Tb H37Rv Research Articles

Targeted Hybridization: Pyrazine‐2‐Carbohydrazide Derivatives as Promising Antitubercular and Antimicrobial Leads

AbstractIn an attempt to identify a new pharmacological lead which should be more potent than the established drug pyrazinamide, a molecular hybridization technique was applied to fuse two pharmacophore moieties: pyrazine‐2‐carbohydrazide (1) and diphenyl ether/piperazinyl derivative (2), to isolate a new series of hybrid molecules. The synthesized compounds were tested in vitro for their antibacterial and antimycobacterial properties against E. coli and S. aureus and Mycobacterium TB H37Rv strains. In comparison to the reference medication (pyrazinamide) these compounds exhibited moderate antitubercular and antibacterial action, with a MIC value of 15.625–125 µg/mL. All the synthesized molecules were characterized through IR, NMR and mass spectrometry. Docking studies were carried out in order to have better understanding related to protein and ligand receptor interactions.

Significant elevated CXCL14 and decreased IL-39 levels in patients with tuberculosis.

To explore the serum levels of IL-39, CXCL14, and IL-19 in patients with tuberculosis (TB) along with their clinical significances and their concentration changes in macrophages after Bacille Calmette-Guérin vaccine (BCG) or Mycobacterium tuberculosis (M. tb) H37Rv stimulation in vitro. The serum levels of IL-39, CXCL14, and IL-19 of 38 TB patients, and 20 healthy staff members were measured by enzyme-linked immunosorbent assay. Moreover, the levels of IL-19, CXCL14, and IL-39 in cultured THP-1 macrophages were detected at 12, 24, and 48 h after stimulation with BCG or M. tb H37Rv strains. It was found the serum level of IL-39 was significantly reduced and CXCL14 was remarkably elevated in TB patients. In vitro, at 48 h after stimulation, IL-39 level of cultured THP-1 macrophages in the H37Rv group was significantly lower than that in the BCG and control groups, and the CXCL14 level of cultured THP-1 macrophages in the H37Rv stimulation group was remarkably higher than that in the control group. Therefore, IL-39 and CXCL14 may be involved the pathogenesis of TB, and serum IL-39 and CXCL14 could potentially serve as a new biomarker of TB.

Synthesis of New Derivatives of Leaf-Eating Ant Toxic Gland Pheromone Compounds and Investigation of their Biological Properties

‌Background: The anti-mycobacterial characteristics of 2-pyrazinoic acid esters have been discovered through recent research. Research has demonstrated that the pyrazine ring and the alkyl part of these compounds interact with the enzyme phytynetase, which in turn inhibits the interaction between NADPH and mycobacterial fatty acids synthase, the enzyme responsible for synthesizing the fatty acids required for the cell wall of mycobacteria. Materials and Methods: Targeted pyrazinoic acid molecules have been synthesized, their structure verified by spectroscopic data, and their anti-mycobacterial activity against Mycobacterium TB H37Rv at a dose of 10 micrograms per milliliter assessed in order to test this theory. Significant anti-mycobacterial capabilities were shown by several compounds, including 3c, 3J, and 3M, which inhibited mycobacterial growth by 45.7%, 45.4%, and 51%, respectively. Findings: The results showed that the compounds exhibited little toxicity and did not inhibit macrophage cell proliferation. Pyrazinamide had significant antibacterial and antifungal activity, despite its lack of fatal action against non-tubercular mycobacteria and both Gram-positive and Gram-negative bacteria. Compound 3B, for example, exhibited excellent antibacterial properties against Gram-positive bacteria, such as S. aureus, with a minimum inhibitory concentration (MIC) of 125 micrograms per milliliter. Conversely, the compounds showed equally potent antibacterial properties against Gram-negative bacteria. Interestingly, compared to Gram-positive bacteria, the ester produced from maltol exhibited greater antibacterial efficacy against Gram-negative bacteria. Compounds synthesized with biodegradable groups also exhibited time-dependent toxicity against K562 leukemia cancer cells in MTT measurements, with compounds 3e and 3J displaying IC50 values of 25 micromolars. Conclusion: After synthesizing pyrazinoic acid monoterpene esters, spectral data was used to confirm their structures. When their biological characteristics were assessed, toxicity studies against K562 leukemia cells produced encouraging results. Through the use of Thio Ester-mediated activation of 2-pyrazinoic acid by 2, 2-dibenzothiazole disulfide and triphenylphosphine, 6-aminopenicillanic acid (6-APA) was coupled with pyrazinoic acid.

