Metabolic insights into HIV/TB co-infection: an untargeted urinary metabolomics approach.

INTRODUCTION: Amid the global health crisis, HIV/TB co-infection presents significant challenges, amplifying the burden on patients and healthcare systems alike. Metabolomics offers an innovative window into the metabolic disruptions caused by co-infection, potentially improving diagnosis and treatment monitoring. AIM: This study uses untargeted metabolomics to investigate the urinary metabolic signature of HIV/TB co-infection, enhancing understanding of the metabolic interplay between these infections. METHODS: Urine samples from South African adults, categorised into four groups - healthy controls, TB-positive, HIV-positive, and HIV/TB co-infected - were analysed using GCxGC-TOFMS. Metabolites showing significant differences among groups were identified through Kruskal-Wallis and Wilcoxon rank sum tests. RESULTS: Various metabolites (n = 23) were modulated across the spectrum of health and disease states represented in the cohorts. The metabolomic profiles reflect a pronounced disruption in biochemical pathways involved in energy production, amino acid metabolism, gut microbiome, and the immune response, suggesting a bidirectional exacerbation between HIV and TB. While both diseases independently perturb the host's metabolism, their co-infection leads to a unique metabolic phenotype, indicative of an intricate interplay rather than a simple additive effect. CONCLUSION: Metabolic profiling revealed a unique metabolic landscape shaped by HIV/TB co-infection. The findings highlight the potential of urinary differential metabolites for co-infection, offering a non-invasive tool for enhancing diagnostic precision and tailoring therapeutic interventions. Future research should focus on expanding sample sizes and integrating longitudinal analyses to build upon these foundational insights, paving the way for metabolomic applications in combating these concurrent pandemics.

Proteomic analysis of granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex by MALDI imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

Betaine and I-LG may have a predictive value for ATB: A causal study in a large European population.

PURPOSE: To analyze the causal relationship between 486 human serum metabolites and the active tuberculosis (ATB) in European population. METHODS: In this study, the causal relationship between human serum metabolites and the ATB was analyzed by integrating the genome-wide association study (GWAS). The 486 human serum metabolites were used as the exposure variable, three different ATB GWAS databases in the European population were set as outcome variables, and single nucleotide polymorphisms were used as instrumental variables for Mendelian Randomization. The inverse variance weighting was estimated causality, the MR-Egger intercept to estimate horizontal pleiotropy, and the combined effects of metabolites were also considered in the meta-analysis. Furthermore, the web-based MetaboAnalyst 6.0 was engaged for enrichment pathway analysis, while R (version 4.3.2) software and Review Manager 5.3 were employed for statistical analysis. RESULTS: A total of 21, 17, and 19 metabolites strongly associated with ATB were found in the three databases after preliminary screening (P < 0.05). The intersecting metabolites across these databases included tryptophan, betaine, 1-linoleoylglycerol (1-monolinolein) (1-LG), 1-eicosatrienoylglycerophosphocholine, and oleoylcarnitine. Among them, betaine (I2 = 24%, P = 0.27) and 1-LG (I2 = 0%, P = 0.62) showed the lowest heterogeneity among the different ATB databases. In addition, the metabolic pathways of phosphatidylethanolamine biosynthesis (P = 0.0068), methionine metabolism (P = 0.0089), betaine metabolism (P = 0.0205) and oxidation of branched-chain fatty acids (P = 0.0309) were also associated with ATB. CONCLUSION: Betaine and 1-LG may be biomarkers or auxiliary diagnostic tools for ATB. They may provide new guidance for medical practice in the early diagnosis and surveillance of ATB. In addition, by interfering with phosphatidylethanolamine biosynthesis, methionine metabolism, betaine metabolism, oxidation of branched-chain fatty acids, and other pathways, it is helpful to develop new anti-tuberculosis drugs and explore the virulence or pathogenesis of ATB at a deeper level, providing an effective reference for future studies.

Discovery of urinary biosignatures for tuberculosis and nontuberculous mycobacteria classification using metabolomics and machine learning.

