RESUMO
Lipid biosynthesis in the pathogen Mycobacterium tuberculosis depends on biotin for posttranslational modification of key enzymes. However, the mycobacterial biotin synthetic pathway is not fully understood. Here, we show that rv1590, a gene of previously unknown function, is required by M. tuberculosis to synthesize biotin. Chemical-generic interaction experiments mapped the function of rv1590 to the conversion of dethiobiotin to biotin, which is catalyzed by biotin synthases (BioB). Biochemical studies confirmed that in contrast to BioB of Escherichia coli, BioB of M. tuberculosis requires Rv1590 (which we named "biotin synthase auxiliary protein" or BsaP), for activity. We found homologs of bsaP associated with bioB in many actinobacterial genomes, and confirmed that BioB of Mycobacterium smegmatis also requires BsaP. Structural comparisons of BsaP-associated biotin synthases with BsaP-independent biotin synthases suggest that the need for BsaP is determined by the [2Fe-2S] cluster that inserts sulfur into dethiobiotin. Our findings open new opportunities to seek BioB inhibitors to treat infections with M. tuberculosis and other pathogens.
Assuntos
Proteínas de Bactérias , Biotina , Mycobacterium tuberculosis , Biotina/metabolismo , Biotina/análogos & derivados , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sulfurtransferases/metabolismo , Sulfurtransferases/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/enzimologia , Escherichia coli/metabolismo , Escherichia coli/genéticaRESUMO
Mycobacterium tuberculosis is the causative agent of tuberculosis. M. tuberculosis can survive in a dormant state within the granuloma, avoiding the host-mounting immune attack. M. tuberculosis bacilli in this state show increased tolerance to antibiotics and stress conditions, and thus the transition of M. tuberculosis to the nonreplicating dormant state acts as an obstacle to tuberculosis treatment. M. tuberculosis in the granuloma encounters hostile environments such as hypoxia, nitric oxide, reactive oxygen species, low pH, and nutrient deprivation, etc., which are expected to inhibit respiration of M. tuberculosis. To adapt to and survive in respiration-inhibitory conditions, it is required for M. tuberculosis to reprogram its metabolism and physiology. In order to get clues to the mechanism underlying the entry of M. tuberculosis to the dormant state, it is important to understand the mycobacterial regulatory systems that are involved in the regulation of gene expression in response to respiration inhibition. In this review, we briefly summarize the information regarding the regulatory systems implicated in upregulation of gene expression in mycobacteria exposed to respiration-inhibitory conditions. The regulatory systems covered in this review encompass the DosSR (DevSR) two-component system, SigF partner switching system, MprBA-SigE-SigB signaling pathway, cAMP receptor protein, and stringent response.
Assuntos
Mycobacterium tuberculosis , Tuberculose , Humanos , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Transdução de Sinais , Respiração , Regulação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Bactérias/metabolismoRESUMO
The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen's ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to create a resource for associating bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb transposon mutant fitness (TnSeq) across the CC panel revealed that many virulence pathways are only required in specific host microenvironments, identifying a large fraction of the pathogen's genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits can be associated with genetic variants distributed across the mouse genome, making the CC a unique population for identifying specific host-pathogen genetic interactions that influence pathogenesis.
Assuntos
Camundongos de Cruzamento Colaborativo/genética , Predisposição Genética para Doença , Variação Genética , Interações Hospedeiro-Patógeno/genética , Mycobacterium tuberculosis/genética , Tuberculose/microbiologia , Animais , Modelos Animais de Doenças , Genótipo , Masculino , Camundongos , Mycobacterium tuberculosis/patogenicidade , FenótipoRESUMO
Bacterial chaperones ClpB and DnaK, homologs of the respective eukaryotic heat shock proteins Hsp104 and Hsp70, are essential in the reactivation of toxic protein aggregates that occur during translation or periods of stress. In the pathogen Mycobacterium tuberculosis (Mtb), the protective effect of chaperones extends to survival in the presence of host stresses, such as protein-damaging oxidants. However, we lack a full understanding of the interplay of Hsps and other stress response genes in mycobacteria. Here, we employ genome-wide transposon mutagenesis to identify the genes that support clpB function in Mtb. In addition to validating the role of ClpB in Mtb's response to oxidants, we show that HtpG, a homolog of Hsp90, plays a distinct role from ClpB in the proteotoxic stress response. While loss of neither clpB nor htpG is lethal to the cell, loss of both through genetic depletion or small molecule inhibition impairs recovery after exposure to host-like stresses, especially reactive nitrogen species. Moreover, defects in cells lacking clpB can be complemented by overexpression of other chaperones, demonstrating that Mtb's stress response network depends upon finely tuned chaperone expression levels. These results suggest that inhibition of multiple chaperones could work in concert with host immunity to disable Mtb.