Untargeted metabolomics analysis reveals Mycobacterium tuberculosis strain H37Rv specifically induces tryptophan metabolism in human macrophages

BackgroundTuberculosis (TB) caused by Mycobacterium tuberculosis (M. tb) remains a global health issue. The characterized virulent M. tb H37Rv, avirulent M. tb H37Ra and BCG strains are widely used as reference strains to investigate the mechanism of TB pathogenicity. Here, we attempted to determine metabolomic signatures associated with the Mycobacterial virulence in human macrophages through comparison of metabolite profile in THP-1-derived macrophages following exposure to the M. tb H37Rv, M. tb H37Ra and BCG strains.ResultsOur findings revealed remarkably changed metabolites in infected macrophages compared to uninfected macrophages. H37Rv infection specifically induced 247 differentially changed metabolites compared to H37Ra or BCG infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed H37Rv specifically induces tryptophan metabolism. Moreover, quantitative PCR (qPCR) results showed that indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2) which converts the tryptophan to a series of biologically second metabolites were up-regulated in H37Rv-infected macrophages compared to H37Ra- or BCG-infected macrophages, confirming the result of enhanced tryptophan metabolism induced by H37Rv infection. These findings indicated that targeting tryptophan (Trp) metabolism may be a potential therapeutic strategy for pulmonary TB.ConclusionsWe identified a number of differentially changed metabolites that specifically induced in H37Rv infected macrophages. These signatures may be associated with the Mycobacterial virulence in human macrophages. The present findings provide a better understanding of the host response associated with the virulence of the Mtb strain.

Synthesis of Substituted -N-(5-((7-Methyl-2-Oxo-2H-Chromen-4-yl)- Methyl)-1,3,4-Thiadiazol-2-yl)-Benzamide Derivatives Using TBTU as Coupling Agent and their Evaluation for Anti Tubercular Activity

Abstract: Tuberculosis remains a highly infectious disease across the world. In the identification of new antitubercular agents, coumarin clubbed thiadiazole amides have been synthesized and evaluated for in vitro antitubercular activity. Owing to the growing concern of chemicals and their impact on the environment, greener and faster reaction conditions needed to be incorporated. Therefore, we used TBTU as a coupling reagent for efficient and facile synthesis of substituted-N-(5-((7-methyl-2-oxo-2Hchromes- 4-yl)-methyl)-1,3, 4-thiadiazol-2-yl)-benzamide 4a-j with good yields up to 95% in mild reaction conditions. All the synthesized compounds were evaluated in vitro for anti-tubercular activity against the H37Rv strain of M. tuberculosis. Compounds 4c, 4d, and 4f were found active at 12.5 μg/mL against M. tb H37Rv. Electron withdrawing substituents present on aromatic side chains showed promising anti-tubercular activity.

Rv1258c acts as a drug efflux pump and growth controlling factor in Mycobacterium tuberculosis

The possible role of efflux pump as a survival mechanism in Mycobacterium tuberculosis (M. tb) is gaining an increasing attention. Previously, Rv1258c (Tap) and its certain mutations confer the clinically relevant drug resistance. In this study, we found new mutations of Rv1258c in G195C, T297P and I328T. Effect of modulating T297P and I328T on the drug resistance by knockout and complement in M. tb H37Rv showed that M. tb ΔRv1258c showed a slightly lower MIC for rifampin, ethambutol, ofloxacin, amikacin, capreomycin and streptomycin than M. tb H37Rv WT and the complement. Rv1258c T297P and Rv1258c I328T showed an increased drug resistance to ethambutol and capreomycin than the complement of Rv1258c WT. Most importantly, M. tb ΔRv1258c exhibited a slow growth in the normal culture medium. TMT-based quantitative proteomics analysis of M. tb ΔRv1258c and WT showed that the knockout of Rv1258c greatly down-regulated the expression of the ribosome system and one of the special five type VII secretion systems, ESX-3, which impaired the bacterial growth. These results indicate that the newly found T297P and I328T mutations of Rv1258c contributed to an increased resistance to ethambutol and capreomycin, and Rv1258c as growth controlling factor influencing the growth of M. tb.

