Studies in mycobactin biosynthesis

Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a single bacterial pathogen, $Mycobacterium$ $tuberculosis$. Current TB chemotherapy remains useful in treating susceptible M. tuberculosis strains, however, the emergence of MDR-TB and XDR-TB demand the development of new drugs. Enzymes involved in mycobactin biosynthesis, low molecular weight iron chelators, do not have mammalian homologues; therefore they are considered potential targets for the development of new anti-TB drugs. The aims of this study were to identify potential inhibitors and to investigate the function of the $mbtG$ and $AmbtE$ and $AMbtF$ genes during mycobactin biosynthesis. The full length of $mbtB$ and the ArCP domain were successfully cloned and post-translationally modified by MtaA, a broad phosphopantetheinyl transferase from $Stigmatella$ $aurantiaca$, using $Escherichia$ $coli$. Inhibitors identified by virtual screening as well as 13 chemically synthesised PAS analogues were initially investigated in whole-cell assay against $Mycobacterium$ $bovis$ BCG Pasteur. Seven of these compounds had interesting growth inhibition under ironsufficient conditions. The $mbtA$ gene was cloned and expressed as soluble protein using $Mycobacterium$ $smegmatis$ mc2155. Preliminary $in$ $vitro$ MbtA assays provided hints of its activity, although, the $KM$ for SAL and ATP have not been determined yet. The $mbtG$ and $ambtE$ genes have been cloned and expressed in E. coli to further investigate their biochemical function in mycobactin biosynthesis

Tween 80 Improves the Acid-Fast Bacilli Quantification in the Magnetic Nanoparticle-Based Colorimetric Biosensing Assay (NCBA)

Despite its reduced sensitivity, sputum smear microscopy (SSM) remains the main diagnostic test for detecting tuberculosis in many parts of the world. A new diagnostic technique, the magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was optimized by evaluating different concentrations of glycan-functionalized magnetic nanoparticles (GMNP) and Tween 80 to improve the acid-fast bacilli (AFB) count. Comparative analysis was performed on 225 sputum smears: 30 with SSM, 107 with NCBA at different GMNP concentrations, and 88 with NCBA-Tween 80 at various concentrations and incubation times. AFB quantification was performed by adding the total number of AFB in all fields per smear and classified according to standard guidelines (scanty, 1+, 2+ and 3+). Smears by NCBA with low GMNP concentrations (≤1.5 mg/mL) showed higher AFB quantification compared to SSM. Cell enrichment of sputum samples by combining NCBA-GMNP, incubated with Tween 80 (5%) for three minutes, improved capture efficiency and increased AFB detection up to 445% over SSM. NCBA with Tween 80 offers the opportunity to improve TB diagnostics, mainly in paucibacillary cases. As this method provides biosafety with a simple and inexpensive methodology that obtains results in a short time, it might be considered as a point-of-care TB diagnostic method in regions where resources are limited

Disruption of the serine/threonine protein kinase H affects phthiocerol dimycocerosates synthesis in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses a complex cell wall that is unique and essential for interaction of the pathogen with its human host. Emerging evidence suggests that the biosynthesis of complex cell-wall lipids is mediated by serine/threonine protein kinases (STPKs). Herein, we show, using in vivo radiolabelling, MS and immunostaining analyses, that targeted deletion of one of the STPKs, pknH, attenuates the production of phthiocerol dimycocerosates (PDIMs), a major M. tuberculosis virulence lipid. Comparative protein expression analysis revealed that proteins in the PDIM biosynthetic pathway are differentially expressed in a deleted pknH strain. Furthermore, we analysed the composition of the major lipoglycans, lipoarabinomannan (LAM) and lipomannan (LM), and found a twofold higher LAM/LM ratio in the mutant strain. Thus, we provide experimental evidence that PknH contributes to the production and synthesis of M. tuberculosis cell-wall components

Precise Null Deletion Mutations of the Mycothiol Synthesis Genes Reveal Their Role in Isoniazid and Ethionamide Resistance in Mycobacterium smegmatis ▿