Nontuberculous mycobacteria (NTM) infection diagnosis remains a challenge due to its overlapping clinical symptoms with tuberculosis (TB), leading to inappropriate treatment. Herein, we employed noninvasive metabolic phenotyping coupled with comprehensive statistical modeling to discover potential biomarkers for the differential diagnosis of NTM infection versus TB. Urine samples from 19 NTM and 35 TB patients were collected, and untargeted metabolomics was performed using rapid liquid chromatography-mass spectrometry. The urine metabolome was analyzed using a combination of univariate and multivariate statistical approaches, incorporating machine learning. Univariate analysis revealed significant alterations in amino acids, especially tryptophan metabolism, in NTM infection compared to TB. Specifically, NTM infection was associated with upregulated levels of methionine but downregulated levels of glutarate, valine, 3-hydroxyanthranilate, and tryptophan. Five machine learning models were used to classify NTM and TB. Notably, the random forest model demonstrated excellent performance [area under the receiver operating characteristic (ROC) curve greater than 0.8] in distinguishing NTM from TB. Six potential biomarkers for NTM infection diagnosis, including methionine, valine, glutarate, 3-hydroxyanthranilate, corticosterone, and indole-3-carboxyaldehyde, were revealed from univariate ROC analysis and machine learning models. Altogether, our study suggested new noninvasive biomarkers and laid a foundation for applying machine learning to NTM differential diagnosis.

Epigenetic modulation of cytokine expression in Mycobacterium tuberculosis-infected monocyte derived-dendritic cells: Implications for tuberculosis diagnosis.

BACKGROUND: To delineate alterations in DNA methylation at high resolution within the genomic profile of monocyte-derived-dendritic cells (mo-DCs) in connection with Mycobacterium tuberculosis (MTB) infection, with particular emphasis on pro/ anti-inflammatory genes. METHODS: In the context of this investigation, mo-DCs were infected by various active strains of MTB (Rifampicin-resistant [RIFR], H37Rv, multidrug-resistant [MDR], and extensively drug-resistant [XDR]). Subsequently, the pro/anti-inflammatory hub gene expression levels within the IL-6, IL-12, IFN-γ, IL-1ß, TNF-α, and IL-10 pathways were evaluated employing real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, the effects of MTB infection on mo-DC protein expression were examined through western blot analysis. The methylation status (%) of TNF-α and IL-10 was considered through Methylation Sensitive-High Resolution Melting (MS-HRM). RESULTS: The results revealed an up-regulation of all pro-inflammatory genes among all groups, with TNF-α exhibiting the highest expression level. Conversely, the anti-inflammatory gene (IL-10) showed a down-regulated expression level. Furthermore, the DNA methylation status (%) of TNF-α decreased significantly among all the groups (P < 0.001), although there were no notable distinctions in the DNA methylation status (%) of IL-10 when compared to the control group (P > 0.05). CONCLUSION: MTB infection induces DNA methylation changes in mo-DCs. The hypo-methylation of TNF-α may induce the up-regulation of this gene. This correlation revealed that the more resistant the MTB strain (XDR) is, the lower the methylation status (%) in the TNF-α gene.

Role of Type I Interferons during Mycobacterium tuberculosis and HIV Infections.

Tuberculosis and AIDS remain two of the most relevant human infectious diseases. The pathogens that cause them, Mycobacterium tuberculosis (Mtb) and HIV, individually elicit an immune response that treads the line between beneficial and detrimental to the host. Co-infection further complexifies this response since the different cytokines acting on one infection might facilitate the dissemination of the other. In these responses, the role of type I interferons is often associated with antiviral mechanisms, while for bacteria such as Mtb, their importance and clinical relevance as a suitable target for manipulation are more controversial. In this article, we review the recent knowledge on how these interferons play distinct roles and sometimes have opposite consequences depending on the stage of the pathogenesis. We highlight the dichotomy between the acute and chronic infections displayed by both infections and how type I interferons contribute to an initial control of each infection individually, while their chronic induction, particularly during HIV infection, might facilitate Mtb primo-infection and progression to disease. We expect that further findings and their systematization will allow the definition of windows of opportunity for interferon manipulation according to the stage of infection, contributing to pathogen clearance and control of immunopathology.

