The nuclear pore protein NUP98 impedes LTR-driven basal gene expression of HIV-1, viral propagation, and infectivity.

Nucleoporins (NUPs) are cellular effectors of human immunodeficiency virus-1 (HIV-1) replication that support nucleocytoplasmic trafficking of viral components. However, these also non-canonically function as positive effectors, promoting proviral DNA integration into the host genome and viral gene transcription, or as negative effectors by associating with HIV-1 restriction factors, such as MX2, inhibiting the replication of HIV-1. Here, we investigated the regulatory role of NUP98 on HIV-1 as we observed a lowering of its endogenous levels upon HIV-1 infection in CD4+ T cells. Using complementary experiments in NUP98 overexpression and knockdown backgrounds, we deciphered that NUP98 negatively affected HIV-1 long terminal repeat (LTR) promoter activity and lowered released virus levels. The negative effect on promoter activity was independent of HIV-1 Tat, suggesting that NUP98 prevents the basal viral gene expression. ChIP-qPCR showed NUP98 to be associated with HIV-1 LTR, with the negative regulatory element (NRE) of HIV-1 LTR playing a dominant role in NUP98-mediated lowering of viral gene transcription. Truncated mutants of NUP98 showed that the attenuation of HIV-1 LTR-driven transcription is primarily contributed by its N-terminal region. Interestingly, the virus generated from the producer cells transiently expressing NUP98 showed lower infectivity, while the virus generated from NUP98 knockdown CD4+ T cells showed higher infectivity as assayed in TZM-bl cells, corroborating the anti-HIV-1 properties of NUP98. Collectively, we show a new non-canonical function of a nucleoporin adding to the list of moonlighting host factors regulating viral infections. Downregulation of NUP98 in a host cell upon HIV-1 infection supports the concept of evolutionary conflicts between viruses and host antiviral factors.

Mycobacterium tuberculosis EspR modulates Th1-Th2 shift by transcriptionally regulating IL-4, steering increased mycobacterial persistence and HIV propagation during co-infection.

Mycobacterium tuberculosis (Mtb) and HIV are known to mutually support each other during co-infection by multiple mechanisms. This synergistic influence could be either by direct interactions or indirectly through secreted host or pathogen factors that work in trans. Mtb secretes several virulence factors to modulate the host cellular environment for its persistence and escaping cell-intrinsic immune responses. We hypothesized that secreted Mtb transcription factors that target the host nucleus can directly interact with host DNA element(s) or HIV LTR during co-infection, thereby modulating immune gene expression, or driving HIV transcription, helping the synergistic existence of Mtb and HIV. Here, we show that the Mtb-secreted protein, EspR, a transcription regulator, increased mycobacterial persistence and HIV propagation during co-infection. Mechanistically, EspR localizes to the nucleus of the host cells during infection, binds to its putative cognate motif on the promoter region of the host IL-4 gene, activating IL-4 gene expression, causing high IL-4 titers that induce a Th2-type microenvironment, shifting the macrophage polarization to an M2 state as evident from CD206 dominant population over CD64. This compromised the clearance of the intracellular mycobacteria and enhanced HIV propagation. It was interesting to note that EspR did not bind to HIV LTR, although its transient expression increased viral propagation. This is the first report of an Mtb transcription factor directly regulating a host cytokine gene. This augments our understanding of the evolution of Mtb immune evasion strategies and unveils how Mtb aggravates comorbidities, such as HIV co-infection, by modulating the immune microenvironment.

HIV-1 Tat commandeers nuclear export of Rev-viral RNA complex by controlling hnRNPA2-mediated splicing.

