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Thiopeptides are ribosomally biosynthesized and post-translationally modified peptides (RiPPs) that potently inhibit the growth of Gram-positive bacteria by targeting multiple steps in protein biosynthesis. The poor pharmacological properties of thiopeptides, particularly their low aqueous solubility, has hindered their development into clinically useful antibiotics. Antimicrobial activity screens of a library of Actinomycetota extracts led to discovery of the novel polyglycosylated thiopeptides persiathiacins A and B from Actinokineospora sp. UTMC 2448. Persiathiacin A is active against methicillin-resistant Staphylococcus aureus and several Mycobacterium tuberculosis strains, including drug-resistant and multidrug-resistant clinical isolates, and does not significantly affect the growth of ovarian cancer cells at concentrations up to 400 µM. Polyglycosylated thiopeptides are extremely rare and nothing is known about their biosynthesis. Sequencing and analysis of the Actinokineospora sp. UTMC 2448 genome enabled identification of the putative persiathiacin biosynthetic gene cluster (BGC). A cytochrome P450 encoded by this gene cluster catalyzes the hydroxylation of nosiheptide in vitro and in vivo, consistent with the proposal that the cluster directs persiathiacin biosynthesis. Several genes in the cluster encode homologues of enzymes known to catalyze the assembly and attachment of deoxysugars during the biosynthesis of other classes of glycosylated natural products. One of these encodes a glycosyl transferase that was shown to catalyze attachment of a D-glucose residue to nosiheptide in vitro. The discovery of the persiathiacins and their BGC thus provides the basis for the development of biosynthetic engineering approaches to the creation of novel (poly)glycosylated thiopeptide derivatives with enhanced pharmacological properties.
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3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.
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Mycobacterium tuberculosis , Animais , Oxidiazóis/farmacologia , Oxidiazóis/química , Tetrazóis/farmacologia , Tetrazóis/química , Testes de Sensibilidade Microbiana , Antituberculosos/farmacologia , Antituberculosos/química , Relação Estrutura-Atividade , Nitrorredutases , MamíferosRESUMO
Intrinsic and Rho-dependent transcription termination mechanisms regulate gene expression and recycle RNA polymerase in bacteria. Both the modes are well studied in Escherichia coli, and a few other organisms. The understanding of Rho function is limited in most other bacteria including mycobacteria. Here, we highlight the dominance of Rho-dependent termination in mycobacteria and validate Rho as a key regulatory factor. The lower abundance of intrinsic terminators, high cellular levels of Rho, and its genome-wide association with a majority of transcriptionally active genes indicate the pronounced role of Rho-mediated termination in Mycobacterium tuberculosis (Mtb). Rho modulates the termination of RNA synthesis for both protein-coding and stable RNA genes in Mtb. Concordantly, the depletion of Rho in mycobacteria impact its growth and enhances the transcription read-through at 3' ends of the transcription units. We demonstrate that MtbRho is catalytically active in the presence of RNA with varied secondary structures. These properties suggest an evolutionary adaptation of Rho as the efficient and preponderant mode of transcription termination in mycobacteria.
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Mycobacterium tuberculosis , Mycobacterium tuberculosis/genética , Estudo de Associação Genômica Ampla , Transcrição Gênica , Escherichia coli/genética , RNA/metabolismoRESUMO
Tuberculosis, and especially multidrug-resistant tuberculosis (MDR-TB), is a major global health threat which emphasizes the need to develop new agents to improve and shorten treatment of this difficult-to-manage infectious disease. Among the new agents, macozinone (PBTZ169) is one of the most promising candidates, showing extraordinary potency in vitro and in murine models against drug-susceptible and drug-resistant Mycobacterium tuberculosis. A previous analytical method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed by our group to support phase I clinical trials of PBTZ169. These plasma sample analyses revealed the presence of several additional metabolites among which the most prominent was H2PBTZ, a reduced species obtained by dearomatization of macozinone, one of the first examples of Meisenheimer Complex (MC) metabolites identified in mammals. Identification of these new metabolites required the optimization of our original method for enhancing the selectivity between isobaric metabolites as well as for ensuring optimal stability for H2PBTZ analyses. Sample preparation methods were also developed for plasma and urine, followed by extensive quantitative validation in accordance with international bioanalytical method recommendations, which include selectivity, linearity, qualitative and quantitative matrix effect, trueness, precision and the establishment of accuracy profiles using ß-expectation tolerance intervals for known and newer analytes. The newly optimized methods have been applied in a subsequent Phase Ib clinical trial conducted in our University Hospital with healthy subjects. H2PBTZ was found to be the most abundant species circulating in plasma, underscoring the importance of measuring accurately and precisely this unprecedented metabolite. Low concentrations were found in urine for all monitored analytes, suggesting extensive metabolism before renal excretion.
