Mapping and characterization of G-quadruplexes in Mycobacterium tuberculosis gene promoter regions

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), one of the top 10 causes of death worldwide in 2015. The recent emergence of strains resistant to all current drugs urges the development of compounds with new mechanisms of action. G-quadruplexes are nucleic acids secondary structures that may form in G-rich regions to epigenetically regulate cellular functions. Here we implemented a computational tool to scan the presence of putative G-quadruplex forming sequences in the genome of Mycobacterium tuberculosis and analyse their association to transcription start sites. We found that the most stable G-quadruplexes were in the promoter region of genes belonging to definite functional categories. Actual G-quadruplex folding of four selected sequences was assessed by biophysical and biomolecular techniques: all molecules formed stable G-quadruplexes, which were further stabilized by two G-quadruplex ligands. These compounds inhibited Mycobacterium tuberculosis growth with minimal inhibitory concentrations in the low micromolar range. These data support formation of Mycobacterium tuberculosis G-quadruplexes in vivo and their potential regulation of gene transcription, and prompt the use of G4 ligands to develop original antitubercular agents

Assessment of treatment response in tuberculosis

Antibiotic treatment of tuberculosis has a duration of several months. There is significant variability of the host immune response and the pharmacokinetic-pharmacodynamic properties of Mycobacterium tuberculosis sub-populations at the site of disease. A limitation of sputum-based measures of treatment response may be sub-optimal detection and monitoring of Mycobacterium tuberculosis sub-populations. Potential biomarkers and surrogate endpoints should be benchmarked against hard clinical outcomes (failure/relapse/death) and may need tailoring to specific patient populations. Here, we assess the evidence supporting currently utilized and future potential host and pathogen-based models and biomarkers for monitoring treatment response in active and latent tuberculosis. Biomarkers for monitoring treatment response in extrapulmonary, pediatric and drug resistant tuberculosis are research priorities

Antituberculosis Activity of Brotowali (Tinospora Crispa) Extract and Fractions Against Mycobacterium Tuberculosis Using Microplate Alamar Blue Assay Method

Tuberculosis (TB), in which caused by pathogenic bacteria, Mycobacterium tuberculosis, has become the major causes of death among all of infectious diseases. The increasing incidence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) has created a need to discover a new antituberculosis drug candidate. The aim of this study was to screen extract and fractions of Tinospora crispa for activity against Mycobacterium tuberculosis H37Rv using the Microplate Alamar Blue Assay (MABA) method. T. crispa extract was prepared by maceration in ethanol (96%) and antituberculosis activity was carried out using MABA method. The result of this study showed that ethanolic extract of T. crispa exhibit antituberculosis activity with minimum inhibition concentration of 12.5 mg/ml

Performance of the 2007 WHO Algorithm to diagnose Smear-negative Pulmonary Tuberculosis in a HIV prevalent setting

The 2007 WHO algorithm for diagnosis of smear-negative pulmonary tuberculosis (PTB) including Mycobacterium tuberculosis (MTB) culture was evaluated in a HIV prevalent area of Kenya

Comparative Computational Analysis of Mycobacterium Species by using Different Techniques in Study

Mycobacterium tuberculosis (MTB) is a pathogenic bacteria species in the genus Mycobacterium and the causative agent of most cases of tuberculosis. It is spread through the air when people who have an active MTB infection cough, sneeze, or otherwise transmit their saliva through the air. Most infections in humans result in an asymptomatic, latent infection, and about one in ten latent infections eventually progresses to active disease, which, if left untreated, kills more than 50% of its victims.  Mycobacterium tuberculosis is a member of the genus ‘tuberculosis’ in which contains various other mycobacterium species also. These species within a gene must have some similarity in them. In spite of this similarity only mycobacterium tuberculosis cause the tuberculosis disease, the remaining does not. This signifies that mycobacterium tuberculosis must be having some specific genes or proteins which are uniquely present only in it and not in the other species. This fact is used in this research and blast program is executed recursively for the comparison between these mycobacterium species. Keywords: BLAST, Mycobacterium Tuberculosis, Nontuberculous mycobacterium group, EEA1, KEG

Antimicrobial effects of folk medicinal plants from the North of Iran against Mycobacterium tuberculosis

