RESUMO
Vγ9Vδ2 T lymphocytes are programmed for broad antimicrobial responses with rapid production of Th1 cytokines even before birth, and thus thought to play key roles against pathogens in infants. The process regulating Vδ2 cell acquisition of cytotoxic potential shortly after birth remains understudied. We observed that perforin production in cord blood Vδ2 cells correlates with phenotypes defined by the concomitant assessment of PD-1 and CD56. Bulk RNA sequencing of sorted Vδ2 cell fractions indicated that transcripts related to cytotoxic activity and NK function are enriched in the subset with the highest proportion of perforin+ cells. Among differentially expressed transcripts, IRF8, previously linked to CD8 T cell effector differentiation and NK maturation, has the potential to mediate Vδ2 cell differentiation towards cytotoxic effectors. Our current and past results support the hypothesis that distinct mechanisms regulate Vδ2 cell cytotoxic function before and after birth, possibly linked to different levels of microbial exposure.
Assuntos
Antígeno CD56 , Linfócitos T CD8-Positivos , Citotoxicidade Imunológica , Receptor de Morte Celular Programada 1 , Receptores de Antígenos de Linfócitos T gama-delta , Subpopulações de Linfócitos T , Humanos , Linfócitos T CD8-Positivos/imunologia , Citocinas/metabolismo , Sangue Fetal , Perforina/genética , Perforina/metabolismo , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/imunologia , Antígeno CD56/metabolismoRESUMO
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a worldwide scourge with more than 10 million people affected yearly. Among the proteins essential for the survival of Mtb, InhA has been and is still clinically validated as a therapeutic target. A new family of direct diaryl ether inhibitors, not requiring prior activation by the catalase peroxidase enzyme KatG, has been designed with the ambition of fully occupying the InhA substrate-binding site. Thus, eleven compounds, featuring three pharmacophores within the same molecule, were synthesized. One of them, 5-(((4-(2-hydroxyphenoxy)benzyl)(octyl)amino)methyl)-2-phenoxyphenol (compound 21), showed good inhibitory activity against InhA with IC50 of 0.70 µM. The crystal structure of compound 21 in complex with InhA/NAD+ showed how the molecule fills the substrate-binding site as well as the minor portal of InhA. This study represents a further step towards the design of new inhibitors of InhA.
Assuntos
Antituberculosos , Imidazóis , Mycobacterium tuberculosis , Sulfonamidas , Tiofenos , Humanos , Antituberculosos/farmacologia , Antituberculosos/química , Éter , Éteres , Sítios de Ligação , Etil-Éteres , Proteínas de Bactérias/metabolismoRESUMO
Tuberculosis is a serious public health problem worldwide. The search for new antibiotics has become a priority, especially with the emergence of resistant strains. A new family of imidazoquinoline derivatives, structurally analogous to triazolophthalazines, which had previously shown good antituberculosis activity, were designed to inhibit InhA, an essential enzyme for Mycobacterium tuberculosis survival. Over twenty molecules were synthesized and the results showed modest inhibitory efficacy against the protein. Docking experiments were carried out to show how these molecules could interact with the protein's substrate binding site. Disappointingly, unlike triazolophthlazines, these imidazoquinoline derivatives showed an absence of inhibition on mycobacterial growth.
Assuntos
Antituberculosos , Proteínas de Bactérias , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis , Oxirredutases , Quinolinas , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/efeitos dos fármacos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Antituberculosos/farmacologia , Antituberculosos/química , Antituberculosos/síntese química , Quinolinas/química , Quinolinas/farmacologia , Imidazóis/química , Imidazóis/farmacologia , Imidazóis/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Relação Estrutura-Atividade , Testes de Sensibilidade Microbiana , Sítios de Ligação , Estrutura MolecularRESUMO
Mycobacterium abscessus is an opportunistic pathogen that mainly colonizes and infects cystic fibrosis patients' lungs. M. abscessus is naturally resistant to many antibiotics such as rifamycin, tetracyclines and ß-lactams. The current therapeutic regimens are not very effective and are mostly based on repurposed drugs used against Mycobacterium tuberculosis infections. Thus, new approaches and novel strategies are urgently needed. This review aims to provide an overview of the latest ongoing findings to fight M. abscessus infections by analyzing emerging and alternative treatments, novel drug delivery strategies, and innovative molecules.