Alkyltriphenylphosphonium turns naphthoquinoneimidazoles into potent membrane depolarizers against mycobacteria.

Due to its central role in energy generation and bacterial viability, mycobacterial bioenergetics is an attractive therapeutic target for anti-tuberculosis drug discovery. Building upon our work on antimycobacterial dioxonaphthoimidazoliums that were activated by a proximal positive charge and generated reactive oxygen species upon reduction by Type II NADH dehydrogenase, we herein studied the effect of a distal positive charge on the antimycobacterial activity of naphthoquinoneimidazoles by incorporating a trialkylphosphonium cation. The potency-enhancing properties of the linker length were affirmed by structure-activity relationship studies. The most active compound against M. tb H37Rv displayed good selectivity index (SI = 34) and strong bactericidal activity in the low micromolar range, which occurred through rapid bacterial membrane depolarization that resulted in depletion of intracellular ATP. Through this work, we demonstrated a switch of the scaffold's mode-of-action via relocation of positive charge while retaining its excellent antibacterial activity and selectivity.

The experimental study of TNF-α & CRP expression in the spinal tuberculosis after instrumentation

IntroductionPreviously, the management of spinal TB was using drugs and external stabilization. Surgical techniques were developed afterwards to clean the infected vertebral segment. The TB treatment approach is now based on immunology because the bacteria Mycobacterium tuberculosis has unique characteristics and the increasing cases of MDR (multiple drug resistant) TB due to mutation processes. TNF-α and CRP has a major role in immune activity of spinal TB. The energy from metal devices composed of ions and particles that have been used in instrumentation is expected to reduce the biomolecular and biocellular activity of the spinal tuberculosis inflammation activity. This study aims to investigate TNFα and CRP value as evaluator of bone inflammation activity in Spinal TB through experimental studies in Laboratory at Veterinary Faculty, Universitas Brawijaya. MethodsWe investigates 40 New Zealand Rabbits which were given TB H37Rv strain infection in the vertebral body. Samples were divided into five groups namely control rabbits, infected rabbits without intervention, infected rabbits treated by instrumentation, infected rabbits given anti-tuberculosis drugs and infected rabbits treated by instrumentation and given drugs. The cytokine levels of TNF-α and CRP were evaluated and compared as the main outcome. ResultThe results showed a notable TNF-α and CRP decrease in infected rabbits given drugs alone and instrumentation alone compared to infected rabbits without intervention. There was a significant TNF-α and CRP decrease in infected rabbits given drugs and treated by instrumentation compared to control rabbits and rabbits who received drugs only. ConclusionInstrumentation can reduce the inflammation activity in spinal tuberculosis by affecting the body's cytokine levels.

The Role of Instrumentation in the Healing Process of Spinal Tuberculosis: An Experimental Study

Introduction: Tuberculosis is still commonly found in many developing countries. Spinal tuberculosis can cause vertebral deformity and neurological disorders. It was discovered thousands years ago and its management was aimed to eradicate infection and maintain the integrity of the vertebrae. Previously, the management of spinal TB was using drugs and external stabilization. Surgical techniques were developed afterwards to clean the infected vertebral segment. Because of the vertebral deformity remained inevitable and had impacts on neurological disorders, new paradigm had been developed by using instrumentation to stabilize the deformity of infected vertebral segment and to restore and maintain neurological function. TGF-β has a major role in angiogenesis in bone healing process. Spinal TB instrumentation uses metal devices composed of ions and particles that can interact each other so it could produce physical and chemical energy that is transmitted to the vertebrae. The energy is expected to enhance the biomolecular and biocellular activity of the body's immune cells so the healing process could be better.
 Methods: An experimental study was carried out on New Zealand Rabbits which were given TB H37Rv strain infection in the vertebral body. Samples were divided into five groups namely control rabbits, infected rabbits without intervention, infected rabbits treated by instrumentation, infected rabbits given anti-tuberculosis drugs and infected rabbits treated by instrumentation and given drugs. Then the cytokine levels of TGF-β were evaluated and compared.
 Results: The results showed a significant TGF- β level increase in infected rabbits given drugs alone and instrumentation alone compared to infected rabbits without intervention. There was a significant TGF- β increase in infected rabbits given drugs and treated by instrumentation compared to control rabbits and rabbits who received drugs only.
 Conclusions: Instrumentation can improve the healing process in spinal tuberculosis by increasing the body's cytokine levels.