Significance of extracellular vesicles in orchestration of immune responses in Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis (M.tb), the causative agent of Tuberculosis, is an intracellular bacterium well known for its ability to subvert host energy and metabolic pathways to maintain its intracellular survival. For this purpose, the bacteria utilize various mechanisms of which extracellular vehicles (EVs) related mechanisms attracted more attention. EVs are nanosized particles that are released by almost all cell types containing active biomolecules from the cell of origin and can target bioactive pathways in the recipient cells upon uptake. It is hypothesized that M.tb dictates the processes of host EV biogenesis pathways, selectively incorporating its molecules into the host EV to direct immune responses in its favor. During infection with Mtb, both mycobacteria and host cells release EVs. The composition of these EVs varies over time, influenced by the physiological and nutritional state of the host environment. Additionally, different EV populations contribute differently to the pathogenesis of disease at various stages of illness participating in a complex interplay between host cells and pathogens. These interactions ultimately influence immune responses and disease outcomes. However, the precise mechanisms and roles of EVs in pathogenicity and disease outcomes remain to be fully elucidated. In this review, we explored the properties and function of EVs in the context of M.tb infection within the host microenvironment and discussed their capacity as a novel therapeutic strategy to combat tuberculosis.

The impact of Mycobacterium tuberculosis on the macrophage cholesterol metabolism pathway.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen capable of adapting and surviving within macrophages, utilizing host nutrients for its growth and replication. Cholesterol is the main carbon source during the infection process of Mtb. Cholesterol metabolism in macrophages is tightly associated with cell functions such as phagocytosis of pathogens, antigen presentation, inflammatory responses, and tissue repair. Research has shown that Mtb infection increases the uptake of low-density lipoprotein (LDL) and cholesterol by macrophages, and enhances de novo cholesterol synthesis in macrophages. Excessive cholesterol is converted into cholesterol esters, while the degradation of cholesterol esters in macrophages is inhibited by Mtb. Furthermore, Mtb infection suppresses the expression of ATP-binding cassette (ABC) transporters in macrophages, impeding cholesterol efflux. These alterations result in the massive accumulation of cholesterol in macrophages, promoting the formation of lipid droplets and foam cells, which ultimately facilitates the persistent survival of Mtb and the progression of tuberculosis (TB), including granuloma formation, tissue cavitation, and systemic dissemination. Mtb infection may also promote the conversion of cholesterol into oxidized cholesterol within macrophages, with the oxidized cholesterol exhibiting anti-Mtb activity. Recent drug development has discovered that reducing cholesterol levels in macrophages can inhibit the invasion of Mtb into macrophages and increase the permeability of anti-tuberculosis drugs. The development of drugs targeting cholesterol metabolic pathways in macrophages, as well as the modification of existing drugs, holds promise for the development of more efficient anti-tuberculosis medications.

Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis.

During tuberculosis (TB), migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell-mediated immunity. However, the mechanisms involved in the delayed migration of DCs during TB are still poorly defined. Here, we found that infection of DCs with Mycobacterium tuberculosis (Mtb) triggers HIF1A-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase inhibitor oxamate and the HIF1A inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB.

Longitudinal metabolomics of human plasma reveal metabolic dynamics and predictive markers of antituberculosis drug-induced liver injury.

