Mesenchymal stem cells-derived extracellular vesicles for therapeutics of renal tuberculosis.

Extrapulmonary tuberculosis with a renal involvement can be a manifestation of a disseminated infection that requires therapeutic intervention, particularly with a decrease in efficacy of conventional regimens. In the present study, we investigated the therapeutic potency of mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) in the complex anti-tuberculosis treatment (ATT). A rabbit model of renal tuberculosis (rTB) was constructed by injecting of the standard strain Mycobacterium tuberculosis H37Rv into the cortical layer of the kidney parenchyma. Isolated rabbit MSC-EVs were intravenously administered once as an addition to standard ATT (isoniazid, pyrazinamide, and ethambutol). The therapeutic efficacy was assessed by analyzing changes of blood biochemical biomarkers and levels of anti- and pro-inflammatory cytokines as well as by renal computed tomography with subsequent histological and morphometric examination. The therapeutic effect of therapy with MSC-EVs was shown by ELISA method that confirmed a statistically significant increase of the anti-inflammatory and decrease of pro-inflammatory cytokines as compared to conventional treatment. In addition, there is a positive trend in increase of ALP level, animal weigh, and normalization of ADA activity that can indicate an improvement of kidney state. A significant reduction of the area of specific and interstitial inflammation indicated positive affect of MSC-EVs that suggests a shorter duration of ATT. The number of MSC-EVs proteins (as identified by mass-spectometry analysis) with anti-microbial, anti-inflammatory and immunoregulatory functions reduced the level of the inflammatory response and the severity of kidney damage (further proved by morphometric analysis). In conclusion, MSC-EVs can be a promising tool for the complex treatment of various infectious diseases, in particularly rTB.

Identification of selective plant-derived natural carotenoid and flavonoids as the potential inhibitors of DHHC-mediated protein S-palmitoylation: an in silico study.

Protein S-palmitoylation mediated by DHHCs is recognized as a distinct and reversible form of lipid modification connected with several health perturbations, including neurodegenerative disorders, cancer, and autoimmune conditions. However, the pharmacological characteristics of current pan-DHHC inhibitors, particularly their toxicity and off-target effects, have hindered their in-depth cellular investigations. The therapeutic properties of the natural compounds, with minimal side effects, allowed us to evaluate them as DHHC-targeting inhibitors. Here, we performed an insilico screening of 115 phytochemicals to assess their interactions with the DHHC20 binding site. Among these compounds, lutein, 5-hydroxyflavone, and 6-hydroxyflavone exhibited higher binding energy (-9.2, -8.5, and -8.5 kcal/mol) in the DHHC20 groove compared to pan-DHHC inhibitor 2-BP (-7.0 kcal/mol). Furthermore, we conducted a 100 ns MD simulation to evaluate the stability of these complexes under physiological conditions. The MDsimulation results indicated that DHHC20 formed a more stable conformation with lutein compared to 5-hydroxyflavone and 6-hyroxyflavone via hydrophobic and H-bond interactions. Conclusively, these results could serve as a promising starting point for exploring the use of these natural molecules as DHHC20 inhibitors.Communicated by Ramaswamy H. Sarma.

Preclinical evaluation of engineered L-asparaginase variants to improve the treatment of Acute Lymphoblastic Leukemia.

