus pranii (MIP) is of specific interest because of its unique immunological and evolutionary significance. Evolutionarily, it is the progenitor of opportunistic pathogens belonging to M. avium complex and is endowed with features that place it between saprophytic and pathogenic species. Herein, we have sequenced the complete MIP genome to understand its unique life style, basis of immunomodulation and habitat diversification in mycobacteria. As a case of massive gene acquisitions, 50.5% of MIP open reading frames (ORFs) are laterally acquired. We show, for the first time for Mycobacterium, that MIP genome has mosaic architecture. These gene acquisitions have led to the enrichment of selected gene families critical to MIP physiology. Comparative genomic analysis indicates a higher antigenic potential of MIP imparting it a unique ability for immunomodulation. Besides, it also suggests an important role of genomic fluidity in habitat diversification within mycobacteria and provides a unique view of evolutionary divergence and putative bottlenecks that might have eventually led to intracellular survival and pathogenic attributes in mycobacteria.
Assuntos
Evolução Molecular , Transferência Genética Horizontal , Genes Bacterianos , Mycobacterium/genética , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Especiação Genética , Genoma Bacteriano , Hemeritrina/genética , Sequências Repetitivas Dispersas , Proteínas de Membrana Transportadoras/genética , Família Multigênica , Mycobacterium/imunologia , Mycobacterium/metabolismo , Plasmídeos/genética , Proteoma/genética , Seleção GenéticaRESUMO
BACKGROUND: Low iron availability in the host upregulates the mbt gene cluster of Mycobacterium tuberculosis, which is responsible for mycobactin biosynthesis. However, the biological significance of mycobactins in the growth of this pathogen and in disease progression has not been elucidated. METHODS: We have disrupted the mbtE gene (Rv2380c) in the mbt cluster to evaluate the importance of mycobactin biosynthesis in the growth and virulence of M. tuberculosis. RESULTS: The mbtE mutant (MtbΔmbtE) was unable to synthesize mycobactins, displayed an altered colony morphology, and was attenuated for growth in broth culture and in macrophages. Transmission electron microscopy revealed that MtbΔmbtE displayed an altered cell wall permeability. The growth characteristics and colony morphology of MtbΔmbtE were similar to wild type when the medium was supplemented with mycobactins or when MtbΔmbtE was genetically complemented with the mbtE gene. Moreover, guinea pigs infected with MtbΔmbtE exhibited a significantly reduced bacillary load and histopathological damage in the organs, in comparison to M. tuberculosis-infected animals. CONCLUSIONS: This study highlights the importance of mycobactins in the growth and virulence of M. tuberculosis and establishes the enzymes of mycobactin biosynthesis as novel targets for the development of therapeutic interventions against tuberculosis.
Assuntos
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Oxazóis/metabolismo , Animais , Carga Bacteriana , Linhagem Celular , Feminino , Deleção de Genes , Genes Bacterianos , Cobaias , Histocitoquímica , Humanos , Monócitos/microbiologia , Família Multigênica , Mycobacterium tuberculosis/crescimento & desenvolvimento , Estatísticas não ParamétricasRESUMO
To identify the crucial residues involved in the self-assembly and function of BfrB, one of the important iron storage proteins of Mycobacterium tuberculosis, we constructed various mutants by employing site-directed mutagenesis. The analysis of mutants led to the identification of "interface hot-spot residues" (R69, L129, and F159) that act as "switch points" for BfrB oligomerization, and our observations show the importance of 4-fold axis residues in assembly formation. Moreover, we demonstrate that single-point mutations Q51A, Q126A, and E135A can enhance the thermal stability of the protein without affecting its assembly. Importantly, a comparative analysis of various mutations revealed that the function of various homologous positions in different ferritins could be at variance; hence, predicting the function of a residue just based on sequence-structure comparisons may not be appropriate. Thus, we report the identification of novel residues in the assembly formation and function of BfrB and show that single-point mutations have a remarkable potential for alteration of multiple properties of ferritins. Besides, "switch residues" or "interface hot spots" identified in this study could also prove to be helpful for the rational design of interfacial inhibitors.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Grupo dos Citocromos b/química , Grupo dos Citocromos b/metabolismo , Ferritinas/química , Ferritinas/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Grupo dos Citocromos b/genética , Ferritinas/genética , Genes Bacterianos , Ferro/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Subunidades ProteicasRESUMO