IMPORTANCE: HIV-infected host cells impose varied degrees of regulation on viral replication, from very high to abortive. Proliferation of HIV in astrocytes is limited when compared to immune cells, such as CD4+ T lymphocytes. Understanding such differential regulation is one of the key questions in the field as these cells permit HIV persistence and rebound viremia, challenging HIV treatment and clinical cure. This study focuses on understanding the molecular mechanism behind such cell-specific disparities. We show that one of the key mechanisms is the regulation of heterogenous nuclear ribonucleoprotein A2, a host factor involved in alternative splicing and RNA processing, by HIV-1 Tat in CD4+ T lymphocytes, not observed in astrocytes. This regulation causes an increase in the levels of unspliced/partially spliced viral RNA and nuclear export of Rev-RNA complexes which results in high viral propagation in CD4+ T lymphocytes. The study reveals a new mechanism imposed by HIV on host cells that determines the fate of infection.

Exacerbating effects of single-dose acute ethanol exposure on neuroinflammation and amelioration by GPR110 (ADGRF1) activation.

BACKGROUND: Neuroinflammation is a widely studied phenomenon underlying various neurodegenerative diseases. Earlier study demonstrated that pharmacological activation of GPR110 in both central and peripheral immune cells cooperatively ameliorates neuroinflammation caused by systemic lipopolysaccharide (LPS) administration. Ethanol consumption has been associated with exacerbation of neurodegenerative and systemic inflammatory conditions. The goal of this study is to determine the effects of single-dose acute ethanol exposure and GPR110 activation on the neuro-inflammation mechanisms. METHODS: For in vivo studies, GPR110 wild type (WT) and knockout (KO) mice at 10-12 weeks of age were given an oral gavage of ethanol (3 g/kg) or maltose (5.4 g/kg) at 1-4 h prior to the injection of LPS (1 mg/kg, i.p.) followed by the GPR110 ligand, synaptamide (5 mg/kg). After 2-24 h, brains were collected for the analysis of gene expression by RT-PCR or protein expression by western blotting and enzyme-linked immunosorbent assay (ELISA). Microglial activation was assessed by western blotting and immunohistochemistry. For in vitro studies, microglia and peritoneal macrophages were isolated from adult WT mice and treated with 25 mM ethanol for 4 h and then with LPS (100 ng/ml) followed by 10 nM synaptamide for 2 h for gene expression and 12 h for protein analysis. RESULTS: Single-dose exposure to ethanol by gavage before LPS injection upregulated pro-inflammatory cytokine expression in the brain and plasma. The LPS-induced Iba-1 expression in the brain was significantly higher after ethanol pretreatment in both WT and GPR110KO mice. GPR110 ligand decreased the mRNA and/or protein expression of these cytokines and Iba-1 in the WT but not in GPR110KO mice. In the isolated microglia and peritoneal macrophages, ethanol also exacerbated the LPS-induced expression of pro-inflammatory cytokines which was mitigated at least partially by synaptamide. The expression of an inflammasome marker NLRP3 upregulated by LPS was further elevated with prior exposure to ethanol, especially in the brains of GPR110KO mice. Both ethanol and LPS reduced adenylate cyclase 8 mRNA expression which was reversed by the activation of GPR110. PDE4B expression at both mRNA and protein level in the brain increased after ethanol and LPS treatment while synaptamide suppressed its expression in a GPR110-dependent manner. CONCLUSION: Single-dose ethanol exposure exacerbated LPS-induced inflammatory responses. The GPR110 ligand synaptamide ameliorated this effect of ethanol by counteracting on the cAMP system, the common target for synaptamide and ethanol, and by regulating NLRP3 inflammasome.

Mycobacterial infection alters host mitochondrial activity.