Tuberculosis (TB) remains the second leading cause of death from a single infectious agent and long-term medication could lead to antituberculosis drug-induced liver injury (ATB-DILI). We established a prospective longitudinal cohort of ATB-DILI with multiple timepoint blood sampling and used untargeted metabolomics to analyze the metabolic profiles of 107 plasma samples from healthy controls and newly diagnosed TB patients who either developed ATB-DILI within 2 months of anti-TB treatment (ATB-DILI subjects) or completed their treatment without any adverse drug reaction (ATB-Ctrl subjects). The untargeted metabolome revealed that 77 metabolites (of 895 total) were significantly changed with ATB-DILI progression. Among them, levels of multiple fatty acids and bile acids significantly increased over time in ATB-DILI subjects. Meanwhile, metabolites of the same class were highly correlated with each other and pathway analysis indicated both fatty acids metabolism and bile acids metabolism were up-regulated with ATB-DILI progression. The targeted metabolome further validated that 5 fatty acids had prediction capability at the early stage of the disease and 6 bile acids had a better diagnostic performance when ATB-DILI occurred. These findings provide evidence indicating that fatty acids metabolism and bile acids metabolism play a vital role during ATB-DILI progression. Our report adds a dynamic perspective better to understand the pathological process of ATB-DILI in clinical settings.

The function of CD36 in Mycobacterium tuberculosis infection.

CD36 is a scavenger receptor that has been reported to function as a signaling receptor that responds to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and could integrate metabolic pathways and cell signaling through its dual functions. Thereby influencing activation to regulate the immune response and immune cell differentiation. Recent studies have revealed that CD36 plays critical roles in the process of lipid metabolism, inflammatory response and immune process caused by Mycobacterium tuberculosis infection. This review will comprehensively investigate CD36's functions in lipid uptake and processing, inflammatory response, immune response and therapeutic targets and biomarkers in the infection process of M. tuberculosis. The study also raised outstanding issues in this field to designate future directions.

Distributable, metabolic PET reporting of tuberculosis.

Tuberculosis remains a large global disease burden for which treatment regimens are protracted and monitoring of disease activity difficult. Existing detection methods rely almost exclusively on bacterial culture from sputum which limits sampling to organisms on the pulmonary surface. Advances in monitoring tuberculous lesions have utilized the common glucoside [18F]FDG, yet lack specificity to the causative pathogen Mycobacterium tuberculosis (Mtb) and so do not directly correlate with pathogen viability. Here we show that a close mimic that is also positron-emitting of the non-mammalian Mtb disaccharide trehalose - 2-[18F]fluoro-2-deoxytrehalose ([18F]FDT) - is a mechanism-based reporter of Mycobacteria-selective enzyme activity in vivo. Use of [18F]FDT in the imaging of Mtb in diverse models of disease, including non-human primates, successfully co-opts Mtb-mediated processing of trehalose to allow the specific imaging of TB-associated lesions and to monitor the effects of treatment. A pyrogen-free, direct enzyme-catalyzed process for its radiochemical synthesis allows the ready production of [18F]FDT from the most globally-abundant organic 18F-containing molecule, [18F]FDG. The full, pre-clinical validation of both production method and [18F]FDT now creates a new, bacterium-selective candidate for clinical evaluation. We anticipate that this distributable technology to generate clinical-grade [18F]FDT directly from the widely-available clinical reagent [18F]FDG, without need for either custom-made radioisotope generation or specialist chemical methods and/or facilities, could now usher in global, democratized access to a TB-specific PET tracer.

Type I interferon exacerbates Mycobacterium tuberculosis induced human macrophage death.

Type I interferons (IFN-I) are implicated in exacerbation of tuberculosis (TB), but the mechanisms are unclear. Mouse macrophages infected with Mycobacterium tuberculosis (Mtb) produce IFN-I, which contributes to their death. Here we investigate whether the same is true for human monocyte-derived macrophages (MDM). MDM prepared by a conventional method markedly upregulate interferon-stimulated genes (ISGs) upon Mtb infection, while MDM prepared to better restrict Mtb do so much less. A mixture of antibodies inhibiting IFN-I signaling prevents ISG induction. Surprisingly, secreted IFN-I are undetectable until nearly two days after ISG induction. These same antibodies do not diminish Mtb-infected MDM death. MDM induce ISGs in response to picogram/mL levels of exogenous IFN-I while depleting similar quantities from the medium. Exogenous IFN-I increase the proportion of dead MDM. We speculate that Mtb-infected MDM produce and respond to minute levels of IFN-I, and that only some of the resultant signaling is susceptible to neutralizing antibodies. Many types of cells may secrete IFN-I in patients with TB, where IFN-I is likely to promote the death of infected macrophages.