We describe here an efficient strategy that employs whole-cell-based screening and further short listing of the compounds by cytotoxicity- and fluorescence-based intracellular assays, resulting in potential bactericidal hits against the growth of Mycobacterium tuberculosis in broth culture as well as in phagosomes. These compounds also inhibited multidrug-resistant strains of M. tuberculosis but showed no or poor inhibition of nonpathogenic mycobacteria or other bacterial species such as Escherichia coli.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Fagossomos/microbiologiaRESUMO
Iron is one of the crucial elements required for the growth of Mycobacterium tuberculosis. However, excess free iron becomes toxic for the cells because it catalyzes the production of reactive oxygen radicals, leading to oxidative damage. Hence, it is essential for the pathogen to have the ability to store intracellular iron in an iron-rich environment and utilize it under iron depletion. M. tuberculosis has two iron storage proteins, namely BfrA (Rv1876; a bacterioferritin) and BfrB (Rv3841; a ferritin-like protein). However, the demonstration of biological significance requires the disruption of relevant genes and the evaluation of the resulting mutant for its ability to survive in the host and cause disease. In this study, we have disrupted bfrA and bfrB of M. tuberculosis and demonstrated that these genes are crucial for the storage and supply of iron for the growth of bacteria and to withstand oxidative stress in vitro. In addition, the bfrA bfrB double mutant (H37Rv ΔbfrA ΔbfrB) exhibited a marked reduction in its ability to survive inside human macrophages. Guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited a marked diminution in the dissemination of the bacilli to spleen compared to that of the parental strain. Moreover, guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited significantly reduced pathological damage in spleen and lungs compared to that of animals infected with the parental strain. Our study clearly demonstrates the importance of these iron storage proteins in the survival and pathogenesis of M. tuberculosis in the host and establishes them as attractive targets for the development of new inhibitors against mycobacterial infections.
Assuntos
Proteínas de Bactérias/metabolismo , Ferritinas/metabolismo , Macrófagos/microbiologia , Viabilidade Microbiana , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologia , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Modelos Animais de Doenças , Feminino , Ferritinas/genética , Cobaias , Humanos , Ferro/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , VirulênciaRESUMO
BACKGROUND: The Guinea pig (Cavia porcellus) is one of the most extensively used animal models to study infectious diseases. However, despite its tremendous contribution towards understanding the establishment, progression and control of a number of diseases in general and tuberculosis in particular, the lack of fully annotated guinea pig genome sequence as well as appropriate molecular reagents has severely hampered detailed genetic and immunological analysis in this animal model. RESULTS: By employing the cross-species hybridization technique, we have developed an oligonucleotide microarray with 44,000 features assembled from different mammalian species, which to the best of our knowledge is the first attempt to employ microarray to study the global gene expression profile in guinea pigs. To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection. A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection. CONCLUSION: We report the development of first comprehensive microarray for studying the global gene expression profile in guinea pigs and validation of its usefulness with tuberculosis as a case study. An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases.
Assuntos
Doenças Transmissíveis/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , CobaiasRESUMO
The recent discovery of fatty acyl-AMP ligases (FAALs) in Mycobacterium tuberculosis (Mtb) provided a new perspective of fatty acid activation. These proteins convert fatty acids to the corresponding adenylates, which are intermediates of acyl-CoA-synthesizing fatty acyl-CoA ligases (FACLs). Presently, it is not evident how obligate pathogens such as Mtb have evolved such new themes of functional versatility and whether the activation of fatty acids to acyladenylates could indeed be a general mechanism. Here, based on elucidation of the first structure of an FAAL protein and by generating loss-of-function and gain-of-function mutants that interconvert FAAL and FACL activities, we demonstrate that an insertion motif dictates formation of acyladenylate. Because FAALs in Mtb are crucial nodes in the biosynthetic network of virulent lipids, inhibitors directed against these proteins provide a unique multipronged approach to simultaneously disrupting several pathways.