The ability of Mycobacterium tuberculosis (M. tb) to hijack host mitochondria and control host immune signaling is the key to its successful infection. Infection of M. tb causes distinct changes in mitochondrial morphology, metabolism, disruption of innate signaling, and cell fate. The alterations in mitochondria are intricately linked to the immunometabolism of host immune cells such as macrophages, dendritic cells, and T cells. Different immune cells are tuned to diverse immunometabolic states that decide their immune response. These changes could be attributed to the several proteins targeted to host mitochondria by M. tb. Bioinformatic analyses and experimental evidence revealed the potential localization of secreted mycobacterial proteins in host mitochondria. Given the central role of mitochondria in the host metabolism, innate signaling, and cell fate, its manipulation by M. tb renders it susceptible to infection. Restoring mitochondrial health can override M. tb-mediated manipulation and thus clear infection. Several reviews are available on the role of different immune cells in tuberculosis infection and M. tb evasion of immune responses; in the present chapter, we discuss the mitochondrial functional alterations in the innate immune signaling of various immune cells driven by differential mitochondrial immunometabolism during M. tb infection and the role of M. tb proteins, which are directly targeted to the host mitochondria and compromise its innate signaling system. Further studies would help in uncovering the molecular mechanisms of M. tb-directed proteins in host mitochondria to conceptualize both host- directed and pathogen- directed interventions in TB disease management.

Wang resin catalysed sonochemical synthesis of pyrazolo[4,3-d]pyrimidinones and 2,3-dihydroquinazolin-4(1H)-ones: Identification of chorismate mutase inhibitors having effects on Mycobacterium tuberculosis cell viability.

The enzyme chorismate mutase (or CM that is vital for the survival of bacteria) is an interesting pharmacological target for the identification of new anti-tubercular agents. The 5,5-disibstituted pyrazolo[4,3-d]pyrimidinone derivatives containing the fragment based on 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide were designed and explored as the potential inhibitors of chorismate mutase. Based on encouraging docking results of two representative molecules evaluated in silico against MtbCM (PDB: 2FP2) the Wang resin catalysed sonochemical synthesis of target N-heteroarenes were undertaken. The methodology involved the reaction of 4-amino-1-methyl-3-propyl-1H-pyrazole-5-carboxamide with the appropriate cyclic/acyclic ketones to afford the desired products in acceptable (51-94%) yields. The methodology was also extended successfully towards the synthesis of 2,2-disubstituted 2,3-dihydroquinazolin-4(1H)-ones in excellent (85-90%) yields. In vitro MTT assay against the RAW 264.7 cell line followed by enzymatic assay against MtbCM identified 3b and 3c as active compounds that showed two H-bonding via their NH (at position 6) and CO group with MtbCM in silico and encouraging (54-57%) inhibition at 30 µM in vitro. Notably, none of the 2,2-disubstituted 2,3-dihydroquinazolin-4(1H)-ones showed any significant inhibition of MtbCM suggesting the favourable role of the pyrazole moiety in case of pyrazolo[4,3-d]pyrimidinones. The favourable role of cyclopentyl ring attached to the pyrazolo[4,3-d]pyrimidinone moiety and that of two methyl groups in place of cyclopentyl ring was also indicated by the SAR study. Besides showing effects against MtbCM in the concentration response study, 3b and 3c showed little or no effects on mammalian cell viability up to 100 µM in an MTT assay but decreased the % Mtb cell viability at 10-30 µM with > 20% decrease at 30 µM in an Alamar Blue Assay. Moreover, no adverse effects were noted for these compounds when tested for teratogenicity and hepatotoxicity in zebrafish at various concentrations. Overall, being the only example of MtbCM inhibitors that showed effects on Mtb cell viability the compound 3b and 3c are of further interest form the view point of discovery and development of new anti-tubercular agents.

Tough Way In, Tough Way Out: The Complex Interplay of Host and Viral Factors in Nucleocytoplasmic Trafficking during HIV-1 Infection.

Human immunodeficiency virus-1 (HIV-1) is a retrovirus that integrates its reverse-transcribed genome as proviral DNA into the host genome to establish a successful infection. The viral genome integration requires safeguarding the subviral complexes, reverse transcription complex (RTC) and preintegration complex (PIC), in the cytosol from degradation, presumably effectively secured by the capsid surrounding these complexes. An intact capsid, however, is a large structure, which raises concerns about its translocation from cytoplasm to nucleus crossing the nuclear membrane, guarded by complex nuclear pore structures, which do not allow non-specific transport of large molecules. In addition, the generation of new virions requires the export of incompletely processed viral RNA from the nucleus to the cytoplasm, an event conventionally not permitted through mammalian nuclear membranes. HIV-1 has evolved multiple mechanisms involving redundant host pathways by liaison with the cell's nucleocytoplasmic trafficking system, failure of which would lead to the collapse of the infection cycle. This review aims to assemble the current developments in temporal and spatial events governing nucleocytoplasmic transport of HIV-1 factors. Discoveries are anticipated to serve as the foundation for devising host-directed therapies involving selective abolishment of the critical interactomes between viral proteins and their host equivalents.