INTRODUCTION: Escherichia coli l-asparaginase (EcA), an integral part of multi-agent chemotherapy protocols of acute lymphoblastic leukemia (ALL), is constrained by safety concerns and the development of anti-asparaginase antibodies. Novel variants with better pharmacological properties are desirable. METHODS: Thousands of novel EcA variants were constructed using protein engineering approach. After preliminary screening, two mutants, KHY-17 and KHYW-17 were selected for further development. The variants were characterized for asparaginase activity, glutaminase activity, cytotoxicity and antigenicity in vitro. Immunogenicity, pharmacokinetics, safety and efficacy were tested in vivo. Binding of the variants to pre-existing antibodies in primary and relapsed ALL patients' samples was evaluated. RESULTS: Both variants showed similar asparaginase activity but approximately 24-fold reduced glutaminase activity compared to wild-type EcA (WT). Cytotoxicity against Reh cells was significantly higher with the mutants, although not toxic to human PBMCs than WT. The mutants showed approximately 3-fold lower IgG and IgM production compared to WT. Pharmacokinetic study in BALB/c mice showed longer half-life of the mutants (KHY-17- 267.28±9.74; KHYW-17- 167.41±14.4) compared to WT (103.24±18). Single and repeat-doses showed no toxicity up to 2000 IU/kg and 1600 IU/kg respectively. Efficacy in ALL xenograft mouse model showed 80-90 % reduction of leukemic cells with mutants compared to 40 % with WT. Consequently, survival was 90 % in each mutant group compared to 10 % with WT. KHYW-17 showed over 2-fold lower binding to pre-existing anti-asparaginase antibodies from ALL patients treated with l-asparaginase. CONCLUSION: EcA variants demonstrated better pharmacological properties compared to WT that makes them good candidates for further development.

4″-Alkyl EGCG Derivatives Induce Cytoprotective Autophagy Response by Inhibiting EGFR in Glioblastoma Cells.

Epidermal growth factor receptor (EGFR)-targeted therapy has been proven vital in the last two decades for the treatment of multiple cancer types, including nonsmall cell lung cancer, glioblastoma, breast cancer and head and neck squamous cell carcinoma. Unfortunately, the majority of approved EGFR inhibitors fall into the drug resistance category because of continuous mutations and acquired resistance. Recently, autophagy has surfaced as one of the emerging underlying mechanisms behind resistance to EGFR-tyrosine kinase inhibitors (TKIs). Previously, we developed a series of 4″-alkyl EGCG (4″-Cn EGCG, n = 6, 8, 10, 12, 14, 16, and 18) derivatives with enhanced anticancer effects and stability. Therefore, the current study hypothesized that 4″-alkyl EGCG might induce cytoprotective autophagy upon EGFR inhibition, and inhibition of autophagy may lead to improved cytotoxicity. In this study, we have observed growth inhibition and caspase-3-dependent apoptosis in 4″-alkyl EGCG derivative-treated glioblastoma cells (U87-MG). We also confirmed that 4″-alkyl EGCG could inhibit EGFR in the cells, as well as mutant L858R/T790M EGFR, through an in vitro kinase assay. Furthermore, we have found that EGFR inhibition with 4″-alkyl EGCG induces cytoprotective autophagic responses, accompanied by the blockage of the AKT/mTOR signaling pathway. In addition, cytotoxicity caused by 4″-C10 EGCG, 4″-C12 EGCG, and 4″-C14 EGCG was significantly increased after the inhibition of autophagy by the pharmacological inhibitor chloroquine. These findings enhance our understanding of the autophagic response toward EGFR inhibitors in glioblastoma cells and suggest a potent combinatorial strategy to increase the therapeutic effectiveness of EGFR-TKIs.

20 years since the approval of first EGFR-TKI, gefitinib: Insight and foresight.

Epidermal growth factor receptor (EGFR) actively involves in modulation of various cancer progression related mechanisms including angiogenesis, differentiation and migration. Therefore, targeting EGFR has surfaced as a prominent approach for the treatment of several types of cancers, including non-small cell lung cancer (NSCLC), pancreatic cancer, glioblastoma. Various first, second and third generation of EGFR tyrosine kinase inhibitors (EGFR-TKIs) have demonstrated effectiveness as an anti-cancer therapeutics. However, rapid development of drug resistance and mutations still remains a major challenge for the EGFR-TKIs therapy. Overcoming from intrinsic and acquired resistance caused by EGFR mutations warrants the further exploration of alternative strategies and discovery of novel inhibitors. In this review, we delve into the breakthrough discoveries have been made in previous 20 years, and discuss the currently ongoing efforts aimed to circumvent the chemo-resistance. We also highlight the new challenges, limitations and future directions for the development of improved therapeutic approaches such as fourth-generation EGFR-TKIs, peptides, nanobodies, PROTACs etc.