Contribution of N-heterocycles towards anti-tubercular drug discovery (2014-2019); predicted and reengineered molecular frameworks.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis, responsible for high death frequency every year all over the world. In this regard, efficient drug-design and discovery towards the prevention of M.tb H37 Rv is of prime concern. Prevention of the infection may include vaccination, and the treatment comprises anti-TB drug regimen. However, the vaccine decreases the risk of tuberculosis infection only to some extent, while drug-resistance limits the efficacy of the existing anti-TB agents. Much improvement has to be achieved to overcome pitfalls such as side effects, high-toxicity, low bioavailability, pharmacokinetics and pharmacodynamics, and hence forth in clinical therapeutics. Amongst heterocyclic compounds, N-heterocycles played a pivotal role in drug-design and discovery. A wide range of microbial diseases are being treated by the N-heterocyclic drugs. The present review comprises description of anti-TB effects of the N-heterocycles such as indoles, triazoles, thiazoles, and pyrazoles. The potent anti-TB activity exerted by the derivatives of these heterocycles is evaluated critically alongside emphasizing structure-activity relationship. Besides, docking studies supporting anti-TB activity is supplemented. Alongside this, based on the potent heterocyclic molecules, the molecular frameworks are designed that would bring about enhanced M. tb H37 Rv inhibitory potencies.

Activation of hypoxia-inducible factor 1 (Hif-1) enhanced bactericidal effects of macrophages to Mycobacterium tuberculosis

BackgroundTuberculosis is chronic infection caused by Mycobacterium tuberculosis (M.tb), which infects specifically macrophages. Hif-1, hypoxia-inducible factor-1, was reported to act as master regulator of killing functions in macrophages. AimTo investigate whether Hif-1 activation would enhance bactericidal effect of macrophages and anti-tuberculosis effect of chemical reagent. MethodsHif-1 and LC3B were detected in tissues from pulmonary tuberculosis. U937, human monocytic leukemia cell line, was stimulated with PMA and differentiated into macrophages. Cells were pretreated with Hif-1 chemical inhibitor YC-1, stimulated with CoCl2 (Hif-1 activator), to detect LC3B with Western blot and confocal microscopy. Cells were infected with M. tb H37Rv strain, stimulated with CoCl2, following rifampine treatment. Expression of autophagy markers was detected using Western blot. IL-6 and TNF-α were detected in cell supernatant with ELISA. Acid-fast staining and CFU assay were performed to evaluate intracellular bacterial load. Results and conclusionsHif-1 and LC3B increased in tissues of pulmonary tuberculosis. Hif-1 activation enhanced autophagy in M. tb infected U937 cells and production of IL-6 and TNF-α. Data from acid-fast staining and CFU indicated that Hif-1 activation enhanced anti-tuberculosis effect of rifampine in macrophages. Conclusively, to activate Hif-1 would strengthen bactericidal effect of macrophages, to further enhance anti-tuberculosis effect of chemical reagent.

Design, synthesis and antimycobacterial evaluation of novel adamantane and adamantanol analogues effective against drug-resistant tuberculosis

