Invariant natural killer T cells recognize glycolipids from pathogenic Gram-positive bacteria.

Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.

Anti-leishmanial, anti-inflammatory and antimicrobial activities of phenolic derivatives from Tibouchina paratropica.

A new phenolic derivative, 2,8-dihydroxy-7H-furo[2,3-f]chromen-7-one (1), together with isoquercitrin (2), was isolated from the aerial parts of Tibouchina paratropica. Compound structures were elucidated by spectroscopic methods. Both compounds show antimicrobial activity towards a panel of bacterial and fungal pathogens, and compound 1 displayed potent anti-parasitic activity against Leishmania donovani (IC50 = 0.809 µg/mL). In addition, an 85% reduction in the secretion of the pro-inflammatory cytokine IL-6 was recorded when macrophages challenged with lipopolysaccharide were exposed to compound 1, but no effect on the anti-inflammatory IL-10 was observed. Compound 2 showed neither anti-parasitic nor anti-inflammatory properties. In addition, no cytotoxic activities were observed against the human-derived macrophage THP-1 cells.

Sociodemographic factors associated with the success or failure of anti-tuberculosis treatment in the Chiapas Highlands, Mexico, 2019-2022.

OBJECTIVE: To 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. METHODS: Retrospective 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. RESULTS: Two 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. CONCLUSIONS: It 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.

The mycobacterial transcriptional regulator whiB7 gene links redox homeostasis and intrinsic antibiotic resistance.

Intrinsic drug resistance in Mycobacterium tuberculosis limits therapeutic options for treating tuberculosis. The mycobacterial transcriptional regulator whiB7 contributes to intrinsic resistance by activating its own expression and many drug resistance genes in response to antibiotics. To investigate whiB7 activation, we constructed a GFP reporter to monitor its expression, and we used it to investigate the whiB7 promoter and to screen our custom library of almost 600 bioactive compounds, including the majority of clinical antibiotics. Results showed whiB7 was transcribed from a promoter that was conserved across mycobacteria and other actinomycetes, including an AT-rich sequence that was likely targeted by WhiB7. Expression was induced by compounds having diverse structures and targets, independent of the ability of whiB7 to mediate resistance, and was dependent on media composition. Pretreatment with whiB7 activators resulted in clinically relevant increases in intrinsic drug resistance. Antibiotic-induced transcription was synergistically increased by the reductant dithiothreitol, an effect mirrored by a whiB7-dependent shift to a highly reduced cytoplasm reflected by the ratio of reduced/oxidized mycothiol. These data provided evidence that intrinsic resistance resulting from whiB7 activation is linked to fundamental changes in cell metabolism.

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.

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.

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.

Genetic Diversity of Mycobacterium tuberculosis Isolates From an Amerindian Population in Chiapas, México.

This is the first report of the genetic diversity of the Mycobacterium tuberculosis complex isolates found in a Mexican-Amerindian setting. In this study, we analyzed isolates collected from the Highlands region of Chiapas, Mexico, by using spoligotyping and whole-genome sequencing analyses. Seventy-three M. tuberculosis isolates were analyzed initially by spoligotyping; no new spoligotypes were identified. Nineteen percent of the isolates were identified as SIT53 (T1) (n = 14), followed by SIT42 (14%, n = 10, LAM9) and SIT119 (11%; n = 8, X1). SIT53, SIT42, and orphan isolates (16.4%, n = 12) constituted about 50% of the isolates studied and were subjected to whole-genome sequencing (WGS) analysis. Most SIT53 (10/12) isolates belonged to the Euro-American sub-lineage 4.8. Most SIT42 isolates (4/7) as .well as most orphan isolates (5/8) belonged to the lineage 4.3.3 LAM group. By comparing the single-nucleotide polymorphism (SNP) patterns of the SIT53 isolates, we found one clone (<7 SNPs) and four clustered isolates (<15 SNPs). In isolates from the SIT42 and orphan groups, we did not find any clones or clusters. This work demonstrates the success of sub-lineage 4.8 to predominate in Mexico and confirms the dominion of sub-lineage 4.3.3 in Central and South America.

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.

Magnetic Nanoparticle-Based Biosensing Assay Quantitatively Enhances Acid-Fast Bacilli Count in Paucibacillary Pulmonary Tuberculosis.

A new method using a magnetic nanoparticle-based colorimetric biosensing assay (NCBA) was compared with sputum smear microscopy (SSM) for the detection of pulmonary tuberculosis (PTB) in sputum samples. Studies were made to compare the NCBA against SSM using sputum samples collected from PTB patients prior to receiving treatment. Experiments were also conducted to determine the appropriate concentration of glycan-functionalized magnetic nanoparticles (GMNP) used in the NCBA and to evaluate the optimal digestion/decontamination solution to increase the extraction, concentration and detection of acid-fast bacilli (AFB). The optimized NCBA consisted of a 1:1 mixture of 0.4% NaOH and 4% N-acetyl-L-cysteine (NALC) to homogenize the sputum sample. Additionally, 10 mg/mL of GMNP was added to isolate and concentrate the AFB. All TB positive sputum samples were identified with an increased AFB count of 47% compared to SSM, demonstrating GMNP's ability to extract and concentrate AFB. Results showed that NCBA increased AFB count compared to SSM, improving the grade from "1+" (in SSM) to "2+". Extending the finding to paucibacillary cases, there is the likelihood of a "scant" grade to become "1+". The assay uses a simple magnet and only costs $0.10/test. NCBA has great potential application in TB control programs.

Ergothioneine Maintains Redox and Bioenergetic Homeostasis Essential for Drug Susceptibility and Virulence of Mycobacterium tuberculosis.

The mechanisms by which Mycobacterium tuberculosis (Mtb) maintains metabolic equilibrium to survive during infection and upon exposure to antimycobacterial drugs are poorly characterized. Ergothioneine (EGT) and mycothiol (MSH) are the major redox buffers present in Mtb, but the contribution of EGT to Mtb redox homeostasis and virulence remains unknown. We report that Mtb WhiB3, a 4Fe-4S redox sensor protein, regulates EGT production and maintains bioenergetic homeostasis. We show that central carbon metabolism and lipid precursors regulate EGT production and that EGT modulates drug sensitivity. Notably, EGT and MSH are both essential for redox and bioenergetic homeostasis. Transcriptomic analyses of EGT and MSH mutants indicate overlapping but distinct functions of EGT and MSH. Last, we show that EGT is critical for Mtb survival in both macrophages and mice. This study has uncovered a dynamic balance between Mtb redox and bioenergetic homeostasis, which critically influences Mtb drug susceptibility and pathogenicity.

Exploring the biological activities of Echeveria leucotricha.

Echeveria leucotricha J. A. Purpus (Crassulaceae) was evaluated for its potential antibacterial, antifungal, antiparasitic, cytotoxic and anti-inflammatory bioactivities. Aerial parts were extracted with hexane, methanol and chloroform, and fractionated accordingly. Biological activity was assessed in vitro against five Gram-positive and four Gram-negative bacteria, four human pathogenic fungi and the protozoan Leishmania donovani. Extracts and fractions showing bioactivities were further investigated for their cytotoxic activities on macrophages. Results show that several extracts and fractions exhibited significant antibacterial, antifungal, and antiparasitic activities, but no anti-inflammatory activity was recorded. Here, we report for the first time, and to the best of our knowledge, these bioactivities, which suggest that this plant can be used in the traditional Mexican medicine.