Milk exosomes elicit a potent anti-viral activity against dengue virus.

BACKGROUND: Exosomes are nano-sized vesicles secreted by various cells into the intra and extracellular space and hence is an integral part of biological fluids including milk. In the last few decades, many research groups have proved the potential of milk exosomes as a sustainable, economical and non-immunogenic drug delivery and therapeutic agent against different pathological conditions. However, its anti-viral properties still remain to be unearthed. METHODS: Here, we have been able to isolate, purify and characterize the milk derived exosomes from Cow (CME) and Goat (GME) and further studied its antiviral properties against Dengue virus (DENV), Newcastle Disease Virus strain Komarov (NDV-K) and Human Immunodeficiency Virus (HIV-1) using an in-vitro infection system. RESULTS: TEM, NTA and DLS analysis validated the appropriate size of the isolated cow and goat milk exosomes (30-150 nm). Real-time PCR and immunoblotting results confirmed the presence of several milk exosomal miRNAs and protein markers. Our findings suggest that GME significantly decreased the infectivity of DENV. In addition, we confirmed that GME significantly reduces DENV replication and reduced the secretion of mature virions. Furthermore, heat inactivation of GME did not show any inhibition on DENV infection, replication, and secretion of mature virions. RNase treatment of GME abrogates the anti-viral properties indicating direct role of exosomes in DENV inhibition. In addition GME inhibited the infectivity of NDV-K, but not HIV-1, suggesting that the GME mediated antiviral activity might be virus specific. CONCLUSION: This study demonstrates the anti-viral properties of milk exosomes and opens new avenues for the development of exosome-based therapies to treat viral diseases.

Staufen-2 functions as a cofactor for enhanced Rev-mediated nucleocytoplasmic trafficking of HIV-1 genomic RNA via the CRM1 pathway.

Nucleocytoplasmic shuttling of viral elements, supported by several host factors, is essential for the replication of the human immunodeficiency virus (HIV). HIV-1 uses a nuclear RNA export pathway mediated by viral protein Rev to transport its Rev response element (RRE)-containing partially spliced and unspliced transcripts aided by the host nuclear RNA export protein CRM1. The factor(s) interacting with the CRM1-Rev complex are potential antiretroviral target(s) and could serve as a retroviral model system to study nuclear export machinery adapted by these viruses. We earlier reported that cellular Staufen-2 interacts with Rev, facilitating viral-RNA export. Here, we identified the formation of a complex between Staufen-2, CRM1 and Rev. Molecular docking and simulations mapped the interacting residues in the RNA-binding Domain 4 of Staufen-2 as R336 and R337, which were experimentally verified to be critical for interactions among Staufen-2, CRM1 and Rev by mutational analysis. Staufen-2 mutants defective in interaction with CRM1 or Rev failed to supplement the Rev-RNA export activity and viral production, demonstrating the importance of these interactions. Rev-dependent reporter assays and proviral DNA-construct transfection-based studies in Staufen-2 knockout cells in the presence of leptomycin-B (LMB) revealed a significant reduction in CRM1-mediated Rev-dependent RNA export with decreased virus production as compared to Staufen-2 knockout background or LMB treatment alone, suggesting the relevance of these interactions in augmenting RNA export activity of Rev. Our observations provide further insights into the mechanistic intricacies of unspliced viral-RNA export to the cytoplasm and support the notion that abrogating such interactions can reduce HIV-1 proliferation.