The treacherous nature of tuberculosis (TB) combined with the ubiquitous presence of the drug-resistant (DR) forms pose this disease as a growing public health menace. Therefore, it is imperative to develop new chemotherapeutic agents with a novel mechanism of action to circumvent the cross-resistance problems. The unique architecture of the Mycobacterium tuberculosis (M. tb) outer envelope plays a predominant role in its pathogenesis, contributing to its intrinsic resistance against available therapeutic agents. The mycobacterial membrane protein large 3 (MmpL3), which is a key player in forging the M. tb rigid cell wall, represents an emerging target for TB drug development. Several indole-2-carboxamides were previously identified in our group as potent anti-TB agents that act as inhibitor of MmpL3 transporter protein. Despite their highly potent in vitro activities, the lingering Achilles heel of these indoleamides can be ascribed to their high lipophilicity as well as low water solubility. In this study, we report our attempt to improve the aqueous solubility of these indole-2-carboxamides while maintaining an adequate lipophilicity to allow effective M. tb cell wall penetration. A more polar adamantanol moiety was incorporated into the framework of several indole-2-carboxamides, whereupon the corresponding analogues were tested for their anti-TB activity against drug-sensitive (DS) M. tb H37Rv strain. Three adamantanol derivatives 8i, 8j and 8l showed nearly 2- and 4-fold higher activity (MIC = 1.32 – 2.89 µM) than ethambutol (MIC = 4.89 µM). Remarkably, the most potent adamantanol analogue 8j demonstrated high selectivity towards DS and DR M. tb strains over mammalian cells [IC50 (Vero cells) ≥ 169 µM], evincing its lack of cytotoxicity. The top eight active compounds 8b, 8d, 8f, 8i, 8j, 8k, 8l and 10a retained their in vitro potency against DR M. tb strains and were docked into the MmpL3 active site. The most potent adamantanol/adamantane-based indoleamides 8j/8k displayed a two-fold surge in potency against extensively DR (XDR) M. tb strains with MIC values of 0.66 and 0.012 µM, respectively. The adamantanol-containing indole-2-carboxamides exhibited improved water solubility both in silico and experimentally, relative to the adamantane counterparts. Overall, the observed antimycobacterial and physicochemical profiles support the notion that adamantanol moiety is a suitable replacement to the adamantane scaffold within the series of indole-2-carboxamide-based MmpL3 inhibitors.

PE_PGRS31-S100A9 Interaction Promotes Mycobacterial Survival in Macrophages Through the Regulation of NF-κB-TNF-α Signaling and Arachidonic Acid Metabolism.

Mycobacterium tuberculosis (M. tb) evades the surveillance of immune responses for survival in macrophages. However, the precise mechanism and toxins/proteins encoded by M. tb involved in the bacterial escape remain elusive. The function of Rv1768 protein (also referred to as PE_PGRS31, belonging to the PE_PGRS family) encoded by the region of deletion 14 (RD-14) in the virulent M. tb H37Rv strain has not, to the best of our knowledge, been reported previously. Here, we found that Rv1768 remarkably promotes bacterial survival in macrophages. Compared to wild type (WT) H37Rv, the Rv1768 deficient strain (H37RvΔ1768) showed significantly decreased colony-forming units in the lungs, spleen, and liver of the murine M. tb infection model. The bacterial burdens of WT H37Rv in WT macrophages and C57BL/6 mice were significantly higher than those in S100A9 deficiency cells and mice, but there were no significant differences for H37RvΔRv1768. Rv1768 binds S100A9 with the proline-glutamic acid domain (PE domain) and blocks the interaction between S100A9 and Toll-like receptor 4 (TLR4), and suppresses TLR4-myeloid differentiation factor 88-nuclear factor-kappa B (NF-κB)-tumor necrosis factor α (TNF-α) signaling in macrophages. Interestingly, Rv1768 binding to S100A9 also disturbs the metabolism of arachidonic acid by activating 5-lipoxygenase, increasing lipotoxin A4, and down-regulating cyclooxygenase-2 and prostaglandin E2 expression, thus, promoting mycobacterial survival. Our results revealed that M. tb Rv1768 promotes mycobacterial survival in macrophages by regulating NF-κB-TNF-α signaling and arachidonic acid metabolism via S100A9. Disturbing the interaction between Rv1768 and S100A9 may be a potential therapeutic target for tuberculosis.

Regulation of Interferon-γ receptor (IFN-γR) expression in macrophages during Mycobacterium tuberculosis infection.

Interferon-gamma (IFN-γ) is a key cytokine that mediates immunity to tuberculosis (TB). Mycobacterium tuberculosis (M. tb) is known to downregulate the surface expression of IFN-γ receptor (IFN-γR) on macrophages and peripheral blood mononuclear cells (PBMCs) of patients with active TB disease. Many M. tb antigens also downmodulate IFN-γR levels in macrophages when compared with healthy controls. In the current study, we aimed at deciphering key factors involved in M. tb mediated downregulation of IFN-γR levels on macrophage surface. Our data showed that both M. tb H37Rv and M. bovis BCG infections mediate downmodulation of IFN-γR on human macrophages. This downmodulation is regulated at the level of TLR signaling pathway, second messengers such as calcium and cellular kinases i.e. PKC and ERK-MAPK, indicating that fine tuning of calcium response is critical to maintaining IFN-γR levels on macrophage surface. In addition, genes in the calcium and cysteine protease pathways which were previously identified by us to play a negative role during M. tb infection, also regulated IFN-γR expression. Thus, modulations in IFN-γR levels by utilizing host machinery may be a key immune suppressive strategy adopted by the TB pathogen to ensure its persistence and thwart host defense.