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Tuberculose Resistente a Múltiplos Medicamentos , Tuberculose , Animais , Humanos , Camundongos , Cromatografia Líquida/métodos , Mamíferos , Piperazinas , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Tuberculose/tratamento farmacológico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Ensaios Clínicos Fase I como AssuntoRESUMO
The Mycobacterium tuberculosis type-7 protein secretion system ESX-1 is a major driver of its virulence. While the functions of most ESX-1 components are characterized, many others remain poorly defined. In this study, we examined the role of EspK, an ESX-1-associated protein that is thought to be dispensable for ESX-1 activity in members of the Mycobacterium tuberculosis complex. We show that EspK is needed for the timely and optimal secretion of EsxA and absolutely essential for EspB secretion in M. tuberculosis Erdman. We demonstrate that only the EsxA secretion defect can be alleviated in EspK-deficient M. tuberculosis by culturing it in media containing detergents like Tween 80 or tyloxapol. Subcellular fractionation experiments reveal EspK is exported by M. tuberculosis in an ESX-1-independent manner and localized to its cell wall. We also show a conserved W-X-G motif in EspK is important for its interaction with EspB and enabling its secretion. The same motif, however, is not important for EspK localization in the cell wall. Finally, we show EspB in EspK-deficient M. tuberculosis tends to adopt higher-order oligomeric conformations, more so than EspB in wild-type M. tuberculosis. These results suggest EspK interacts with EspB and prevents it from assembling prematurely into macromolecular complexes that are presumably too large to pass through the membrane-spanning ESX-1 translocon assembly. Collectively, our findings indicate M. tuberculosis EspK has a far more active role in ESX-1-mediated secretion than was previously appreciated and underscores the complex nature of this secretion apparatus. IMPORTANCE Mycobacterium tuberculosis uses its ESX-1 system to secrete EsxA and EspB into a host to cause disease. We show that EspK, a protein whose role in the ESX-1 machinery was thought to be nonessential, is needed by M. tuberculosis for optimal EsxA and EspB secretion. Culturing EspK-deficient M. tuberculosis with detergents alleviates EsxA but not EspB secretion defects. We also show that EspK, which is exported by M. tuberculosis in an ESX-1-independent manner to the cell wall, interacts with and prevents EspB from assembling into large structures inside the M. tuberculosis cell that are nonsecretable. Collectively, our observations demonstrate EspK is an active component of the ESX-1 secretion machinery of the tubercle bacillus.