Assuntos
Endopeptidase Clp/metabolismo , Mycobacterium tuberculosis/metabolismo , Estresse Fisiológico/fisiologia , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/genéticaRESUMO
As ß-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb's survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these ß-lactams to target diverse classes of proteins.
RESUMO
The synthesis, absolute stereochemical configuration, complete biological characterization, mechanism of action and resistance, and pharmacokinetic properties of ( S)-(-)-acidomycin are described. Acidomycin possesses promising antitubercular activity against a series of contemporary drug susceptible and drug-resistant M. tuberculosis strains (minimum inhibitory concentrations (MICs) = 0.096-6.2 µM) but is inactive against nontuberculosis mycobacteria and Gram-positive and Gram-negative pathogens (MICs > 1000 µM). Complementation studies with biotin biosynthetic pathway intermediates and subsequent biochemical studies confirmed acidomycin inhibits biotin synthesis with a Ki of approximately 1 µM through the competitive inhibition of biotin synthase (BioB) and also stimulates unproductive cleavage of S-adenosyl-l-methionine (SAM) to generate the toxic metabolite 5'-deoxyadenosine. Cell studies demonstrate acidomycin selectively accumulates in M. tuberculosis providing a mechanistic basis for the observed antibacterial activity. The development of spontaneous resistance by M. tuberculosis to acidomycin was difficult, and only low-level resistance to acidomycin was observed by overexpression of BioB. Collectively, the results provide a foundation to advance acidomycin and highlight BioB as a promising target.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Sulfurtransferases/antagonistas & inibidores , Tiazolidinas/farmacologia , Tuberculose/microbiologia , Animais , Antituberculosos/síntese química , Antituberculosos/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Produtos Biológicos/síntese química , Produtos Biológicos/química , Produtos Biológicos/farmacologia , Biotina/biossíntese , Caproatos/síntese química , Caproatos/química , Caproatos/farmacologia , Farmacorresistência Bacteriana , Humanos , Cinética , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Sulfurtransferases/química , Sulfurtransferases/genética , Sulfurtransferases/metabolismo , Tiazolidinas/síntese química , Tiazolidinas/química , Tuberculose/tratamento farmacológicoRESUMO
Thyroid cancer is the fifth most common cancer diagnosed in women worldwide. Notwithstanding advancements in the prognosis and treatment of thyroid cancer, 10-20% of thyroid cancer patients develops chemotherapeutic resistance and experience relapse. According to previous reports and TCGA database, MUC15 (MUCIN 15) upregulation is highly correlated with thyroid cancer progression. However, the role of MUC15 in tumor progression and metastasis is unclear. This study aimed to investigate factors mediating cancer stemness in thyroid cancer. MUC15 plays an important role in sphere formation, as an evident from the expression of stemness markers including SOX2, KLF4, ALDH1A3, and IL6. Furthermore, ectopic expression of MUC15 activated extracellular signal-regulated kinase (ERK) signaling via G-protein-coupled receptor (GPCR)/cyclic AMP (cAMP) and integrin/focal adhesion kinase pathways. Interestingly, ectopic expression of MUC15 did not affect RAF/mitogen-activated protein kinase kinase (MEK)-mediated ERK activation. The present findings may provide novel insights into the development of diagnostic, prognostic, and therapeutic applications of MUC15 in thyroid cancer.