Role of PE/PPE proteins of Mycobacterium tuberculosis in triad of host mitochondria, oxidative stress and cell death.

The PE and PPE family proteins of Mycobacterium tuberculosis (Mtb) is exclusively found in pathogenic Mycobacterium species, comprising approximately 8-10 % of the Mtb genome. These emerging virulent factors have been observed to play pivotal roles in Mtb pathogenesis and immune evasion through various strategies. These immunogenic proteins are known to modulate the host immune response and cell-death pathways by targeting the powerhouse of the cell, the mitochondria to support Mtb survival. In this article, we are focused on how PE/PPE family proteins target host mitochondria to induce mitochondrial perturbations, modulate the levels of cellular ROS (Reactive oxygen species) and control cell death pathways. We observed that the time of expression of these proteins at different stages of infection is crucial for elucidating their impact on the cell death pathways and eventually on the outcome of infection. This article focuses on understanding the contributions of the PE/PPE proteins by unravelling the triad of host mitochondria, oxidative stress and cell death pathways that facilitate the Mtb persistence. Understanding the role of these proteins in host cellular pathways and the intricate mechanisms paves the way for the development of novel therapeutic strategies to combat TB infections.

Mycobacterium tuberculosis infection induces a novel type of cell death: Ferroptosis.

Ferroptosis is a lipid peroxidation-driven and iron-dependent form of programmed cell death, which is involved in a variety of physical processes and multiple diseases. Numerous reports have demonstrated that ferroptosis is closely related to the pathophysiological processes of Mycobacterium tuberculosis (M. tuberculosis) infection and is characterized by the accumulation of excess lipid peroxides on the cell membrane. In this study, the various functions of ferroptosis, and the therapeutic strategies and diagnostic biomarkers of tuberculosis, were summarized. Notably, this review provides insights into the molecular mechanisms and functions of M. tuberculosis-induced ferroptosis, suggesting potential future therapeutic and diagnostic markers for tuberculosis.

The hsa_circ_0002371/hsa-miR-502-5p/ATG16L1 axis modulates the survival of intracellular Mycobacterium tuberculosis and autophagy in macrophages.

Circular RNAs (circRNAs) play a critical role in pathological mechanisms of Mycobacterium tuberculosis (Mtb) and can be used as a new biomarker for active tuberculosis (ATB) diagnosis. Therefore, we identified significantly dysregulated circRNAs in ATB patients and healthy controls (HC) and explored their molecular mechanism. We found that hsa_circ_0002371 was significantly up-regulated in PBMCs of ATB patients and Mycobacterium tuberculosis H37Rv- or Mycobacterium bovis bacillus Calmette Guerin (BCG)-infected THP-1 cells. Functional experiments demonstrated that hsa_circ_0002371 inhibited autophagy in BCG-infected THP-1 cells and promoted intracellular BCG survival rate. In terms of mechanism, hsa_circ_0002371 facilitated the expression of hsa-miR-502-5p, as shown by bioinformatics and dual-luciferase reporter gene analysis, respectively. Notably, hsa-miR-502-5p inhibited autophagy via suppressing autophagy related 16 like 1 (ATG16L1) in BCG-infected macrophages and thus promoting intracellular BCG growth. In summation, hsa_circ_0002371 increased the suppression of hsa-miR-502-5p on ATG16L1 and inhibited autophagy to promote Mtb growth in macrophages. In Conclusion, our data suggested that hsa_circ_0002371 was significantly up-regulated in the PBMCs of ATB patients compared with HC. The hsa_circ_0002371/hsa-miR-502-5p/ATG16L1 axis promoted the survival of intracellular Mtb and inhibited autophagy in macrophages. Our findings suggested hsa_circ_0002371 could act as a potential diagnostic biomarker and therapeutic target.