Assuntos
Fibrose Cística , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Humanos , Fibrose Cística/tratamento farmacológico , Antibacterianos/farmacologia , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , beta-Lactamas/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
Hydrazone compounds represent an important area of research that includes, among others, synthetic approaches and biological studies. A series of 17 hydrazones have been synthesized by mechanochemical means. The fragments chosen were phenolic and furanyl aldehydes coupled with 12 heterocyclic hydrazines or hydrazinamides. All compounds can be obtained quantitatively when operating on a planetary ball mill and a maximum reaction time of 180 min (6 cycles of 30 min each). Complete spectroscopic analyses of hydrazones revealed eight compounds (3-5, 8-11, 16) present in one geometric form, six compounds (1, 2, 13-15) present in two isomeric forms, and three compounds (6, 7, 12) where one rotation is restricted giving rise to two different forms. The single crystal X-ray structure of one of the hydrazones bearing the isoniazid fragment (8) indicates a crystal lattice consisting of two symmetry-independent molecules with different geometries. All compounds obtained were tested for anti-infectious and antibacterial activities. Four compounds (1, 3, 5 and 8) showed good activity against Mycobacterium tuberculosis, and one (7) was very potent against Staphylococcus aureus. Most interesting, this series of compounds displayed very promising antileishmanial activity. Among all, compound 9 exhibited an IC50 value of 0.3 µM on the Leishmania donovani intramacrophage amastigote in vitro model and a good selectivity index, better than miltefosine, making it worth evaluating in vivo.
Assuntos
Antiprotozoários , Hidrazonas , Hidrazonas/farmacologia , Hidrazonas/química , Aldeídos , Amidas , Hidrazinas , Antibacterianos/farmacologia , Antiprotozoários/farmacologia , Antiprotozoários/química , Relação Estrutura-AtividadeRESUMO
Tuberculosis (TB) is one of the leading causes of death worldwide, due to a single pathogen, Mycobacterium tuberculosis. To eradicate TB, management of drug-resistant strains is fundamental, therefore, the identification and characterization of drug targets is pivotal. In this work we aim at describing the relationships with the well-known drug target DprE1 and DprE2, working in association for the biosynthesis of the arabinogalactan precursor, essential component of mycobacterial cell wall. We demonstrated that the enzymes behave as a stable heterodimeric complex, once co-expressed into the same system. This complex showed improved catalytic properties, compared to the singularly expressed enzymes, demonstrating that co-expression is fundamental to achieve the proper folding of the active sites. Our results represent an important step forward in deciphering the functional properties of these enzymes, and lay the foundations for structural studies, useful for development of more specific inhibitors helpful to contrast the spreading of drug-resistant strains.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Humanos , Racemases e Epimerases , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB), one of the most life-threatening communicable diseases, which causes 10 million new cases each year and results in an estimated 1 [...].
Assuntos
Doenças Transmissíveis , Mycobacterium tuberculosis , Tuberculose , Humanos , Tuberculose/tratamento farmacológico , Tuberculose/microbiologiaRESUMO
Avermectins are macrocyclic lactones with anthelmintic activity. Recently, they were found to be effective against Mycobacterium tuberculosis, which accounts for one third of the worldwide deaths from antimicrobial resistance. However, their anti-mycobacterial mode of action remains to be elucidated. The activity of selamectin was determined against a panel of M. tuberculosis mutants. Two strains carrying mutations in DprE1, the decaprenylphosphoryl-ß-D-ribose oxidase involved in the synthesis of mycobacterial arabinogalactan, were more susceptible to selamectin. Biochemical assays against the Mycobacterium smegmatis DprE1 protein confirmed this finding, and docking studies predicted a binding site in a loop that included Leu275. Sequence alignment revealed variants in this position among mycobacterial species, with the size and hydrophobicity of the residue correlating with their MIC values; M. smegmatis DprE1 variants carrying these point mutations validated the docking predictions. However, the correlation was not confirmed when M. smegmatis mutant strains were constructed and MIC phenotypic assays performed. Likewise, metabolic labeling of selamectin-treated M. smegmatis and M. tuberculosis cells with 14C-labeled acetate did not reveal the expected lipid profile associated with DprE1 inhibition. Together, our results confirm the in vitro interactions of selamectin and DprE1 but suggest that selamectin could be a multi-target anti-mycobacterial compound.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Antiparasitários/farmacologia , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Ivermectina/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Sítios de Ligação , Relação Dose-Resposta a Droga , Descoberta de Drogas , Ivermectina/farmacologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Relação Estrutura-AtividadeRESUMO