Mycothiol (MSH; AcCys-GlcN-Ins) is the glutathione analogue for mycobacteria. Mutations in MSH biosynthetic genes have been associated with resistance to isoniazid (INH) and ethionamide (ETH) in mycobacteria, but rigorous genetic studies are lacking, and those that have been conducted have yielded different results. In this study, we constructed independent null deletion mutants for all four genes involved in the MSH biosynthesis pathway (mshA, mshB, mshC, and mshD) in Mycobacterium smegmatis and made complementing constructs in integrating plasmids. The resulting set of strains was analyzed for levels of MSH, INH resistance, and ETH resistance. The mshA and mshC single deletion mutants were devoid of MSH production and resistant to INH, whereas the mshB deletion mutant produced decreased levels of MSH yet was sensitive to INH, suggesting that MSH biosynthesis is essential for INH susceptibility in M. smegmatis. Further evidence supporting this conclusion was generated by deleting the gene encoding the MSH S-conjugate amidase (mca) from the ΔmshB null mutant. This double mutant, ΔmshB Δmca, completely abolished MSH production and was resistant to INH. The mshA, mshC, and mshB single deletion mutants were also resistant to ETH, indicating that ETH resistance is modulated by the level of MSH in M. smegmatis. Surprisingly, the mshD deletion mutant lacked MSH production but was sensitive to both INH and ETH. The drug sensitivity was likely mediated by the compensated synthesis of N-formyl-Cys-GlcN-Ins, previously demonstrated to substitute for MSH in an mshD mutant of M. smegmatis. We conclude that MSH or N-formyl-Cys-GlcN-Ins is required for susceptibility to INH or ETH in M. smegmatis

S1 Data -

ObjectiveTo estimate the incidence rate of tuberculosis (TB) in the Highlands (Tsotsil-Tseltal) region of Chiapas and to analyze sociodemographic factors that might influence the success of anti-TB treatment from the period of January 2019 to June 2022.MethodsRetrospective study in which the TB databases of the National Epidemiological Surveillance System (SINAVE) were analyzed. TB incidence rates were calculated based on the number of registered TB cases and estimated annual populations. The success-failure of anti-TB treatment was analyzed according to sociodemographic indicators, degree of concentration of indigenous population of the municipality of residence and admission to SINAVE.ResultsTwo hundred thirty-three cases were analyzed. The variables associated to a lower success rate of treatment against TB were: living in a municipality with high-very high concentration of indigenous population, being indigenous, having a primary school education or lower, and agricultural occupation. The number of TB diagnosed from 2020–2022 and the incidence rates from 2020–2021 decreased significantly compared to 2019.ConclusionsIt is necessary to strengthen the follow-up of TB cases in the region, mainly in areas with high-very high indigenous concentration, in people with low levels of education and engaged in agricultural work.</div

Demographic and socioeconomic characteristics of the studied population (n = 233) registered with tuberculosis in the Highlands Region V Tsotsil-Tseltal of Chiapas, Mexico, during the period January 2019 to June 2022.

Demographic and socioeconomic characteristics of the studied population (n = 233) registered with tuberculosis in the Highlands Region V Tsotsil-Tseltal of Chiapas, Mexico, during the period January 2019 to June 2022.</p

Tuberculosis incidence rates per 100,000 inhabitants in the Highlands Region V Tsotsil-Tseltal of Chiapas, Mexico, period 2019–2021.

Tuberculosis incidence rates per 100,000 inhabitants in the Highlands Region V Tsotsil-Tseltal of Chiapas, Mexico, period 2019–2021.</p

Antimicrobial, Antiparasitic, Anti-Inflammatory, and Cytotoxic Activities of Lopezia racemosa

The present study investigates the potential benefits of the Mexican medicinal plant Lopezia racemosa (Onagraceae). Extracts and fractions from aerial parts of this plant were assessed to determine their antibacterial, antifungal, antiparasitic, anti-inflammatory and cytotoxic activities in vitro. Aerial parts of the plant were extracted with various solvents and fractionated accordingly. Extracts and fractions were tested against a panel of nine bacterial and four fungal species. The antiparasitic activity was tested against Leishmania donovani, whereas the anti-inflammatory activity of the compounds was determined by measuring the secretion of interleukin-6 from human-derived macrophages. The same macrophage cell line was used to investigate the cytotoxicity of the compounds. Various extracts and fractions showed antibacterial, antifungal, antiparasitic, and anti-inflammatory activities. The hexanic fraction HF 11-14b was the most interesting fraction with antimicrobial, and anti-inflammatory activities. The benefit of L. racemosa as a traditional medicinal plant was confirmed as shown by its antibacterial, antifungal and anti-inflammatory activities. To the best of our knowledge, this is the first study reporting the biological activities of L. racemosa, including antiparasitic and anti-inflammatory activities

Percentages of success and non-success of anti-tuberculosis treatment in people affected by tuberculosis in the Highlands Region V Tsotsil-Tseltal of Chiapas, Mexico, period 2019–2022^a.

Percentages of success and non-success of anti-tuberculosis treatment in people affected by tuberculosis in the Highlands Region V Tsotsil-Tseltal of Chiapas, Mexico, period 2019–2022a.</p