RESUMO
Here we demonstrated that the inhibition of electron flux through the respiratory electron transport chain (ETC) by either the disruption of the gene for the major terminal oxidase (aa3 cytochrome c oxidase) or treatment with KCN resulted in the induction of ald encoding alanine dehydrogenase in Mycobacterium smegmatis A decrease in functionality of the ETC shifts the redox state of the NADH/NAD+ pool toward a more reduced state, which in turn leads to an increase in cellular levels of alanine by Ald catalyzing the conversion of pyruvate to alanine with the concomitant oxidation of NADH to NAD+ The induction of ald expression under respiration-inhibitory conditions in M. smegmatis is mediated by the alanine-responsive AldR transcriptional regulator. The growth defect of M. smegmatis by respiration inhibition was exacerbated by inactivation of the ald gene, suggesting that Ald is beneficial to M. smegmatis in its adaptation and survival under respiration-inhibitory conditions by maintaining NADH/NAD+ homeostasis. The low susceptibility of M. smegmatis to bcc1 complex inhibitors appears to be, at least in part, attributable to the high expression level of the bd quinol oxidase in M. smegmatis when the bcc1-aa3 branch of the ETC is inactivated.IMPORTANCE We demonstrated that the functionality of the respiratory electron transport chain is inversely related to the expression level of the ald gene encoding alanine dehydrogenase in Mycobacterium smegmatis Furthermore, the importance of Ald in NADH/NAD+ homeostasis during the adaptation of M. smegmatis to severe respiration-inhibitory conditions was demonstrated in this study. On the basis of these results, we propose that combinatory regimens including both an Ald-specific inhibitor and respiration-inhibitory antitubercular drugs such as Q203 and bedaquiline are likely to enable a more efficient therapy for tuberculosis.
Assuntos
Alanina Desidrogenase/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Mycobacterium smegmatis/enzimologia , Consumo de Oxigênio/fisiologia , Alanina Desidrogenase/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Imidazóis/farmacologia , Testes de Sensibilidade Microbiana , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , NAD/metabolismo , Piperidinas/farmacologia , Piridinas/farmacologiaRESUMO
Successful drug treatment for tuberculosis (TB) depends on the unique contributions of its component drugs. Drug resistance poses a threat to the efficacy of individual drugs and the regimens to which they contribute. Biologically and chemically validated targets capable of replacing individual components of current TB chemotherapy are a major unmet need in TB drug development. We demonstrate that chemical inhibition of the bacterial biotin protein ligase (BPL) with the inhibitor Bio-AMS (5'-[N-(d-biotinoyl)sulfamoyl]amino-5'-deoxyadenosine) killed Mycobacterium tuberculosis (Mtb), the bacterial pathogen causing TB. We also show that genetic silencing of BPL eliminated the pathogen efficiently from mice during acute and chronic infection with Mtb Partial chemical inactivation of BPL increased the potency of two first-line drugs, rifampicin and ethambutol, and genetic interference with protein biotinylation accelerated clearance of Mtb from mouse lungs and spleens by rifampicin. These studies validate BPL as a potential drug target that could serve as an alternate frontline target in the development of new drugs against Mtb.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Tuberculose/metabolismo , Animais , Biotinilação/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Sulfurtransferases/metabolismo , Tuberculose/tratamento farmacológicoRESUMO
5'-O-[N-(Salicyl)sulfamoyl]adenosine (Sal-AMS, 1) is a nucleoside antibiotic that inhibits incorporation of salicylate into siderophores required for bacterial iron acquisition and has potent activity against Mycobacterium tuberculosis (Mtb). Cinnolone analogues exemplified by 5 were designed to replace the acidic acyl-sulfamate functional group of 1 (pKa = 3) by a more stable sulfonamide linkage (pKa = 6.0) in an attempt to address potential metabolic liabilities and improve membrane permeability. We showed 5 potently inhibited the mycobacterial salicylate ligase MbtA (apparent Ki = 12 nM), blocked production of the salicylate-capped siderophores in whole-cell Mtb, and exhibited excellent antimycobacterial activity under iron-deficient conditions (minimum inhibitor concentration, MIC = 2.3 µM). To provide additional confirmation of the mechanism of action, we demonstrated the whole-cell activity of 5 could be fully antagonized by the addition of exogenous salicylate to the growth medium. Although the total polar surface area (tPSA) of 5 still exceeds the nominal threshold value (140 Å) typically required for oral bioavailability, we were pleasantly surprised to observe introduction of the less acidic and conformationally constrained cinnolone moiety conferred improved drug disposition properties as evidenced by the 7-fold increase in volume of distribution in Sprague-Dawley rats.