Chitosan-Biotin-Conjugated pH-Responsive Ru(II) Glucose Nanogel: A Dual Pathway of Targeting Cancer Cells and Self-Drug Delivery.

The current study paves the way for improved chemotherapy by creating pH-responsive nanogels (NGs) (GC1 and GC2) loaded with synthetic ruthenium(II) arene complexes to increase biological potency. NGs are fabricated by the conjugation of chitosan (CTS)-biotin biopolymers that selectively target the cancer cells as CTS has the pH-responsive property, which helps in releasing the drug in cancer cells having pH ∼ 5.5, and biotin provides the way to target the cancer cells selectively due to the overexpression of integrin. The synthesized compounds and NGs were thoroughly characterized using various spectroscopic and analytical techniques such as NMR, electrospray ionization-mass spectrometry, Fourier transform infrared, UV-vis, scanning electron microscopy, transmission electron microscopy, X-ray photoelectron spectroscopy, rheology, Brunauer-Emmett-Teller, and others. NGs displayed exceptional increased efficacy toward cancerous cells with IC50 values ranging from 7.50 to 18.86 µM via induced apoptosis in three human cancer cell lines. Apart from its potency, NGs were found to be highly selective toward cancer cells. Moreover, based on the results of immunoblot analysis, it was observed that the synthesized compounds exhibit a significant increase in the expression of cleaved caspase-3 and a decrease in the expression of the antiapoptotic protein BCL-XL. Interestingly, the complexes were discovered to have the additional capability of catalyzing the conversion of NADH to NAD+, leading to the generation of radical oxygen species within the cells. Additionally, it was discovered that NG-induced apoptosis depends on ROS production and DNA binding. A narrower range of LD50 values (1185.93 and 823.03 µM) was seen after administering NGs to zebrafish embryos in vivo. The results support the use of drug-loaded NGs as potential chemotherapeutic and chemopreventive agents for human cancer cells.

Role of EGFR and FASN in breast cancer progression.

Breast cancer (BC) emerged as one of the life-threatening diseases among females. Despite notable improvements made in cancer detection and treatment worldwide, according to GLOBACAN 2020, BC is the fifth leading cancer, with an estimated 1 in 6 cancer deaths, in a majority of countries. However, the exact cause that leads to BC progression still needs to be determined. Here, we reviewed the role of two novel biomarkers responsible for 50-70% of BC progression. The first one is epidermal growth factor receptor (EGFR) which belongs to the ErbB tyrosine kinases family, signalling pathways associated with it play a significant role in regulating cell proliferation and division. Another one is fatty acid synthase (FASN), a key enzyme responsible for the de novo lipid synthesis required for cancer cell development. This review presents a rationale for the EGFR-mediated pathways, their interaction with FASN, communion of these two biomarkers with BC, and improvements to overcome drug resistance caused by them.

Multifaceted Carbon Dots: toward pH-Responsive Delivery of 5-Fluorouracil for In Vitro Antiproliferative Activity.

The synthesis of smart hybrid material to assimilate diagnosis and treatment is crucial in nanomedicine. Herein, we present a simple and facile method to synthesize multitalented blue-emissive nitrogen-doped carbon dots N@PEGCDs. The as-prepared carbon dots N@PEGCDs show enhanced biocompatibility, small size, high fluorescence, and high quantum yield. The N@PEGCDs are used as a drug carrier for 5-fluorouracil (5-FU) with more release at acidic pH. Furthermore, the mode of action of drug-loaded CD (5FU-N@PEGCDs) has also been explored by performing wound healing assay, DCFDA assay for ROS generation, and Hoechst staining. The drug loaded with carbon dots showed less toxicity to normal cells compared to cancer cells, making it a perfect candidate to be studied for designing next-generation drug delivery systems.

Studies on anticancer properties with varying co-ligands in a Ru(II) arene benzimidazole system.