The Structural and Molecular Mechanisms of Mycobacterium tuberculosis Translational Elongation Factor Proteins.

Targeting translation factor proteins holds promise for developing innovative anti-tuberculosis drugs. During protein translation, many factors cause ribosomes to stall at messenger RNA (mRNA). To maintain protein homeostasis, bacteria have evolved various ribosome rescue mechanisms, including the predominant trans-translation process, to release stalled ribosomes and remove aberrant mRNAs. The rescue systems require the participation of translation elongation factor proteins (EFs) and are essential for bacterial physiology and reproduction. However, they disappear during eukaryotic evolution, which makes the essential proteins and translation elongation factors promising antimicrobial drug targets. Here, we review the structural and molecular mechanisms of the translation elongation factors EF-Tu, EF-Ts, and EF-G, which play essential roles in the normal translation and ribosome rescue mechanisms of Mycobacterium tuberculosis (Mtb). We also briefly describe the structure-based, computer-assisted study of anti-tuberculosis drugs.

Dual neutrophil subsets exacerbate or suppress inflammation in tuberculosis via IL-1ß or PD-L1.

Neutrophils can be beneficial or deleterious during tuberculosis (TB). Based on the expression of MHC-II and programmed death ligand 1 (PD-L1), we distinguished two functionally and transcriptionally distinct neutrophil subsets in the lungs of mice infected with mycobacteria. Inflammatory [MHC-II-, PD-L1lo] neutrophils produced inflammasome-dependent IL-1ß in the lungs in response to virulent mycobacteria and "accelerated" deleterious inflammation, which was highly exacerbated in IFN-γR-/- mice. Regulatory [MHC-II+, PD-L1hi] neutrophils "brake" inflammation by suppressing T-cell proliferation and IFN-γ production. Such beneficial regulation, which depends on PD-L1, is controlled by IFN-γR signaling in neutrophils. The hypervirulent HN878 strain from the Beijing genotype curbed PD-L1 expression by regulatory neutrophils, abolishing the braking function and driving deleterious hyperinflammation in the lungs. These findings add a layer of complexity to the roles played by neutrophils in TB and may explain the reactivation of this disease observed in cancer patients treated with anti-PD-L1.

Serine protease Rv2569c facilitates transmission of Mycobacterium tuberculosis via disrupting the epithelial barrier by cleaving E-cadherin.

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.

Metabolic changes enhance necroptosis of type 2 diabetes mellitus mice infected with Mycobacterium tuberculosis.

Previously, we found that Mycobacterium tuberculosis (Mtb) infection in type 2 diabetes mellitus (T2DM) mice enhances inflammatory cytokine production which drives pathological immune responses and mortality. In the current study, using a T2DM Mtb infection mice model, we determined the mechanisms that make T2DM mice alveolar macrophages (AMs) more inflammatory upon Mtb infection. Among various cell death pathways, necroptosis is a major pathway involved in inflammatory cytokine production by T2DM mice AMs. Anti-TNFR1 antibody treatment of Mtb-infected AMs from T2DM mice significantly reduced expression of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) (necroptosis markers) and IL-6 production. Metabolic profile comparison of Mtb-infected AMs from T2DM mice and Mtb-infected AMs of nondiabetic control mice indicated that 2-ketohexanoic acid and deoxyadenosine monophosphate were significantly abundant, and acetylcholine and pyridoxine (Vitamin B6) were significantly less abundant in T2DM mice AMs infected with Mtb. 2-Ketohexanoic acid enhanced expression of TNFR1, RIPK3, MLKL and inflammatory cytokine production in the lungs of Mtb-infected nondiabetic mice. In contrast, pyridoxine inhibited RIPK3, MLKL and enhanced expression of Caspase 3 (apoptosis marker) in the lungs of Mtb-infected T2DM mice. Our findings demonstrate that metabolic changes in Mtb-infected T2DM mice enhance TNFR1-mediated necroptosis of AMs, which leads to excess inflammation and lung pathology.