RESUMO
Chemotherapy for tuberculosis (TB) is lengthy and could benefit from synergistic adjuvant therapeutics that enhance current and novel drug regimens. To identify genetic determinants of intrinsic antibiotic susceptibility in Mycobacterium tuberculosis, we applied a chemical genetic interaction (CGI) profiling approach. We screened a saturated transposon mutant library and identified mutants that exhibit altered fitness in the presence of partially inhibitory concentrations of rifampin, ethambutol, isoniazid, vancomycin, and meropenem, antibiotics with diverse mechanisms of action. This screen identified the M. tuberculosis cell envelope to be a major determinant of antibiotic susceptibility but did not yield mutants whose increase in susceptibility was due to transposon insertions in genes encoding efflux pumps. Intrinsic antibiotic resistance determinants affecting resistance to multiple antibiotics included the peptidoglycan-arabinogalactan ligase Lcp1, the mycolic acid synthase MmaA4, the protein translocase SecA2, the mannosyltransferase PimE, the cell envelope-associated protease CaeA/Hip1, and FecB, a putative iron dicitrate-binding protein. Characterization of a deletion mutant confirmed FecB to be involved in the intrinsic resistance to every antibiotic analyzed. In contrast to its predicted function, FecB was dispensable for growth in low-iron medium and instead functioned as a critical mediator of envelope integrity.
Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/genética , Parede Celular/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/efeitos dos fármacos , Serina Proteases/genética , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Parede Celular/genética , Parede Celular/metabolismo , Etambutol/farmacologia , Galactanos/biossíntese , Perfilação da Expressão Gênica , Humanos , Bombas de Íon/deficiência , Bombas de Íon/genética , Isoniazida/farmacologia , Ligases/genética , Ligases/metabolismo , Manosiltransferases/genética , Manosiltransferases/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Meropeném , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Ácidos Micólicos/metabolismo , Peptidoglicano/biossíntese , Rifampina/farmacologia , Serina Proteases/metabolismo , Tienamicinas/farmacologia , Vancomicina/farmacologiaRESUMO
The pyridoxal 5'-phosphate (PLP)-dependent transaminase BioA catalyzes the second step in the biosynthesis of biotin in Mycobacterium tuberculosis (Mtb) and is an essential enzyme for bacterial survival and persistence in vivo. A promising BioA inhibitor 6 containing an N-aryl, N'-benzoylpiperazine scaffold was previously identified by target-based whole-cell screening. Here, we explore the structure-activity relationships (SAR) through the design, synthesis, and biological evaluation of a systematic series of analogues of the original hit using a structure-based drug design strategy, which was enabled by cocrystallization of several analogues with BioA. To confirm target engagement and discern analogues with off-target activity, each compound was evaluated against wild-type (WT) Mtb in biotin-free and -containing medium as well as BioA under- and overexpressing Mtb strains. Conformationally constrained derivative 36 emerged as the most potent analogue with a KD of 76 nM against BioA and a minimum inhibitory concentration of 1.7 µM (0.6 µg/mL) against Mtb in biotin-free medium.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Biotina/biossíntese , Mycobacterium tuberculosis/efeitos dos fármacos , Piperazinas/farmacologia , Fosfato de Piridoxal/metabolismo , Transaminases/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Biocatálise , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/metabolismo , Piperazinas/síntese química , Piperazinas/química , Relação Estrutura-Atividade , Transaminases/metabolismoRESUMO
Terrabacter carboxydivorans is able to grow aerobically at low concentrations of carbon monoxide (CO) as a sole source of carbon and energy. The genes for carbon monoxide dehydrogenase (CO-DH) were cloned from T. carboxydivorans and analyzed. The operon encoding T. carboxydivorans CO-DH was composed of three structural genes with the transcriptional order of cutB, cutC and cutA, as well as an additional accessory gene (orf4). Phylogenetic analysis of CutA revealed that T. carboxydivorans CO-DH was classified into a group distinct from previously characterized CO-DHs. Expression of antisense RNA for the cutB or cutA gene in T. carboxydivorans led to a decrease in CO-DH activity, confirming that cutBCA genes are the functional genes encoding CO-DH. The CO-DH operon was expressed even in the absence of CO and further inducible by CO. In addition, CO-DH synthesis was increased in the stationary phase compared to the exponential phase during heterotrophic growth on glucose and glycerol. Point mutations of a partially inverted repeat sequence (TCGGA-N6-GCCCA) in the upstream region of the cutB gene almost abolished expression of the CO-DH operon, indicating that the inverted-repeat sequence might be a cis-acting regulatory site for the positive regulation of the CO-DH operon.