Immunogenic profiling of Mycobacterium tuberculosis DosR protein Rv0569 reveals its ability to switch on Th1 based immunity.

Mycobacterium tuberculosis (M.tb) is a multifaceted bacterial pathogen known to infect more than 2 billion people globally. However, a majority of the individuals (>90%) show no overt clinical symptoms of active Tuberculosis (TB) and, it is reported that M.tb in these individuals resides in the latent form. Therefore, a huge burden of latently infected population poses serious threat to the human health. Inconsistent efficacy of BCG vaccine and poor understanding of latency-associated determinants contribute to the failure of combating M.tb. The discovery of DosR as the master regulator of dormancy, opened new avenues to understand the pathophysiology of the bacterium. Though the specific functions of various DosR genes are yet to be discovered, they have been reported as potent T-cell activators and could elicit strong protective immune responses. Rv0569 is a DosR-encoded conserved hypothetical protein overexpressed during dormancy. However, it is not clearly understood how this protein modulates the host immune response. In the present study, we have demonstrated that Rv0569 has a high antigenic index and induces enhanced secretion of Th1 cytokines IL-12p40 and TNF-α as compared to Th2 cytokine IL-10 in macrophages. Mechanistically, Rv0569 induced the transcription of these pro-inflammatory signatures through the activation of NF-κB pathway. Further, immunization of mice with DosR protein Rv0569 switched the immune response towards Th1-biased cytokine pattern, characterized by the enhanced production of IFN-γ, IL-12p40, and TNF-α. Rv0569 augmented the expansion of antigen-specific IFN-γ and IL-2 producing effector CD4+and CD8+ T-cells which are hallmarks of Th1 biased protective immunity. Additionally, IgG2a/IgG1 and IgG2b/IgG1 ratio in the serum of immunized mice further confirmed the ability of Rv0569 to skew Th1 biased immune response. In conclusion, we emphasize that Rv0569 has the ability to generate signals to switch on Th1-dominated responses and further suggest that it could be a potential vaccine candidate against latent M.tb infection.

ArgD of Mycobacterium tuberculosis is a functional N-acetylornithine aminotransferase with moonlighting function as an effective immune modulator.

Mycobacterium tuberculosis (M. tuberculosis) encodes an essential enzyme acetyl ornithine aminotransferase ArgD (Rv1655) of arginine biosynthetic pathway which plays crucial role in M. tuberculosis growth and survival. ArgD catalyzes the reversible conversion of N-acetylornithine and 2 oxoglutarate into glutamate-5-semialdehyde and L-glutamate. It also possesses succinyl diaminopimelate aminotransferase activity and can thus carry out the corresponding step in lysine biosynthesis. These essential roles played by ArgD in amino acid biosynthetic pathways highlight it as an important metabolic chokepoint thus an important drug target. We showed that M. tuberculosis ArgD rescues the growth of ΔargD E. coli grown in minimal media validating its functional importance. Phylogenetic analysis of M. tuberculosis ArgD showed homology with proteins in gram positive bacteria, pathogenic and non-pathogenic mycobacteria suggesting the essentiality of this protein. ArgD is a secretory protein that could be utilized by M. tuberculosis to modulate host innate immunity as its moonlighting function. In-silico analysis predicted it to be a highly antigenic protein. The recombinant ArgD protein when exposed to macrophage cells induced enhanced production of pro-inflammatory cytokines TNF, IL6 and IL12 in a dose dependent manner. ArgD also induced the increased production of innate immune effector molecule NOS2 and NO in macrophages. We also demonstrated ArgD mediated activation of the canonical NFkB pathway. Notably, we also show that ArgD is a specific TLR4 agonist involved in the activation of pro-inflammatory signaling for sustained production of effector cytokines. Intriguingly, ArgD protein treatment activated macrophages to acquire the M1 phenotype through the increased surface expression of MHCII and costimulatory molecules CD80 and CD86. ArgD induced robust B-cell response in immunized mice, validating its antigenicity potential as predicted by the in-silico analysis. These properties of M. tuberculosis ArgD signify its functional plasticity that could be exploited as a possible drug target to combat tuberculosis.