Recently in the field of chemotherapeutics, to combat the side effects of cisplatin, ruthenium complexes have been investigated extensively. In this work, a bidentate benzimidazole-based ligand, HL [HL = 2-(1H-benzo[d]imidazol-2-yl)-6-methoxyphenol], was utilized to obtain three Ru(II) arene complexes having a generalized formula [Ru(η6-p-cym)(L)(X)] or [Ru(η6-p-cym)(L)(X)]+ (where p-cym = p-cymene). The co-ligand X (X = (i) Cl, (ii) PPh3 = triphenyl phosphine, and (iii) PTA = 1,3,5-triaza-7-phosphaadamantane) was varied in order to study the effect it has on the antitumor activity of the compounds. The synthesized compounds were thoroughly characterized using different analytical techniques, including ESI-MS, NMR, FTIR, UV-Vis, and fluorescence spectroscopy. A fluorescence quenching experiment with serum albumin proteins revealed good interactions between the complexes and HSA and BSA. An analysis of their lipophilic character via the shake flask method and a stability study using UV spectroscopy were conducted as well. The anticancer properties of the synthesized compounds were further explored by conducting a DNA binding study using absorption spectroscopy and fluorometric titration with DAPI to check the mode of binding with DNA. Interestingly, the complexes were also found to catalyze the oxidation of NADH to NAD+, giving rise to radical species in the cells. An immunoblot analysis strongly suggested that all three complexes can remarkably upregulate the expression of cleaved caspase-3 and downregulate the expression of the anti-apoptotic protein BCLXL. It is important to note that such studies are yet to be reported for similar benzimidazole-based ruthenium complexes and therefore present a new direction for the investigation of antitumor ruthenium-based metallodrugs. Furthermore, Hoechst and AO/EtBr staining was used to analyze the morphological changes of the compound-treated cancer cells due to apoptosis, which was also confirmed by the IC50 values obtained from the colorimetric assay (MTT) against different cancer cell lines.

Detection of Peroxisomal Proteins During Mycobacterial Infection.

Peroxisomes are ubiquitous organelles with essential roles in lipid and reactive oxygen species (ROS) metabolism. They are involved in modulating the immune responses during microbial infection, thus having major impact on several bacterial and viral infectious diseases including tuberculosis. Intracellular pathogens such as Mycobacterium tuberculosis (M. tb) employ various strategies to suppress the host oxidative stress mechanisms to avoid killing by the host. Peroxisome-mediated ROS balance is crucial for innate immune responses to M. tb. Therefore, peroxisomes represent promising targets for host-directed therapeutics to tuberculosis. Here, we present protocols used in our laboratory for the culture of M. tb and detection of peroxisomal proteins in M. tb infected macrophages.

Structure-based design and synthesis of a novel long-chain 4''-alkyl ether derivative of EGCG as potent EGFR inhibitor: in vitro and in silico studies.

Herein, we report the discovery of a novel long-chain ether derivative of (-)-epigallocatechin-3-gallate (EGCG), a major green tea polyphenol as a potent EGFR inhibitor. A series of 4''-alkyl EGCG derivatives have been synthesized via regio-selectively alkylating the 4'' hydroxyl group in the D-ring of EGCG and tested for their antiproliferative activities against high (A431), moderate (HeLa), and low (MCF-7) EGFR-expressing cancer cell lines. The most potent compound, 4''-C14 EGCG showed the lowest IC50 values across all the tested cell lines. 4''-C14 EGCG was also found to be significantly more stable than EGCG under physiological conditions (PBS at pH 7.4). Further western blot analysis and imaging data revealed that 4''-C14 EGCG induced cell death in A431 cells with shrunken nuclei, nuclear fragmentation, membrane blebbing, and increased population of apoptotic cells where BAX upregulation and BCLXL downregulation were observed. In addition, autophosphorylation of EGFR and its downstream signalling proteins Akt and ERK were markedly inhibited by 4''-C14 EGCG. MD simulation and the MM/PBSA analysis disclosed the binding mode of 4''-C14 EGCG in the ATP-binding site of EGFR kinase domain. Taken together, our findings demonstrate that 4''-C14 EGCG can act as a promising potent EGFR inhibitor with enhanced stability.

Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal ß-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11ß, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

Mycobacterium tuberculosis Phosphoribosyltransferase Promotes Bacterial Survival in Macrophages by Inducing Histone Hypermethylation in Autophagy-Related Genes.

Mycobacterium tuberculosis (Mtb) inhibits autophagy to promote its survival in host cells. However, the molecular mechanisms by which Mtb inhibits autophagy are poorly understood. Here, we report a previously unknown mechanism in which Mtb phosphoribosyltransferase (MtbPRT) inhibits autophagy in an mTOR, negative regulator of autophagy, independent manner by inducing histone hypermethylation (H3K9me2/3) at the Atg5 and Atg7 promoters by activating p38-MAPK- and EHMT2 methyltransferase-dependent signaling pathways. Additionally, we find that MtbPRT induces EZH2 methyltransferase-dependent H3K27me3 hypermethylation and reduces histone acetylation modifications (H3K9ac and H3K27ac) by upregulating histone deacetylase 3 to inhibit autophagy. In summary, this is the first demonstration that Mtb inhibits autophagy by inducing histone hypermethylation in autophagy-related genes to promote intracellular bacterial survival.

Mycobacterium tuberculosis EsxL induces TNF-α secretion through activation of TLR2 dependent MAPK and NF-κB pathways.

Mycobacterium tuberculosis (Mtb) employs distinct strategies to circumvent host immune responses during the infection process. Various Mtb cell-wall associated and secretory proteins are known to play a critical role in the orchestration of host innate immune responses through modulation of signaling pathways. Mtb genome encodes for 23 (EsxA-EsxW) proteins belonging to the ESAT-6 like family; however, most of them are functionally unknown. Here, we show that Mtb EsxL induces tumor necrosis factor-alpha (TNF-α) production by activating nuclear translocation of nuclear factor-κB (NF-κB) via interaction with Toll-like Receptor 2 (TLR2). Blocking or silencing of TLR2 abrogated nuclear translocation of NF-kB and TNF-α production. Treatment with recombinant purified EsxL (rEsxL) activated mitogen-activated protein kinase (MAPK) pathway by inducing the phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase (p38) pathways. At the same time, inhibition of ERK and p38 down-regulated the expression of TNF-α in rEsxL exposed murine macrophages. Besides TNF-α, EsxL also induced the production of IL-6 proinflammatory cytokine. Taken together, these results suggest that EsxL is able to induce TNF-α secretion via TLR2 through activation of NF-κB and MAPK signaling. This study will help in deducing therapeutic strategies for better control of the disease.

Identification and evaluation of immunogenic MHC-I and MHC-II binding peptides from Mycobacterium tuberculosis.

Due to several limitations of the only available BCG vaccine, to generate adequate protective immune responses, it is important to develop potent and cost-effective vaccines against tuberculosis (TB). In this study, we have used an immune-informatics approach to identify potential peptide based vaccine targets against TB. The proteome of Mycobacterium tuberculosis (Mtb), the causative agent of TB, was analyzed for secretory or surface localized antigenic proteins as potential vaccine candidates. The T- and B-cell epitopes as well as MHC molecule binding efficiency were identified and mapped in the modelled structures of the selected proteins. Based on antigenicity score and molecular dynamic simulation (MD) studies two peptides namely Pep-9 and Pep-15 were analyzed, modelled and docked with MHC-I and MHC-II structures. Both peptides exhibited no cytotoxicity and were able to induce proinflammatory cytokine secretion in stimulated macrophages. The molecular docking, MD and in-vitro studies of the predicted B and T-cell epitopes of Pep-9 and Pep-15 peptides with the modelled MHC structures exhibited strong binding affinity and antigenic properties, suggesting that the complex is stable, and that these peptides can be considered as a potential candidates for the development of vaccine against TB.

Plant-derived natural polyphenols as potential antiviral drugs against SARS-CoV-2 via RNA-dependent RNA polymerase (RdRp) inhibition: an in-silico analysis.