Identification of pyrvinium pamoate as an anti-tuberculosis agent in vitro and in vivo by SOSA approach amongst known drugs

ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb) bacteria, is a leading infectious cause of mortality worldwide. The emergence of drug-resistant M. tb has made control of TB more difficult. The selective optimization of side activities (SOSA) approach uses old drugs for new pharmacological targets. In the present study by using SOSA approach, we have successfully identified pyrvinium pamoate (PP) which is capable of inhibiting the growth of mycobacteria, including M. tb H37Rv, Mycobacterium smegmatis, Bacille Calmette-Guérin (BCG), M. tb H37Ra, and drug-resistant M. tb clinical isolates in vitro from 1280 known drugs library. The MIC99 of PP, the minimum inhibitory concentration that inhibits more than 99% of M. tb H37Rv and the drug-resistant M. tb clinical isolates, ranges from 1.55 to 4.8 µg/mL. Importantly, PP could reduce the bacterial colony-forming units (CFUs) in lung, spleen and liver tissues, and effectively inhibit inflammatory response in M. tb H37Rv, multidrug-resistant (MDR) M. tb and extensively drug-resistant (XDR) M.tb-infected mice. Our results clearly show that the PP has the potential application for treatment of TB.

Characteristic genes in THP‑1 derived macrophages infected with Mycobacterium tuberculosis H37Rv strain identified by integrating bioinformatics methods.

Mycobacterium tuberculosis (M. tb) is a highly successful pathogen that has co-existed with humans for 1,000's of years. As the cornerstone of the immune system, macrophages are a key part of innate immunity. They ingest and degrade foreign substances including aging cells and microorganisms, coordinate the inflammatory process, and are the first line of defense against M. tb infection. Recent advances in cellular mycobacteriology have indicated that M. tb uses an remarkably complex strategy to disrupt macrophage function, in order to counteract the antimicrobial mechanisms of the innate and adaptive immune responses, thereby achieving immune escape. With the popularity of microarray technology, a variety of public platforms have provided a variety of gene expression data associated with physiological and disease conditions. Meta-analysis can systematically and quantitatively analyze multiple independent data concerning the same disease, greatly improving the statistical significance and credibility of the gene expression data analysis performed. In the present study, 6 microarray expression datasets of human acute monocytic leukemia THP-1 cell line infected by M. tb H37Rv strain were collected from the GEO database. A total of 4 high-quality datasets were identified using meta-analysis methods in R language, and 306 differentially expressed genes with statistical significance were obtained. Then, a protein-protein interaction (PPI) network of these differentially expressed genes was constructed on the Search Tool for the Retrieval of Interacting Genes/Proteins Database online tool and visualized by Cytoscape v. 3.6.1 software. Using CentiScape and MCODE plugin in the Cytoscape software to mine the functional modules associated with M. tb infection process, 32 characteristic genes were identified. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis was performed on the 32 characteristic genes, and it was demonstrated that these genes were primarily associated with the type I interferon (IFN) pathway. In the established model of THP-1-derived macrophages infected by M. tb, the actual differential expression levels of IFN-stimulated gene 15 (ISG15), 2′-5-oligoadenylate synthetase like (OASL), IFN regulatory factor 7 (IRF7) and DExD/H-box helicase 58 (DDX58), the first 4 genes of the 32 characteristic genes, were verified by reverse transcription quantitative polymerase chain reaction. The results were consistent with the results of microarray analysis. The association between ISG15, OASL and IRF7 and TB infection was also verified. Although a number of studies have identified that the type I IFN pathway may assist M. tb to achieve immune escape, the present study used a meta-analysis of microarray data and PPI network analysis to examine some of the novel genes identified in the IFN pathway. The results furthered the understanding of the molecular mechanisms of the TB immune response and provided a novel perspective for future therapeutic goals.