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Mycobacterium tuberculosis , Tuberculose , Sistemas de Secreção Tipo VII , Proteínas de Bactérias/metabolismo , Detergentes/metabolismo , Humanos , Mycobacterium tuberculosis/metabolismo , Tuberculose/microbiologia , Sistemas de Secreção Tipo VII/metabolismoRESUMO
Type II NADH dehydrogenase (NDH-2) is an essential component of electron transfer in many microbial pathogens but has remained largely unexplored as a potential drug target. Previously, quinolinyl pyrimidines were shown to inhibit Mycobacterium tuberculosis NDH-2, as well as the growth of the bacteria [Shirude, P. S.; ACS Med. Chem. Lett. 2012, 3, 736-740]. Here, we synthesized a number of novel quinolinyl pyrimidines and investigated their properties. In terms of inhibition of the NDH-2 enzymes from M. tuberculosis and Mycobacterium smegmatis, the best compounds were of similar potency to previously reported inhibitors of the same class (half-maximal inhibitory concentration (IC50) values in the low-µM range). However, a number of the compounds had much better activity against Gram-negative pathogens, with minimum inhibitory concentrations (MICs) as low as 2 µg/mL. Multivariate analyses (partial least-squares (PLS) and principle component analysis (PCA)) showed that overall ligand charge was one of the most important factors in determining antibacterial activity, with patterns that varied depending on the particular bacterial species. In some cases (e.g., mycobacteria), there was a clear correlation between the IC50 values and the observed MICs, while in other instances, no such correlation was evident. When tested against a panel of protozoan parasites, the compounds failed to show activity that was not linked to cytotoxicity. Further, a strong correlation between hydrophobicity (estimated as clogâ¯P) and cytotoxicity was revealed; more hydrophobic analogues were more cytotoxic. By contrast, antibacterial MIC values and cytotoxicity were not well correlated, suggesting that the quinolinyl pyrimidines can be optimized further as antimicrobial agents.
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Mycobacterium tuberculosis , NADH Desidrogenase , Testes de Sensibilidade Microbiana , NAD , Pirimidinas/farmacologiaRESUMO
Leprosy is a chronic dermato-neurological disease caused by Mycobacterium leprae, an obligate intracellular bacterium. Timely detection is a challenge in leprosy diagnosis, relying on clinical examination and trained health professionals. Furthermore, adequate care and transmission control depend on early and reliable pathogen detection. Here, we describe a qPCR test for routine diagnosis of leprosy-suspected patients. The reaction simultaneously amplifies two specific Mycobacterium leprae targets (16S rRNA and RLEP), and the human 18S rRNA gene as internal control. The limit of detection was estimated to be 2.29 copies of the M. leprae genome. Analytical specificity was evaluated using a panel of 20 other skin pathogenic microorganisms and Mycobacteria, showing no cross-reactivity. Intra- and inter-operator Cp variation was evaluated using dilution curves of M. leprae DNA or a synthetic gene, and no significant difference was observed between three operators in two different laboratories. The multiplex assay was evaluated using 97 patient samples with clinical and histopathological leprosy confirmation, displaying high diagnostic sensitivity (91%) and specificity (100%). Validation tests in an independent panel of 50 samples confirmed sensitivity and specificity of 97% and 98%, respectively. Importantly, assay performance remained stable for at least five months. Our results show that the newly developed multiplex qPCR effectively and specifically detects M. leprae DNA in skin samples, contributing to an efficient diagnosis that expedites the appropriate treatment.
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Hanseníase/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , DNA Bacteriano/genética , Feminino , Humanos , Indicadores e Reagentes/normas , Lactente , Hanseníase/microbiologia , Masculino , Pessoa de Meia-Idade , Técnicas de Diagnóstico Molecular/normas , Reação em Cadeia da Polimerase Multiplex/normas , Mycobacterium leprae/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/normas , Sensibilidade e Especificidade , Adulto JovemRESUMO
Humans are considered as the main host for Mycobacterium leprae1, the aetiological agent of leprosy, but spillover has occurred to other mammals that are now maintenance hosts, such as nine-banded armadillos and red squirrels2,3. Although naturally acquired leprosy has also been described in captive nonhuman primates4-7, the exact origins of infection remain unclear. Here we describe leprosy-like lesions in two wild populations of western chimpanzees (Pan troglodytes verus) in Cantanhez National Park, Guinea-Bissau and Taï National Park, Côte d'Ivoire, West Africa. Longitudinal monitoring of both populations revealed the progression of disease symptoms compatible with advanced leprosy. Screening of faecal and necropsy samples confirmed the presence of M. leprae as the causative agent at each site and phylogenomic comparisons with other strains from humans and other animals show that the chimpanzee strains belong to different and rare genotypes (4N/O and 2F). These findings suggest that M. leprae may be circulating in more wild animals than suspected, either as a result of exposure to humans or other unknown environmental sources.