Encapsidation of Staufen-2 Enhances Infectivity of HIV-1.

Staufen, the RNA-binding family of proteins, affects various steps in the Human Immuno-Deficiency Virus (HIV-1) replication cycle. While our previous study established Staufen-2-HIV-1 Rev interaction and its role in augmenting nucleocytoplasmic export of RRE-containing viral RNA, viral incorporation of Staufen-2 and its effect on viral propagation were unknown. Here, we report that Staufen-2 interacts with HIV-1 Gag and is incorporated into virions and that encapsidated Staufen-2 boosted viral infectivity. Further, Staufen-2 gets co-packaged into virions, possibly by interacting with host factors Staufen-1 or antiviral protein APOBEC3G, which resulted in different outcomes on the infectivity of Staufen-2-encapsidated virions. These observations suggest that encapsidated host factors influence viral population dynamics and infectivity. With the explicit identification of the incorporation of Staufen proteins into HIV-1 and other retroviruses, such as Simian Immunodeficiency Virus (SIV), we propose that packaging of RNA binding proteins, such as Staufen, in budding virions of retroviruses is probably a general phenomenon that can drive or impact the viral population dynamics, infectivity, and evolution.

Dodging the Host Interferon-Stimulated Gene Mediated Innate Immunity by HIV-1: A Brief Update on Intrinsic Mechanisms and Counter-Mechanisms.

Host restriction factors affect different phases of a viral life cycle, contributing to innate immunity as the first line of defense against viruses, including HIV-1. These restriction factors are constitutively expressed, but triggered upon infection by interferons. Both pre-integration and post-integration events of the HIV-1 life cycle appear to play distinct roles in the induction of interferon-stimulated genes (ISGs), many of which encode antiviral restriction factors. However, HIV-1 counteracts the mechanisms mediated by these restriction factors through its encoded components. Here, we review the recent findings of pathways that lead to the induction of ISGs, and the mechanisms employed by the restriction factors such as IFITMs, APOBEC3s, MX2, and ISG15 in preventing HIV-1 replication. We also reflect on the current understanding of the counter-mechanisms employed by HIV-1 to evade innate immune responses and overcome host restriction factors. Overall, this mini-review provides recent insights into the HIV-1-host cross talk bridging the understanding between intracellular immunity and research avenues in the field of therapeutic interventions against HIV-1.

HIV co-receptor-tropism: cellular and molecular events behind the enigmatic co-receptor switching.

Recognition of cell-surface receptors and co-receptors is a crucial molecular event towards the establishment of HIV infection. HIV exists as several variants that differentially recognize the principal co-receptors, CCR5 and CXCR4, in different cell types, known as HIV co-receptor-tropism. The relative levels of these variants dynamically adjust to the changing host selection pressures to infect a vast repertoire of cells in a stage-specific manner. HIV infection sets in through immune cells such as dendritic cells, macrophages, and T-lymphocytes in the acute stage, while a wide range of other cells, including astrocytes, glial cells, B-lymphocytes, and epithelial cells, are infected during chronic stages. A change in tropism occurs during the transition from acute to a chronic phase, termed as co-receptor switching marked by a change in disease severity. The cellular and molecular events leading to co-receptor switching are poorly understood. This review aims to collate our present understanding of the dynamics of HIV co-receptor-tropism vis-à-vis host and viral factors, highlighting the cellular and molecular events involved therein. We present the possible correlations between virus entry, cell tropism, and co-receptor switching, speculating its consequences on disease progression, and proposing new scientific pursuits to help in an in-depth understanding of HIV biology.

Loop 1 of APOBEC3C Regulates its Antiviral Activity against HIV-1.