Human Vγ9Vδ2 T cells respond to several diverse pathogens by sensing microbial cholesterol intermediates. Unlike CD4 T cells, they are poised for rapid Th1-like responses even before birth, which allows them to play a key role in the first line of defense against pathogens in early life. However, their regulation and functional maturation during infancy (in particular the acquisition of cytotoxic potential) remain understudied. We thus characterized their responses to cholesterol intermediates and Bacille Calmette-Guérin in a cohort of African neonates and 12-month-old infants. Infant Vδ2 lymphocytes exhibited intermediate or adult-like expression of markers associated with differentiation or function, intermediate proliferative responses, and adult-like cytotoxic potential. The enhancement of Vδ2 cell cytotoxic potential coincided with decreasing PD-1 and increasing NKG2A expression. Our results are consistent with the hypothesis that switching from a PD-1+ to a NKG2A+ phenotype during infancy indicates a shift in mechanisms regulating Vδ2 T cell function.
Assuntos
Sangue Fetal/citologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Adulto , Fatores Etários , Diferenciação Celular/fisiologia , Células Cultivadas , Cordocentese , Feminino , Expressão Gênica/genética , Humanos , Lactente , Recém-Nascido , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Malaui/epidemiologia , Masculino , Subfamília C de Receptores Semelhantes a Lectina de Células NK/imunologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptor de Morte Celular Programada 1/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Linfócitos T/imunologiaRESUMO
Some nontuberculous mycobacteria (NTM) are considered opportunistic pathogens. Nevertheless, NTM infections are increasing worldwide, becoming a major public health threat. Furthermore, there is no current specific drugs to treat these infections, and the recommended regimens generally lack efficacy, emphasizing the need for novel antibacterial compounds. In this paper, we focused on the essential mycolic acids transporter MmpL3, which is a well-characterized target of several antimycobacterial agents, to identify new compounds active against Mycobacterium abscessus (Mab). From the crystal structure of MmpL3 in complex with known inhibitors, through an in silico approach, we developed a pharmacophore that was used as a three-dimensional filter to identify new putative MmpL3 ligands within databases of known drugs. Among the prioritized compounds, mefloquine showed appreciable activity against Mab (MIC = 16 µg/mL). The compound was confirmed to interfere with mycolic acids biosynthesis, and proved to also be active against other NTMs, including drug-resistant clinical isolates. Importantly, mefloquine is a well-known antimalarial agent, opening the possibility of repurposing an already approved drug, which is a useful strategy to reduce the time and cost of disclosing novel drug candidates.
Assuntos
Antibacterianos/farmacologia , Antimaláricos/farmacologia , Mefloquina/farmacologia , Mycobacterium abscessus/metabolismo , Ácidos Micólicos/metabolismoRESUMO
Two macrocyclic derivatives based on the triclosan frame were designed and synthesized as inhibitors of Mycobacterium tuberculosis InhA enzyme. One of the two molecules M02 displayed promising inhibitory activity against InhA enzyme with an IC50 of 4.7 µM. Molecular docking studies of these two compounds were performed and confirmed that M02 was more efficient as inhibitor of InhA activity. These molecules are the first macrocyclic direct inhibitors of InhA enzyme able to bind into the substrate pocket. Furthermore, these biaryl ether compounds exhibited antitubercular activities comparable to that of triclosan against M. tuberculosis H37Rv strain.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Compostos Macrocíclicos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Oxirredutases/antagonistas & inibidores , Triclosan/farmacologia , Antituberculosos/síntese química , Antituberculosos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Oxirredutases/metabolismo , Relação Estrutura-Atividade , Triclosan/síntese química , Triclosan/químicaRESUMO
Sensing and response to changes in nutrient availability are essential for the lifestyle of environmental and pathogenic bacteria. Serine/threonine protein kinase G (PknG) is required for virulence of the human pathogen Mycobacterium tuberculosis, and its putative substrate GarA regulates the tricarboxylic acid cycle in M. tuberculosis and other Actinobacteria by protein-protein binding. We sought to understand the stimuli that lead to phosphorylation of GarA, and the roles of this regulatory system in pathogenic and non-pathogenic bacteria. We discovered that M. tuberculosis lacking garA was severely attenuated in mice and macrophages and furthermore that GarA lacking phosphorylation sites failed to restore the growth of garA deficient M. tuberculosis in macrophages. Additionally we examined the impact of genetic disruption of pknG or garA upon protein phosphorylation, nutrient utilization and the intracellular metabolome. We found that phosphorylation of GarA requires PknG and depends on nutrient availability, with glutamate and aspartate being the main stimuli. Disruption of pknG or garA caused opposing effects on metabolism: a defect in glutamate catabolism or depletion of intracellular glutamate, respectively. Strikingly, disruption of the phosphorylation sites of GarA was sufficient to recapitulate defects caused by pknG deletion. The results suggest that GarA is a cellular target of PknG and the metabolomics data demonstrate that the function of this signaling system is in metabolic regulation. This function in amino acid homeostasis is conserved amongst the Actinobacteria and provides an example of the close relationship between metabolism and virulence.