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Hanseníase/veterinária , Pan troglodytes/microbiologia , Animais , Autopsia/veterinária , Côte d'Ivoire , Fezes/microbiologia , Genótipo , Guiné-Bissau , Humanos , Hanseníase/microbiologia , Mycobacterium leprae/genética , Mycobacterium leprae/isolamento & purificação , FilogeniaRESUMO
BACKGROUND: Hansen's disease (leprosy), widespread in medieval Europe, is today mainly prevalent in tropical and subtropical regions with around 200,000 new cases reported annually. Despite its long history and appearance in historical records, its origins and past dissemination patterns are still widely unknown. Applying ancient DNA approaches to its major causative agent, Mycobacterium leprae, can significantly improve our understanding of the disease's complex history. Previous studies have identified a high genetic continuity of the pathogen over the last 1500 years and the existence of at least four M. leprae lineages in some parts of Europe since the Early Medieval period. RESULTS: Here, we reconstructed 19 ancient M. leprae genomes to further investigate M. leprae's genetic variation in Europe, with a dedicated focus on bacterial genomes from previously unstudied regions (Belarus, Iberia, Russia, Scotland), from multiple sites in a single region (Cambridgeshire, England), and from two Iberian leprosaria. Overall, our data confirm the existence of similar phylogeographic patterns across Europe, including high diversity in leprosaria. Further, we identified a new genotype in Belarus. By doubling the number of complete ancient M. leprae genomes, our results improve our knowledge of the past phylogeography of M. leprae and reveal a particularly high M. leprae diversity in European medieval leprosaria. CONCLUSIONS: Our findings allow us to detect similar patterns of strain diversity across Europe with branch 3 as the most common branch and the leprosaria as centers for high diversity. The higher resolution of our phylogeny tree also refined our understanding of the interspecies transfer between red squirrels and humans pointing to a late antique/early medieval transmission. Furthermore, with our new estimates on the past population diversity of M. leprae, we gained first insights into the disease's global history in relation to major historic events such as the Roman expansion or the beginning of the regular transatlantic long distance trade. In summary, our findings highlight how studying ancient M. leprae genomes worldwide improves our understanding of leprosy's global history and can contribute to current models of M. leprae's worldwide dissemination, including interspecies transmissions.
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Mycobacterium leprae , Europa (Continente) , Genoma Bacteriano/genética , Humanos , Hanseníase/genética , Mycobacterium leprae/genética , Dinâmica PopulacionalRESUMO
Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of pknL and adjacent regulator MSMEG_4242 in rough colony M. smegmatis mc2155 produced both smooth and rough colonies. Upon restreaking rough colonies, smooth colonies appeared at a frequency of ~ 1/250. Smooth mutants did not form biofilms, showed increased sliding motility and anomalous lipids on thin-layer chromatography, identified by mass spectrometry as lipooligosaccharides and perhaps also glycopeptidolipids. RNA-seq and Sanger sequencing revealed that all smooth mutants had inactivated lsr2 genes due to mutations and different IS1096 insertions. When complemented with lsr2, the colonies became rough, anomalous lipids disappeared and sliding motility decreased. Smooth mutants showed increased expression of IS1096 transposase TnpA and MSMEG_4727, which encodes a protein similar to PKS5. When MSMEG_4727 was deleted, smooth pknL/MSMEG_4242/lsr2 mutants reverted to rough, formed good biofilms, their motility decreased slightly and their anomalous lipids disappeared. Rough delpknL/del4242 mutants formed poor biofilms and showed decreased, aberrant sliding motility and both phenotypes were complemented with the two deleted genes. Inactivation of lsr2 changes colony morphology from rough to smooth, augments sliding motility and increases expression of MSMEG_4727 and other enzymes synthesizing lipooligosaccharides, apparently preventing biofilm formation. Similar morphological phase changes occur in other mycobacteria, likely reflecting environmental adaptations. PknL and MSMEG_4242 regulate lipid components of the outer cell envelope and their absence selects for lsr2 inactivation. A regulatory, phosphorylation cascade model is proposed.