The sudden outburst of Coronavirus disease (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) poses a massive threat to global public health. Currently, no therapeutic drug or vaccine exists to treat COVID-19. Due to the time taking process of new drug development, drug repurposing might be the only viable solution to tackle COVID-19. RNA-dependent RNA polymerase (RdRp) catalyzes SARS-CoV-2 RNA replication and hence, is an obvious target for antiviral drug design. Interestingly, several plant-derived polyphenols effectively inhibit the RdRp of other RNA viruses. More importantly, polyphenols have been used as dietary supplementations for a long time and played beneficial roles in immune homeostasis. We were curious to study the binding of polyphenols with SARS-CoV-2 RdRp and assess their potential to treat COVID-19. Herein, we made a library of polyphenols that have shown substantial therapeutic effects against various diseases. They were successfully docked in the catalytic pocket of RdRp. The investigation reveals that EGCG, theaflavin (TF1), theaflavin-3'-O-gallate (TF2a), theaflavin-3'-gallate (TF2b), theaflavin 3,3'-digallate (TF3), hesperidin, quercetagetin, and myricetin strongly bind to the active site of RdRp. Further, a 150-ns molecular dynamic simulation revealed that EGCG, TF2a, TF2b, TF3 result in highly stable bound conformations with RdRp. The binding free energy components calculated by the MM-PBSA also confirm the stability of the complexes. We also performed a detailed analysis of ADME prediction, toxicity prediction, and target analysis for their druggability. Overall, our results suggest that EGCG, TF2a, TF2b, TF3 can inhibit RdRp and represent an effective therapy for COVID-19.Communicated by Ramaswamy H. Sarma.

Differential Roles of the Calcium Ion Channel TRPV4 in Host Responses to Mycobacterium tuberculosis Early and Late in Infection.

Mycobacterium tuberculosis subverts host immunity to proliferate within host tissues. Non-selective transient receptor potential (TRP) ion channels are involved in host responses and altered upon bacterial infections. Altered expression and localization of TRPV4 in macrophages upon virulent M. tuberculosis infection together with differential distribution of TRPV4 in human tuberculosis (TB) granulomas indicate a role of TRPV4 in TB. Compared with wild-type mice, Trpv4-deficient littermates showed transiently higher mycobacterial burden and reduced proinflammatory responses. In the absence of TRPV4, activation failed to render macrophages capable of controlling mycobacteria. Surprisingly, Trpv4-deficient mice were superior to wild-type ones in controlling M. tuberculosis infection in the chronic phase. Thus, Trpv4 is important in host responses to mycobacteria, although with opposite functions early versus late in infection. Ameliorated chronic infection in the absence of Trpv4 and its expression in human TB lesions indicate TRPV4 as putative target for host-directed therapy.

Mycobacterium tuberculosis Rv3034c regulates mTORC1 and PPAR-γ dependant pexophagy mechanism to control redox levels in macrophages.

Mycobacterium tuberculosis survives inside the macrophages by employing several host immune evasion strategies. Here, we reported a novel mechanism in which M. tuberculosis acetyltransferase, encoded by Rv3034c, induces peroxisome homeostasis to regulate host oxidative stress levels to facilitate intracellular mycobacterial infection. Presence of M. tuberculosis Rv3034c induces the expression of peroxisome biogenesis and proliferation factors such as Pex3, Pex5, Pex19, Pex11b, Fis-1 and DLP-1; while depletion of Rv3034c decreased the expression of these molecules, thereby selective degradation of peroxisomes via pexophagy. Further studies revealed that M. tuberculosis Rv3034c inhibit induction of pexophagy mechanism by down-regulating the expression of pexophagy associated proteins (p-AMPKα, p-ULK-1, Atg5, Atg7, Beclin-1, LC3-II, TFEB and Keap-1) and adaptor molecules (NBR1 and p62). Inhibition was found to be dependent on the phosphorylation of mTORC1 and activation of peroxisome proliferator activated receptor-γ. In order to maintain intracellular homeostasis during oxidative stress, M. tuberculosis Rv3034c was found to induce degradation of dysfunctional and damaged peroxisomes through activation of Pex14 in infected macrophages. In conclusion, this is the first report which demonstrated that M. tuberculosis acetyltransferase regulate peroxisome homeostasis in response to intracellular redox levels to favour mycobacterial infection in macrophage.