Assuntos
Actinomycetales/enzimologia , Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Complexos Multienzimáticos/genética , Actinomycetales/genética , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/metabolismo , Monóxido de Carbono/metabolismo , Regulação Bacteriana da Expressão Gênica , Complexos Multienzimáticos/metabolismo , Óperon , Oxirredução , FilogeniaRESUMO
For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. IMPORTANCE: Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.
Assuntos
Elementos de DNA Transponíveis , Genes Essenciais , Genoma Bacteriano , Mutagênese Insercional , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/genética , Biblioteca Gênica , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
Carbon monoxide dehydrogenase (CO-DH) in Mycobacterium sp. strain JC1 is a key enzyme for the carboxydotrophic growth, when carbon monoxide (CO) is supplied as a sole source of carbon and energy. This enzyme is also known to act as nitric oxide dehydrogenase (NO-DH) for the detoxification of NO. Several accessory genes such as cutD, cutE, cutF, cutG, cutH, and cutI, are clustered together with two copies of the CO-DH structural genes (cutB1C1A1 and cutB2C2A2) in Mycobacterium sp. strain JC1 and are well conserved in carboxydotrophic mycobacteria. Transcription of the CO-DH structural and accessory genes was demonstrated to be increased significantly by acidified sodium nitrate as a source of NO. A cutI deletion (ΔcutI) mutant of Mycobacterium sp. strain JC1 was generated to identity the function of CutI. Lithoautotrophic growth of the ΔcutI mutant was severely affected in mineral medium supplemented with CO, while the mutant grew normally with glucose. Western blotting, CO-DH activity staining, and CO-DH-specific enzyme assay revealed a significant decrease in the cellular level of CO-DH in the ΔcutI mutant. Northern blot analysis and promoter assay showed that expression of the cutB1 and cutB2 genes was significantly reduced at the transcriptional level in the ΔcutI mutant, compared to that of the wildtype strain. The ΔcutI mutant was much more susceptible to NO than was the wild type.
Assuntos
Aldeído Oxirredutases/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Complexos Multienzimáticos/genética , Mycobacterium/genética , Transcrição Gênica , Aldeído Oxirredutases/química , Aldeído Oxirredutases/metabolismo , Proteínas de Bactérias/química , Monóxido de Carbono/metabolismo , Clonagem Molecular , Meios de Cultura , Deleção de Genes , Glucose/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Mycobacterium/efeitos dos fármacos , Mycobacterium/enzimologia , Mycobacterium/crescimento & desenvolvimento , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/farmacologiaRESUMO
The PLP-dependent transaminase (BioA) of Mycobacterium tuberculosis and other pathogens that catalyzes the second step of biotin biosynthesis is a now well-validated target for antibacterial development. Fragment screening by differential scanning fluorimetry has been performed to discover new chemical scaffolds and promote optimization of existing inhibitors. Calorimetry confirms binding of six molecules with high ligand efficiency. Thermodynamic data identifies which molecules bind with the enthalpy driven stabilization preferred in compounds that represent attractive starting points for future optimization. Crystallographic characterization of complexes with these molecules reveals the dynamic nature of the BioA active site. Different side chain conformational states are stabilized in response to binding by different molecules. A detailed analysis of conformational diversity in available BioA structures is presented, resulting in the identification of two states that might be targeted with molecular scaffolds incorporating well-defined conformational attributes. This new structural data can be used as part of a scaffold hopping strategy to further optimize existing inhibitors or create new small molecules with improved therapeutic potential.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Transaminases/química , Transaminases/metabolismo , Tuberculose/tratamento farmacológico , Sítios de Ligação , Varredura Diferencial de Calorimetria , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , TermodinâmicaRESUMO
Biotin biosynthesis is essential for survival and persistence of Mycobacterium tuberculosis (Mtb) in vivo. The aminotransferase BioA, which catalyzes the antepenultimate step in the biotin pathway, has been established as a promising target due to its vulnerability to chemical inhibition. We performed high-throughput screening (HTS) employing a fluorescence displacement assay and identified a diverse set of potent inhibitors including many diversity-oriented synthesis (DOS) scaffolds. To efficiently select only hits targeting biotin biosynthesis, we then deployed a whole-cell counterscreen in biotin-free and biotin-containing medium against wild-type Mtb and in parallel with isogenic bioA Mtb strains that possess differential levels of BioA expression. This counterscreen proved crucial to filter out compounds whose whole-cell activity was off target as well as identify hits with weak, but measurable whole-cell activity in BioA-depleted strains. Several of the most promising hits were cocrystallized with BioA to provide a framework for future structure-based drug design efforts.