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Latent Mycobacterium tuberculosis infection presents one of the largest challenges for tuberculosis control and novel antimycobacterial drug development. A series of pyrano[3,2-b]indolone-based compounds was designed and synthesized via an original eight-step scheme. The synthesized compounds were evaluated for their in vitro activity against M. tuberculosis strains H37Rv and streptomycin-starved 18b (SS18b), representing models for replicating and nonreplicating mycobacteria, respectively. Compound 10a exhibited good activity with MIC99 values of 0.3 and 0.4 µg/mL against H37Rv and SS18b, respectively, as well as low toxicity, acceptable intracellular activity, and satisfactory metabolic stability and was selected as the lead compound for further studies. An analysis of 10a-resistant M. bovis mutants disclosed a cross-resistance with pretomanid and altered relative amounts of different forms of cofactor F420 in these strains. Complementation experiments showed that F420-dependent glucose-6-phosphate dehydrogenase and the synthesis of mature F420 were important for 10a activity. Overall these studies revealed 10a to be a prodrug that is activated by an unknown F420-dependent enzyme in mycobacteria.
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Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Antituberculosos/farmacologia , Humanos , Mycobacterium tuberculosis/genéticaRESUMO
In ancient DNA research, the degraded nature of the samples generally results in poor yields of highly fragmented DNA; targeted DNA enrichment is thus required to maximize research outcomes. The three commonly used methods - array-based hybridization capture and in-solution capture using either RNA or DNA baits - have different characteristics that may influence the capture efficiency, specificity and reproducibility. Here we compare their performance in enriching pathogen DNA of Mycobacterium leprae and Treponema pallidum from 11 ancient and 19 modern samples. We find that in-solution approaches are the most effective method in ancient and modern samples of both pathogens and that RNA baits usually perform better than DNA baits.
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DNA Antigo/análise , Mycobacterium leprae/genética , Hibridização de Ácido Nucleico/métodos , Treponema pallidum/genética , Humanos , Reprodutibilidade dos TestesRESUMO
Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, is the world's leading cause of death from an infectious disease. One of the main features of this pathogen is the complex and dynamic lipid composition of the cell envelope, which adapts to the variable host environment and defines the fate of infection by actively interacting with and modulating immune responses. However, while much has been learned about the enzymes of the numerous lipid pathways, little knowledge is available regarding the proteins and metabolic signals regulating lipid metabolism during M. tuberculosis infection. In this work, we constructed and characterized a FasR-deficient mutant in M. tuberculosis and demonstrated that FasR positively regulates fas and acpS expression. Lipidomic analysis of the wild type and mutant strains revealed complete rearrangement of most lipid components of the cell envelope, with phospholipids, mycolic acids, sulfolipids, and phthiocerol dimycocerosates relative abundance severely altered. As a consequence, replication of the mutant strain was impaired in macrophages leading to reduced virulence in a mouse model of infection. Moreover, we show that the fasR mutant resides in acidified cellular compartments, suggesting that the lipid perturbation caused by the mutation prevented M. tuberculosis inhibition of phagolysosome maturation. This study identified FasR as a novel factor involved in regulation of mycobacterial virulence and provides evidence for the essential role that modulation of lipid homeostasis plays in the outcome of M. tuberculosis infection.
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The impact of tuberculosis and of anti-tuberculosis therapy on composition and modification of human lung microbiota has been the object of several investigations. However, no clear outcome has been presented so far and the relationship between M. tuberculosis pulmonary infection and the resident lung microbiota remains vague. In this work we describe the results obtained from a multicenter study of the microbiota of sputum samples from patients with tuberculosis or unrelated lung diseases and healthy donors recruited in Switzerland, Italy and Bangladesh, with the ultimate goal of discovering a microbiota-based biomarker associated with tuberculosis. Bacterial 16S rDNA amplification, high-throughput sequencing and extensive bioinformatic analyses revealed patient-specific flora and high variability in taxon abundance. No common signature could be identified among the individuals enrolled except for minor differences which were not consistent among the different geographical settings. Moreover, anti-tuberculosis therapy did not cause any important variation in microbiota diversity, thus precluding its exploitation as a biomarker for the follow up of tuberculosis patients undergoing treatment.