Assuntos
Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Metabolômica , Mycobacterium tuberculosis , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Ácido Aspártico/metabolismo , Proteínas de Bactérias/genética , Ácido Glutâmico/metabolismo , Homeostase , Macrófagos/microbiologia , Camundongos , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Tuberculose/microbiologia , VirulênciaRESUMO
Nontuberculous mycobacteria (NTM) have recently emerged as important pathogens among cystic fibrosis (CF) patients worldwide. Mycobacterium abscessus is becoming the most worrisome NTM in this cohort of patients and recent findings clarified why this pathogen is so prone to this disease. M. abscessus drug therapy takes up to 2 years and its failure causes an accelerated lung function decline. The M. abscessus colonization of lung alveoli begins with smooth strains producing glycopeptidolipids and biofilm, whilst in the invasive infection, "rough" mutants are responsible for the production of trehalose dimycolate, and consequently, cording formation. Human-to-human M. abscessus transmission was demonstrated among geographically separated CF patients by whole-genome sequencing of clinical isolates worldwide. Using a M. abscessus infected CF zebrafish model, it was demonstrated that CFTR (cystic fibrosis transmembrane conductance regulator) dysfunction seems to have a specific role in the immune control of M. abscessus infections only. This pathogen is also intrinsically resistant to many drugs, thanks to its physiology and to the acquisition of new mechanisms of drug resistance. Few new compounds or drug formulations active against M. abscessus are present in preclinical and clinical development, but recently alternative strategies have been investigated, such as phage therapy and the use of ß-lactamase inhibitors.
Assuntos
Doenças Transmissíveis Emergentes , Fibrose Cística , Farmacorresistência Bacteriana Múltipla/imunologia , Infecções por Mycobacterium não Tuberculosas , Mycobacterium abscessus , Alvéolos Pulmonares , Animais , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/imunologia , Doenças Transmissíveis Emergentes/patologia , Fibrose Cística/epidemiologia , Fibrose Cística/imunologia , Fibrose Cística/patologia , Regulador de Condutância Transmembrana em Fibrose Cística/imunologia , Modelos Animais de Doenças , Humanos , Infecções por Mycobacterium não Tuberculosas/epidemiologia , Infecções por Mycobacterium não Tuberculosas/imunologia , Infecções por Mycobacterium não Tuberculosas/patologia , Mycobacterium abscessus/imunologia , Mycobacterium abscessus/patogenicidade , Alvéolos Pulmonares/imunologia , Alvéolos Pulmonares/microbiologia , Peixe-ZebraRESUMO
The Mycobacterium tuberculosis rv2466c gene encodes an oxidoreductase enzyme annotated as DsbA. It has a CPWC active-site motif embedded within its thioredoxin fold domain and mediates the activation of the prodrug TP053, a thienopyrimidine derivative that kills both replicating and nonreplicating bacilli. However, its mode of action and actual enzymatic function in M. tuberculosis have remained enigmatic. In this study, we report that Rv2466c is essential for bacterial survival under H2O2 stress. Further, we discovered that Rv2466c lacks oxidase activity; rather, it receives electrons through the mycothiol/mycothione reductase/NADPH pathway to activate TP053, preferentially via a dithiol-disulfide mechanism. We also found that Rv2466c uses a monothiol-disulfide exchange mechanism to reduce S-mycothiolated mixed disulfides and intramolecular disulfides. Genetic, phylogenetic, bioinformatics, structural, and biochemical analyses revealed that Rv2466c is a novel mycothiol-dependent reductase, which represents a mycoredoxin cluster of enzymes within the DsbA family different from the glutaredoxin cluster to which mycoredoxin-1 (Mrx1 or Rv3198A) belongs. To validate this DsbA-mycoredoxin cluster, we also characterized a homologous enzyme of Corynebacterium glutamicum (NCgl2339) and observed that it demycothiolates and reduces a mycothiol arsenate adduct with kinetic properties different from those of Mrx1. In conclusion, our work has uncovered a DsbA-like mycoredoxin that promotes mycobacterial resistance to oxidative stress and reacts with free mycothiol and mycothiolated targets. The characterization of the DsbA-like mycoredoxin cluster reported here now paves the way for correctly classifying similar enzymes from other organisms.