APOBEC3 deaminases (A3s) provide mammals with an anti-retroviral barrier by catalyzing dC-to-dU deamination on viral ssDNA. Within primates, A3s have undergone a complex evolution via gene duplications, fusions, arms race, and selection. Human APOBEC3C (hA3C) efficiently restricts the replication of viral infectivity factor (vif)-deficient Simian immunodeficiency virus (SIVΔvif), but for unknown reasons, it inhibits HIV-1Δvif only weakly. In catarrhines (Old World monkeys and apes), the A3C loop 1 displays the conserved amino acid pair WE, while the corresponding consensus sequence in A3F and A3D is the largely divergent pair RK, which is also the inferred ancestral sequence for the last common ancestor of A3C and of the C-terminal domains of A3D and A3F in primates. Here, we report that modifying the WE residues in hA3C loop 1 to RK leads to stronger interactions with substrate ssDNA, facilitating catalytic function, which results in a drastic increase in both deamination activity and in the ability to restrict HIV-1 and LINE-1 replication. Conversely, the modification hA3F_WE resulted only in a marginal decrease in HIV-1Δvif inhibition. We propose that the two series of ancestral gene duplications that generated A3C, A3D-CTD and A3F-CTD allowed neo/subfunctionalization: A3F-CTD maintained the ancestral RK residues in loop 1, while diversifying selection resulted in the RK â†’ WE modification in Old World anthropoids' A3C, possibly allowing for novel substrate specificity and function.

Mycobacterial Control of Host Mitochondria: Bioenergetic and Metabolic Changes Shaping Cell Fate and Infection Outcome.

Mitochondria, are undoubtedly critical organelle of a eukaryotic cell, which provide energy and offer a platform for most of the cellular signaling pathways that decide cell fate. The role of mitochondria in immune-metabolism is now emerging as a crucial process governing several pathological states, including infection, cancer, and diabetes. Mitochondria have therefore been a vulnerable target for several bacterial and viral pathogens to control host machinery for their survival, replication, and dissemination. Mycobacterium tuberculosis, a highly successful human pathogen, persists inside alveolar macrophages at the primary infection site, applying several strategies to circumvent macrophage defenses, including control of host mitochondria. The infection perse and specific mycobacterial factors that enter the host mitochondrial milieu perturb mitochondrial dynamics and function by disturbing mitochondrial membrane potential, shifting bioenergetics parameters such as ATP and ROS, orienting the host cell fate and thereby infection outcome. In the present review, we attempt to integrate the available information and emerging dogmas to get a holistic view of Mycobacterium tuberculosis infection vis-a-vis mycobacterial factors that target host mitochondria and changes therein in terms of morphology, dynamics, proteomic, and bioenergetic alterations that lead to a differential cell fate and immune response determining the disease outcome. We also discuss critical host factors and processes that are overturned by Mycobacterium tuberculosis, such as cAMP-mediated signaling, redox homeostasis, and lipid droplet formation. Further, we also present alternate dogmas as well as the gaps and limitations in understanding some of the present research areas, which can be further explored by understanding some critical processes during Mycobacterium tuberculosis infection and the reasons thereof. Toward the end, we propose to have a set of guidelines for pursuing investigations to maintain uniformity in terms of early and late phase, MOI of infection, infection duration and incubation periods, the strain of mycobacteria, passage numbers, and so on, which all work as probable variables toward different readouts. Such a setup would, therefore, help in the smooth integration of information across laboratories toward a better understanding of the disease and possibilities of host-directed therapy.

ROS-associated immune response and metabolism: a mechanistic approach with implication of various diseases.

The immune system plays a pivotal role in maintaining the defense mechanism against external agents and also internal danger signals. Metabolic programming of immune cells is required for functioning of different subsets of immune cells under different physiological conditions. The field of immunometabolism has gained ground because of its immense importance in coordination and balance of immune responses. Metabolism is very much related with production of energy and certain by-products. Reactive oxygen species (ROS) are generated as one of the by-products of various metabolic pathways. The amount, localization of ROS and redox status determine transcription of genes, and also influences the metabolism of immune cells. This review discusses ROS, metabolism of immune cells at different cellular conditions and sheds some light on how ROS might regulate immunometabolism.