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Escarro/microbiologia , Tuberculose/microbiologia , Adulto , Idoso , Bangladesh , Biomarcadores/sangue , DNA Ribossômico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Itália , Masculino , Pessoa de Meia-Idade , RNA Ribossômico 16S , Suíça , Tuberculose/sangue , Adulto JovemRESUMO
The EspB protein of Mycobacterium tuberculosis is a 60 kDa virulence factor, implicated in conjugation and exported by the ESX-1 system of which it may also be a component. Previous attempts to obtain high-resolution maps of EspB by cryo-electron microscopic examination of single particles have been thwarted by severe orientation bias of the particles. This was overcome by using detergent as a surfactant thereby allowing reconstruction of the EspB structure at 3.37 Å resolution. The final structure revealed the N-terminal domain of EspB to be organized as a cylindrical heptamer with dimensions of 90 Å x 90 Å and a central channel of 45 Å diameter whereas the C-terminal domain was unstructured. New atomic insight was obtained into the helical packing required for protomer interactions and the overall electrostatic potential. The external surface is electronegatively charged while the channel is lined with electropositive patches. EspB thus has many features of a pore-like transport protein that might allow the passage of an ESX-1 substrate such as the 35 Å diameter EsxA-EsxB heterodimer or B-form DNA consistent with its proposed role in DNA uptake.
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Screening of a diversity-oriented compound library led to the identification of two 6,11-dioxobenzo[f]pyrido[1,2-a]indoles (DBPI) that displayed low micromolar bactericidal activity against the Erdman strain of Mycobacterium tuberculosis in vitro. The activity of these hit compounds was limited to tubercle bacilli, including the nonreplicating form, and to Mycobacterium marinum. On hit expansion and investigation of the structure activity relationship, selected modifications to the dioxo moiety of the DBPI scaffold were either neutral or led to reduction or abolition of antimycobacterial activity. To find the target, DBPI-resistant mutants of M. tuberculosis Erdman were raised and characterized first microbiologically and then by whole genome sequencing. Four different mutations, all affecting highly conserved residues, were uncovered in the essential gene rv0338c (ispQ) that encodes a membrane-bound protein, named IspQ, with 2Fe-2S and 4Fe-4S centers and putative iron-sulfur-binding reductase activity. With the help of a structural model, two of the mutations were localized close to the 2Fe-2S domain in IspQ and another in transmembrane segment 3. The mutant genes were recessive to the wild type in complementation experiments and further confirmation of the hit-target relationship was obtained using a conditional knockdown mutant of rv0338c in M. tuberculosis H37Rv. More mechanistic insight was obtained from transcriptome analysis, following exposure of M. tuberculosis to two different DBPI; this revealed strong upregulation of the redox-sensitive SigK regulon and genes induced by oxidative and thiol-stress. The findings of this investigation pharmacologically validate a novel target in tubercle bacilli and open a new vista for tuberculosis drug discovery.
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Proteínas Ferro-Enxofre , Mycobacterium tuberculosis , Tuberculose , Humanos , Indóis , Proteínas Ferro-Enxofre/genética , Proteínas Ferro-Enxofre/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , OxirreduçãoRESUMO
Mycobacterium leprae, the causative agent of leprosy, is an unculturable bacterium with a considerably reduced genome (3.27 Mb) compared to homologues mycobacteria from the same ancestry. In 2001, the genome of M. leprae was first described and subsequently four genotypes (1-4) and 16 subtypes (A-P) were identified providing means to study global transmission patterns for leprosy. In order to understand the role of asymptomatic carriers we investigated M. leprae carriage as well as infection in leprosy patients (n = 60) and healthy household contacts (HHC; n = 250) from Bangladesh using molecular detection of the bacterial element RLEP in nasal swabs (NS) and slit skin smears (SSS). In parallel, to study M. leprae genotype distribution in Bangladesh we explored strain diversity by whole genome sequencing (WGS) and Sanger sequencing. In the studied cohort in Bangladesh, M. leprae DNA was detected in 33.3% of NS and 22.2% of SSS of patients with bacillary index of 0 whilst in HHC 18.0% of NS and 12.3% of SSS were positive. The majority of the M. leprae strains detected in this study belonged to genotype 1D (55%), followed by 1A (31%). Importantly, WGS allowed the identification of a new M. leprae genotype, designated 1B-Bangladesh (14%), which clustered separately between the 1A and 1B strains. Moreover, we established that the genotype previously designated 1C, is not an independent subtype but clusters within the 1D genotype. Intraindividual differences were present between the M. leprae strains obtained including mutations in hypermutated genes, suggesting mixed colonization/infection or in-host evolution. In summary, we observed that M. leprae is present in asymptomatic contacts of leprosy patients fueling the concept that these individuals contribute to the current intensity of transmission. Our data therefore emphasize the importance of sensitive and specific tools allowing post-exposure prophylaxis targeted at M. leprae-infected or -colonized individuals.