Background: Medicinal plants have been used traditionally in Golestan province (north of Iran), against Mycobacterium tuberculosis or the clinical signs of tuberculosis (TB). Objectives: This study aimed to define the inhibitory effects of ethanolic extracts of six of these medicinal plants against Mycobacterium tuberculosis. Materials and Methods: Peganum harmala (seed extract), Punica granatum (peel extract), Digitalis sp. (leaf extract), fruit extract of Citrus lemon, Rosa canina and Berberis vulgaris were extracted in ethanol and their activity against M. tuberculosis isolates were determined by the agar diffusion method. The zone of inhibition (at 200 to 1.6 mg/mL) was measured and the results were compared with isoniazid and rifampin as standard positive controls. Also the concentration of vitamin C of each the extracts was evaluated. Results: The ethanolic extract of Peganum harmala seed and Punica granatum peel exhibited potential activity against all M. tuberculosis isolates with mean inhibitory zone of 18.7 and 18.8 mm, at 200 mg/mL concentration. The mean inhibitory zone around isoniazid and rifampinwere 19.2 and 18.8 mm. Ethanolic extract of Citrus lemon showed moderate inhibitory activity only against sensitive (non MDR; non multi drug resistant) strains of M. tuberculosis, and Digitalis sp. showed inhibitory effects on five isolates. Ascorbic acid content was 43.3 mg/dL in Punica granatum and Digitalis sp. and only 9.1 mg/dL in ethanolic extract of Peganum harmala. Conclusions: The highest content of vitamin C was observed in the extract of Punica granatum, which was observed to be highly active against Mycobacterium tuberculosis, while the P. harmala must have contained other phytochemical constituents that contributed to the anti-tuberculosis effects of this plant. Our findings showed that ethanolic extracts of P. granatum and P. harmala had anti-TB effects comparable to isoniazid and rifampin and can be good candidates for novel and safe natural products against tuberculosis. © 2015, Pediatric Infections Research Center

Tetrahydropyrazolo[1,5-a]Pyrimidine-3-Carboxamide and N-Benzyl-6′,7′-Dihydrospiro[Piperidine-4,4′-Thieno[3,2-c]Pyran] analogues with bactericidal efficacy against Mycobacterium tuberculosis targeting MmpL3

Mycobacterium tuberculosis is a major human pathogen and the causative agent for the pulmonary disease, tuberculosis (TB). Current treatment programs to combat TB are under threat due to the emergence of multi-drug and extensively-drug resistant TB. As part of our efforts towards the discovery of new anti-tubercular leads, a number of potent tetrahydropyrazolo[1,5-a]pyrimidine-3-ca​rboxamide(THPP) and N-benzyl-6′,7′-dihydrospiro[piperidine-4,​4′-thieno[3,2-c]pyran](Spiro) analogues were recently identified against Mycobacterium tuberculosis and Mycobacterium bovis BCG through a high-throughput whole-cell screening campaign. Herein, we describe the attractive in vitro and in vivo anti-tubercular profiles of both lead series. The generation of M. tuberculosis spontaneous mutants and subsequent whole genome sequencing of several resistant mutants identified single mutations in the essential mmpL3 gene. This ‘genetic phenotype’ was further confirmed by a ‘chemical phenotype’, whereby M. bovis BCG treated with both the THPP and Spiro series resulted in the accumulation of trehalose monomycolate. In vivo efficacy evaluation of two optimized THPP and Spiro leads showed how the compounds were able to reduce >2 logs bacterial cfu counts in the lungs of infected mice

Platensimycin Activity against Mycobacterial β-Ketoacyl-ACP Synthases

Background - There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin. Methods and Findings - We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 µg/ml) and against Mycobacterium tuberculosis (MIC = 12 µg/ml). Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against β-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 µg/ml, to 30 and 124 µg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH. Significance - Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues

Biosynthesis of mycobacterial arabinogalactan: identification of a novel (13)arabinofuranosyltransferase

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis, Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum, with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT-C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG (13)-linked arabinofuranosyl (Araf) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, arabinofuranosyltransferase C (aftC). Enzyme assays using the sugar donor -D-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) and a newly synthesized linear (15)-linked Ara5 neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching (13) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target

Isolation of Mycobacterium tuberculosis complex (MTBC) from dairy cows in China

Eleven thousand five hundred and eighty non-blood samples from dairy cows were subjected to mycobacterium culture and genotyping. As a result, a total of 142 isolates of Mycobacterium tuberculosis complex (MBTC) were identified. Among them, 65 were Mycobacterium tuberculosis, while 77 Mycobacterium bovis. The genotype of M. tuberculosis strains was mainly Beijing family. In addition, the isolation rates of MTBC were 33.89% for lung lymph nodes, 2.81% for nasal swabs, and 3.95% for pharyngeal swabs from cattle positive to tuberculin skin test, respectively. This evidence implied that M. tuberculosis infection in cattle is a new risk to public health and should be paid more attention.Key words: Mycobacterium tuberculosis complex, cows, tuberculosis, zoonosis