Aerosol immunization by alginate coated mycobacterium (BCG/MIP) particles provide enhanced immune response and protective efficacy than aerosol of plain mycobacterium against M.tb. H37Rv infection in mice

BackgroundWith the aim of preparing a more effective, safe and economical vaccine for tuberculosis, inhalable live mycobacterium formulations were evaluated.MethodsAlginate particles in the size range of 2–4 μm were prepared by encapsulating live Bacille Calmette–Guérin (BCG) and “Mycobacterium indicus pranii” (MIP). These particles were characterized for their size, stability and release profile. Mice were immunized with liquid aerosol or dry powder aerosol (DPA) alginate encapsulated mycobacterium particles and their in-vitro recall response and infection with mycobacterium H37Rv were investigated.ResultsIt was found that the DPA of alginate encapsulated mycobacterium particles invoked superior immune response and provided higher protection in mice than the liquid aerosol. The BCG encapsulated in alginate particles (BEAP) and MIP encapsulated in alginate particles (MEAP) were engulfed by bone marrow dendritic cells (BMDCs) and co-localized with lysosome. The MEAP/BEAP activated BMDCs exhibited higher chemotaxis movement and had enhanced ability of antigen presentation to T cells.The in-vitro recall response of BEAP/MEAP immunized mice when compared in terms of proliferation index and Interferon gamma (IFN-gamma) released by splenocytes and mediastinal lymph node cells was found to be higher than mice immunized by liquid aerosol of BCG/MIP. Finally, different groups of immunized mice were infected with M. tb H37Rv and after 16 weeks the Colony forming units (CFUs) in lung and spleen estimated. The bacilli burden in the BEAP/MEAP immunized mice was significantly less than the respective liquid aerosol immunized mice and the histopathology of BEAP/MEAP immunized mice lungs showed very little damage.ConclusionsThese inhale-able vaccines formulation of alginate coated live mycobacterium are more immunogenic as compared to the aerosol of bacilli and they provide better protection in mice when infected with H37Rv.

Discovery and evaluation of novel nitrodihydroimidazooxazoles as promising anti-tuberculosis agents.

New analogues of antitubercular drug Delamanid were prepared, seeking drug candidates with enhanced aqueous solubility and high efficacy. The strategy involved replacement of phenoxy linker proximal to the 2-nitroimidazooxazole of Delamanid by piperidine fused 5 or 6-membered ring heterocycles (ring A). The new compounds were all more hydrophilic than Delamanid, and several class of analogues showed remarkable activities against M. bovis. And among these series, the tetrahydro-naphthyridine-linked nitroimidazoles displayed excellent antimycobacterial activity against both replicating (MABA) and nonreplicating (LORA) M. tb H37Rv and low cytotoxicity. Compared to Delamanid, these new compounds (6, 7, 45) demonstrated dramatically improved physicochemical properties and are suitable for further in vitro and in vivo evaluation.

Liver macrophages controls Mycobacterium tuberculosis growth by enhancing autophagy

Abstract Alveolar macrophages are the first cells to come in contact with TB pathogen in lung, but unable to eliminate Mycobacterium tuberculosis (M. tb) completely and serves as niche. In the current study, we compared M. tb growth and cytokine production by murine alveolar and liver macrophages. We found three-fold higher CFU in alveolar macrophages compared to liver macrophages (5 ± 1.7 × 106 vs. 1.776 ± 0.5 ×106 CFU) indicating that liver macrophages are more efficient in inhibiting M. tb growth. In contrast, M. tb H37Rv infected alveolar and liver macrophages produced equal amounts of TNF-α, IL-6, IL-10 & IL-1β. There is no significant difference in the apoptosis and M1/M2 paradigm of M. tb infected alveolar and liver macrophages. Flow cytometry analysis revealed the percentage of LC-3B+ M. tb infected liver macrophages were two-fold higher compared to percentage of LC-3B+ M. tb infected alveolar macrophage. This was confirmed by real time PCR. We also noted distinctly enhanced LC-3B puncta formation in infected liver macrophages as examined by confocal microscopy. We found significantly enhanced M. tb CFU in liver macrophages transfected with LC-3B, ATG-5, ATG-7 and Beclin-1 siRNA compared to liver macrophages transfected with control siRNA (p<0.01; 0.006; 0.01&0.002) respectively. Our results suggest that autophagy is involved in better restriction of M. tb growth by liver macrophages. Studies are underway to compare metabolic changes of control and M. tb H37Rv infected alveolar and liver macrophages and in vivo relevance to our current findings using mouse model of M. tb infection.

Oxadiazolone derivatives, new promising multi-target inhibitors against M. tuberculosis

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc26230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC50, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.