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Human settlement of Madagascar traces back to the beginning of the first millennium with the arrival of Austronesians from Southeast Asia, followed by migrations from Africa and the Middle East. Remains of these different cultural, genetic, and linguistic legacies are still present in Madagascar and other islands of the Indian Ocean. The close relationship between human migration and the introduction and spread of infectious diseases, a well-documented phenomenon, is particularly evident for the causative agent of leprosy, Mycobacterium leprae. In this study, we used whole-genome sequencing (WGS) and molecular dating to characterize the genetic background and retrace the origin of the M. leprae strains circulating in Madagascar (n = 30) and the Comoros (n = 3), two islands where leprosy is still considered a public health problem and monitored as part of a drug resistance surveillance program. Most M. leprae strains (97%) from Madagascar and Comoros belonged to a new genotype as part of branch 1, closely related to single nucleotide polymorphism (SNP) type 1D, named 1D-Malagasy. Other strains belonged to the genotype 1A (3%). We sequenced 39 strains from nine other countries, which, together with previously published genomes, amounted to 242 genomes that were used for molecular dating. Specific SNP markers for the new 1D-Malagasy genotype were used to screen samples from 11 countries and revealed this genotype to be restricted to Madagascar, with the sole exception being a strain from Malawi. The overall analysis thus ruled out a possible introduction of leprosy by the Austronesian settlers and suggests a later origin from East Africa, the Middle East, or South Asia.
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The emergence of multi-drug (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) is a major threat to the global management of tuberculosis (TB) worldwide. New chemical entities are of need to treat drug-resistant TB. In this study, the mode of action of new, potent quinazoline derivatives was investigated against Mycobacterium tuberculosis (M. tb). Four derivatives 11626141, 11626142, 11626252 and 11726148 showed good activity (MIC ranging from 0.02-0.09 µg/mL) and low toxicity (TD50 ≥ 5µg/mL) in vitro against M. tb strain H37Rv and HepG2 cells, respectively. 11626252 was the most selective compound from this series. Quinazoline derivatives were found to target cytochrome bc1 by whole-genome sequencing of mutants selected with 11626142. Two resistant mutants harboured the transversion T943G (Trp312Gly) and the transition G523A (Gly175Ser) in the cytochrome bc1 complex cytochrome b subunit (QcrB). Interestingly, a third mutant QuinR-M1 contained a mutation in the Rieske iron-sulphur protein (QcrA) leading to resistance to quinazoline and other QcrB inhibitors, the first report of cross-resistance involving QcrA. Modelling of both QcrA and QcrB revealed that all three resistance mutations are located in the stigmatellin pocket, as previously observed for other QcrB inhibitors such as Q203, AX-35, and lansoprazole sulfide (LPZs). Further analysis of the mode of action in vitro revealed that 11626252 exposure leads to ATP depletion, a decrease in the oxygen consumption rate and also overexpression of the cytochrome bd oxidase in M. tb. Our findings suggest that quinazoline-derived compounds are a new and attractive chemical entity for M. tb drug development targeting two separate subunits of the cytochrome bc1 complex.