Mycobacterium tuberculosis LprE Suppresses TLR2-Dependent Cathelicidin and Autophagy Expression to Enhance Bacterial Survival in Macrophages.

Despite representing a very important class of virulence proteins, the role of lipoproteins in the pathogenesis of Mycobacterium tuberculosis remains elusive. In this study, we investigated the role of putative lipoprotein LprE in the subversion of host immune responses using the M. tuberculosis CDC1551 LprE (LprE Mtb ) mutant (Mtb∆LprE). We show that deletion of LprE Mtb results in reduction of M. tuberculosis virulence in human and mouse macrophages due to upregulation of vitamin D3-responsive cathelicidin expression through the TLR2-dependent p38-MAPK-CYP27B1-VDR signaling pathway. Conversely, episomal expression of LprE Mtb in Mycobacterium smegmatis improved bacterial survival. Infection in siTLR2-treated or tlr2-/- macrophages reduced the survival of LprE Mtb expressing M. tuberculosis and M. smegmatis because of a surge in the expression of cathelicidin. Infection with the LprE Mtb mutant also led to accumulation of autophagy-related proteins (LC3, Atg-5, and Beclin-1) and augmented recruitment of phagosomal (EEA1 and Rab7) and lysosomal (LAMP1) proteins, thereby resulting in the reduction of the bacterial count in macrophages. The inhibition of phago-lysosome fusion by LprE Mtb was found to be due to downregulation of IL-12 and IL-22 cytokines. Altogether, our data indicate that LprE Mtb is an important virulence factor that plays a crucial role in mycobacterial pathogenesis in the context of innate immunity.

Mouse Bone Marrow Sca-1+ CD44+ Mesenchymal Stem Cells Kill Avirulent Mycobacteria but Not Mycobacterium tuberculosis through Modulation of Cathelicidin Expression via the p38 Mitogen-Activated Protein Kinase-Dependent Pathway.

Mycobacterium tuberculosis primarily infects lung macrophages. However, a recent study showed that M. tuberculosis also infects and persists in a dormant form inside bone marrow mesenchymal stem cells (BM-MSCs) even after successful antibiotic therapy. However, the mechanism(s) by which M. tuberculosis survives in BM-MSCs is still not known. Like macrophages, BM-MSCs do not contain a well-defined endocytic pathway, which is known to play a central role in the clearance of internalized mycobacteria. Here, we studied the fate of virulent and avirulent mycobacteria in Sca-1+ CD44+ BM-MSCs. We found that BM-MSCs were able to kill avirulent Mycobacterium smegmatis and Mycobacterium bovis BCG but not the pathogenic species M. tuberculosis Further mechanistic studies revealed that pathogenic M. tuberculosis dampens the antibacterial response of BM-MSCs by downregulating the expression of the cationic antimicrobial peptide cathelicidin. In contrast, avirulent mycobacteria were effectively killed by inducing the Toll-like receptor 2/4 (TLR2/4) pathway-dependent expression of cathelicidin, while small interfering RNA (siRNA)-mediated cathelicidin silencing increased the survival of M. bovis BCG in BM-MSCs. We also showed that M. bovis BCG infection caused increased expression levels of MyD88, phospho-interleukin-1 receptor-associated kinase 4 (pIRAK-4), and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Further downstream investigations demonstrated that IRAK-4-p38 activation increased the nuclear translocation of NF-κB, which subsequently induced the expression of cathelicidin and the cytokine interleukin-1ß (IL-1ß), resulting in the decreased survival of M. bovis BCG. On the other hand, inhibition of TLR2/4, pIRAK-4, p38, and NF-κB nuclear translocation decreased cathelicidin and IL-1ß expression levels and therefore increased the survival of avirulent mycobacteria. This is the first report that demonstrates that virulent mycobacteria manipulate the TLR2/4-MyD88-IRAK-4-p38-NF-κB-Camp-IL-1ß pathway to survive inside bone marrow stem cells.