Assuntos
Biotina/biossíntese , Mycobacterium tuberculosis/metabolismo , Antituberculosos/química , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Biotina/antagonistas & inibidores , Calorimetria , Cristalografia por Raios X , Desenho de Fármacos , Ensaios de Triagem em Larga Escala , Ligação de Hidrogênio , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Transaminases/antagonistas & inibidores , Transaminases/metabolismoRESUMO
UNLABELLED: Mycobacterium tuberculosis depends on aerobic respiration for growth and utilizes an aa3-type cytochrome c oxidase for terminal electron transfer. Cytochrome c maturation in bacteria requires covalent attachment of heme to apocytochrome c, which occurs outside the cytoplasmic membrane. We demonstrate that in M. tuberculosis the thioredoxin-like protein Rv3673c, which we named CcsX, is required for heme insertion in cytochrome c. Inactivation of CcsX resulted in loss of c-type heme absorbance, impaired growth and virulence of M. tuberculosis, and induced cytochrome bd oxidase. This suggests that the bioenergetically less efficient bd oxidase can compensate for deficient cytochrome c oxidase activity, highlighting the flexibility of the M. tuberculosis respiratory chain. A spontaneous mutation in the active site of vitamin K epoxide reductase (VKOR) suppressed phenotypes of the CcsX mutant and abrogated the activity of the disulfide bond-dependent alkaline phosphatase, which shows that VKOR is the major disulfide bond catalyzing protein in the periplasm of M. tuberculosis. IMPORTANCE: Mycobacterium tuberculosis requires oxygen for growth; however, the biogenesis of respiratory chain components in mycobacteria has not been explored. Here, we identified a periplasmic thioredoxin, CcsX, necessary for heme insertion into cytochrome c. We investigated the consequences of disrupting cytochrome c maturation (CCM) for growth and survival of M. tuberculosis in vitro and for its pathogenesis. Appearance of a second-site suppressor mutation in the periplasmic disulfide bond catalyzing protein VKOR indicates the strong selective pressure for a functional cytochrome c oxidase. The observation that M. tuberculosis is able to partially compensate for defective CCM by upregulation of the cytochrome bd oxidase exposes a functional role of this alternative terminal oxidase under normal aerobic conditions and during pathogenesis. This suggests that targeting both oxidases simultaneously might be required to effectively disrupt respiration in M. tuberculosis.
Assuntos
Proteínas de Bactérias/metabolismo , Citocromos c/metabolismo , Mycobacterium tuberculosis/metabolismo , Tuberculose/microbiologia , Animais , Proteínas de Bactérias/genética , Citocromos c/genética , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Feminino , Heme/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crescimento & desenvolvimento , Oxigênio/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
Although extensive studies on the oxidation of carbon monoxide (CO) in aerobic carboxydotrophic bacteria have been carried out for over 30 years, utilization of CO as a source of carbon and energy by mycobacteria was recognized only recently. Studies on pathogenic and nonpathogenic mycobacteria have revealed that the basis for CO utilization in these bacteria is different in many aspects from that of other aerobic carboxydobacteria. We review the basis for CO utilization in mycobacterial carboxydobacteria, which is unique from physiological, biochemical, molecular, genetic and phylogenetic points of view.
Assuntos
Monóxido de Carbono/metabolismo , Metabolismo Energético , Redes e Vias Metabólicas , Mycobacterium/genética , Mycobacterium/metabolismoRESUMO
BioA catalyzes the second step of biotin biosynthesis, and this enzyme represents a potential target to develop new antitubercular agents. Herein we report the design, synthesis, and biochemical characterization of a mechanism-based inhibitor (1) featuring a 3,6-dihydropyrid-2-one heterocycle that covalently modifies the pyridoxal 5'-phosphate (PLP) cofactor of BioA through aromatization. The structure of the PLP adduct was confirmed by MS/MS and X-ray crystallography at 1.94 Å resolution. Inactivation of BioA by 1 was time- and concentration-dependent and protected by substrate. We used a conditional knock-down mutant of M. tuberculosis to demonstrate the antitubercular activity of 1 correlated with BioA expression, and these results provide support for the designed mechanism of action.