Assuntos
Ácidos Graxos/metabolismo , Mycobacterium tuberculosis/metabolismo , Actinobacteria/enzimologia , Acil Coenzima A/biossíntese , Sequência de Aminoácidos , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Ligases/antagonistas & inibidores , Ligases/química , Ligases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The Mycobacterium tuberculosis genome sequence reveals remarkable absence of many nucleoid-associated proteins (NAPs), such as HNS, Hfq or DPS. In order to characterize the nucleoids of M. tuberculosis, we have attempted to identify NAPs, and report an interesting finding that a chaperonin-homolog, GroEL1, is nucleoid associated. We report that M. tuberculosis GroEL1 binds DNA with low specificity but high affinity, suggesting that it might have naturally evolved to bind DNA. We are able to demonstrate that GroEL1 can effectively function as a DNA-protecting agent against DNase I or hydroxyl-radicals. Moreover, Atomic Force Microscopic studies reveal that GroEL1 can condense a large DNA into a compact structure. We also provide in vivo evidences that include presence of GroEL1 in purified nucleoids, in vivo crosslinking followed by Southern hybridizations and immunofluorescence imaging in M. tuberculosis confirming that GroEL1: DNA interactions occur in natural biological settings. These findings therefore reveal that M. tuberculosis GroEL1 has evolved to be associated with nucleoids.
Assuntos
Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mycobacterium tuberculosis/genética , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Sítios de Ligação , Chaperonina 60/análise , Chaperonina 60/química , Cromossomos Bacterianos/química , DNA/química , DNA/metabolismo , DNA/ultraestrutura , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/química , Conformação de Ácido NucleicoRESUMO
The lengthy TB chemotherapeutic regimen, resulting in the emergence of drug resistance strains, poses a serious problem in the cure of the disease. Further, one-quarter of the world's population is infected with dormant M.tb, which creates a lifetime risk of reactivation. M.tb has a remarkable tendency to escape the host immune responses by hiding in unconventional niches. Recent studies have shown that bone-marrow mesenchymal stem cells (BM-MSCs) can serve as a reservoir of the pathogen and have been suggested to keep them beyond the reach of anti-TB drugs. In this study, we have shown that M.tb infects and grows inside BM-MSCs and were unresponsive to the anti-TB drugs rifampicin and isoniazid when compared to the pathogen residing inside THP-1 macrophages. It was further shown that the ABCG2 efflux pumps of the BM-MSCs were upregulated upon exposure to rifampicin, which may be the contributing factor for the antibiotic unresponsiveness of the bacteria inside these cells. Subsequently, it was shown that inhibition of ABCG2 efflux pumps along with administration of anti-TB drugs led to an increased susceptibility and consequently an enhanced killing of the M.tb inside BM-MSCs. These findings for the first time show that the MIC99 values of anti-TB drugs increase many folds for the M.tb residing in BM-MSCs as compared to M.tb residing inside macrophages and the involvement of ABCG2 efflux pumps in this phenomenon. Our study substantiates that these BM-MSCs acts as a useful niche for M.tb wherein they can survive by escaping the antibiotic assault that can be attributed to the host ABCG2 efflux pumps. Inhibiting these efflux pumps can be an attractive adjunctive chemotherapy to eliminate the bacteria from this protective niche.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Antituberculosos/farmacologia , Antituberculosos/uso terapêutico , Tuberculose Latente/tratamento farmacológico , Macrófagos/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/patogenicidade , Farmacorresistência Bacteriana , Humanos , Isoniazida/farmacologia , Isoniazida/uso terapêutico , Macrófagos/microbiologia , Macrófagos/patologia , Células-Tronco Mesenquimais/microbiologia , Rifampina/farmacologia , Rifampina/uso terapêuticoRESUMO
The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the single- and double-ring variants is mediated by phosphorylation.