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pró-Fármacos/farmacologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Pirimidinas/farmacologia , Ativação Metabólica , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Cisteína/metabolismo , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Drogas em Investigação/química , Drogas em Investigação/metabolismo , Drogas em Investigação/farmacologia , Deleção de Genes , Conformação Molecular , Simulação de Acoplamento Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Oxirredução , Filogenia , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Conformação Proteica , Isomerases de Dissulfetos de Proteínas/química , Isomerases de Dissulfetos de Proteínas/genética , Pirimidinas/química , Pirimidinas/metabolismo , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
Whole cell based screens to identify hits against Mycobacterium tuberculosis (Mtb), carried out under replicating and non-replicating (NRP) conditions, resulted in the identification of multiple, novel but structurally related spiropiperidines with potent antitubercular properties. These compounds could be further classified into three classes namely 3-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[indene-1,4'-piperidine] (abbr. spiroindenes), 4-(3-aryl-1,2,4-oxadiazol-5-yl)-1'-alkylspiro[chromene-2,4'-piperidine] (abbr. spirochromenes) and 1'-benzylspiro[indole-1,4'-piperidin]-2(1H)-one (abbr. spiroindolones). Spiroindenes showed ⩾ 4 log10 kill (at 2-12 µM) on replicating Mtb, but were moderately active under non replicating conditions. Whole genome sequencing efforts of spiroindene resistant mutants resulted in the identification of I292L mutation in MmpL3 (Mycobacterial membrane protein Large), required for the assembly of mycolic acid into the cell wall core of Mtb. MIC modulation studies demonstrated that the mutants were cross-resistant to spirochromenes but not to spiroindolones. This Letter describes lead identification efforts to improve potency while reducing the lipophilicity and hERG liabilities of spiroindenes. Additionally, as deduced from the SAR studies, we provide insights regarding the new chemical opportunities that the spiroindolones can offer to the TB drug discovery initiatives.
Assuntos
Antituberculosos/farmacologia , Piperidinas/farmacologia , Compostos de Espiro/farmacologia , Animais , Antituberculosos/síntese química , Antituberculosos/farmacocinética , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Genoma Bacteriano , Ensaios de Triagem em Larga Escala , Hipóxia , Lipídeos/química , Metaloproteinase 13 da Matriz/biossíntese , Metaloproteinase 13 da Matriz/genética , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Piperidinas/síntese química , Piperidinas/farmacocinética , Compostos de Espiro/síntese química , Compostos de Espiro/farmacocinética , Relação Estrutura-AtividadeRESUMO
The cell envelope of Mycobacterium tuberculosis contains glycans and lipids of peculiar structure that play prominent roles in the biology and pathogenesis of tuberculosis. Consequently, the chemical structure and biosynthesis of the cell wall have been intensively investigated in order to identify novel drug targets. Here, we validate that the function of phosphatidyl-myo-inositol mannosyltransferase PimA is vital for M. tuberculosis in vitro and in vivo. PimA initiates the biosynthesis of phosphatidyl-myo-inositol mannosides by transferring a mannosyl residue from GDP-Man to phosphatidyl-myo-inositol on the cytoplasmic side of the plasma membrane. To prove the essential nature of pimA in M. tuberculosis, we constructed a pimA conditional mutant by using the TetR-Pip off system and showed that downregulation of PimA expression causes bactericidality in batch cultures. Consistent with the biochemical reaction catalyzed by PimA, this phenotype was associated with markedly reduced levels of phosphatidyl-myo-inositol dimannosides, essential structural components of the mycobacterial cell envelope. In addition, the requirement of PimA for viability was clearly demonstrated during macrophage infection and in two different mouse models of infection, where a dramatic decrease in viable counts was observed upon silencing of the gene. Notably, depletion of PimA resulted in complete clearance of the mouse lungs during both the acute and chronic phases of infection. Altogether, the experimental data highlight the importance of the phosphatidyl-myo-inositol mannoside biosynthetic pathway for M. tuberculosis and confirm that PimA is a novel target for future drug discovery programs.
Assuntos
Proteínas de Bactérias/metabolismo , Manosiltransferases/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/crescimento & desenvolvimento , Tuberculose/microbiologia , Animais , Proteínas de Bactérias/genética , Feminino , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Manosiltransferases/genética , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/genética , Fosfatidilinositóis/biossínteseRESUMO
Alpha-ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α-ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non-pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient-dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genes Bacterianos , Ácidos Cetoglutáricos/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico , Regulação Bacteriana da Expressão Gênica , Ácido Glutâmico/metabolismo , Humanos , Macrófagos/microbiologia , Mutagênese Sítio-Dirigida , Mycobacterium smegmatis/metabolismo , Fenótipo , Fosforilação , Proteínas Recombinantes/metabolismoRESUMO
AIMS: The incidence of lung infections is increasing worldwide in individuals suffering from cystic fibrosis and chronic obstructive pulmonary diseases. Mycobacterium abscessus is associated to chronic lung deterioration in these populations. The intrinsic resistance of M. abscessus to most conventional antibiotics jeopardizes treatment success rates. To date no single drug has been developed targeting specifically M. abscessus. Our objective was to characterize the pyrithione-core drug-like small molecule named VOMG as a new compound active against M. abscessus and other pathogens. METHODS: We used a multidisciplinary approach including microbiological, chemical, biochemical and transcriptomics procedures to validate VOMG as a promising anti-M. abscessus drug candidate. RESULTS: We report for the first time the in vitro and in vivo bactericidal activity of VOMG against M. abscessus and other pathogens. Besides being active against M. abscessus biofilm, the compound showed a favourable pharmacology (ADME-Tox) profile. Frequency of resistance studies were unable to isolate resistant mutants. VOMG inhibits cell division, particularly the FtsZ enzyme. CONCLUSIONS: VOMG is a new drug-like molecule discovered against M. abscessus inhibiting cell division with broad spectrum activity against other microbial pathogens.
RESUMO
Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a 'non-culturable' state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The 'non-culturable' mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. 'Non-culturable' cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and 'non-culturable' M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments.
RESUMO
Tuberculosis is one of the deadliest infectious diseases in the world, and the increased number of multidrug-resistant and extensively drug-resistant strains is a reason for concern. We have previously reported a series of substituted 5-(2-aminothiazol-4-yl)isoxazole-3-carboxamides with growth inhibitory activity against Mycobacterium tuberculosis strains and low propensity to be substrate of efflux pumps. Encouraged by these preliminary results, we have undertaken a medicinal chemistry campaign to determine the metabolic fate of these compounds and to delineate a reliable body of Structure-Activity Relationships. Keeping intact the (thiazol-4-yl)isoxazole-3-carboxamide core, as it is deemed to be the pharmacophore of the molecule, we have extensively explored the structural modifications able to confer good activity and avoid rapid clearance. Also, a small set of analogues based on isostere manipulation of the 2-aminothiazole were prepared and tested, with the aim to disclose novel antitubercular chemotypes. These studies, combined, were instrumental in designing improved compounds such as 42g and 42l, escaping metabolic degradation by human liver microsomes and, at the same time, maintaining good antitubercular activity against both drug-susceptible and drug-resistant strains.