Rewiring of Metabolic Network in Mycobacterium tuberculosis During Adaptation to Different Stresses.

Metabolic adaptation of Mycobacterium tuberculosis (M. tuberculosis) to microbicidal intracellular environment of host macrophages is fundamental to its pathogenicity. However, an in-depth understanding of metabolic adjustments through key reaction pathways and networks is limited. To understand how such changes occur, we measured the cellular metabolome of M. tuberculosis subjected to four microbicidal stresses using liquid chromatography-mass spectrometric multiple reactions monitoring (LC-MRM/MS). Overall, 87 metabolites were identified. The metabolites best describing the separation between stresses were identified through multivariate analysis. The coupling of the metabolite measurements with existing genome-scale metabolic model, and using constraint-based simulation led to several new concepts and unreported observations in M. tuberculosis; such as (i) the high levels of released ammonia as an adaptive response to acidic stress was due to increased flux through L-asparaginase rather than urease activity; (ii) nutrient starvation-induced anaplerotic pathway for generation of TCA intermediates from phosphoenolpyruvate using phosphoenolpyruvate kinase; (iii) quenching of protons through GABA shunt pathway or sugar alcohols as possible mechanisms of early adaptation to acidic and oxidative stresses; and (iv) usage of alternate cofactors by the same enzyme as a possible mechanism of rewiring metabolic pathways to overcome stresses. Besides providing new leads and important nodes that can be used for designing intervention strategies, the study advocates the strength of applying flux balance analyses coupled with metabolomics to get a global picture of complex metabolic adjustments.

Lupeol alters viability of SK-RC-45 (Renal cell carcinoma cell line) by modulating its mitochondrial dynamics.

Renal cell carcinoma (RCC) is the most common kidney cancer leading to 140,000 deaths per year. Among all RCCs 80% evolve from the epithelial proximal tubular cells within the kidney. There is a high tendency of developing chemoresistance and resistance to radiation therapy in most RCC patients. Therefore, kidney resection is considered as the most effective treatments for patients having localized RCC. There is a high tendency of post-operative recurrence among 20-40% of the patients and this recurrence is not curable. It is also clear that modern medicine has no curative treatment options against metastatic RCC. Lupeol [lup-20(29)-en-3ß-ol] is a pentacyclic triterpenoid compound naturally found in various edible fruits and in many traditionally used medicinal plants, and has been demonstrated as effective against highly metastatic melanoma and prostate cancers. The present study was designed to evaluate the effect of lupeol to RCC with molecular details. Treatment with lupeol on SK-RC-45 (a RCC cell line) with the LC50 dose of 40µM (for 48 h) induces mitochondrial hyper fission which eventually leads to apoptosis while SK-RC-45 counteracts by enhancing autophagy-mediated selective removal of fragmented mitochondria. This is the first study which concurrently reports the effects of lupeol on RCC and its effect on the mitochondrial dynamics of a cell. Herein, we conclude that lupeol has potential to be an effective agent against RCC with the modulation of mitochondrial dynamics.

Metabolomics Studies To Decipher Stress Responses in Mycobacterium smegmatis Point to a Putative Pathway of Methylated Amine Biosynthesis.

Mycobacterium smegmatis, the saprophytic soil mycobacterium, is routinely used as a surrogate system to study the human pathogen Mycobacterium tuberculosis It has also been reported as an opportunistic pathogen in immunocompromised hosts. In addition, it can exist in several ecological setups, thereby suggesting its capacity to adapt to a variety of environmental cues. In this study, we employed untargeted proton nuclear magnetic resonance (1H-NMR)-based metabolomics to identify metabolites and metabolic pathways critical for early adaptive responses to acidic stress, oxidative stress, and nutrient starvation in Mycobacterium smegmatis We identified 31, 20, and 46 metabolites that showed significant changes in levels in response to acidic, oxidative, and nutrient starvation stresses, respectively. Pathway analyses showed significant perturbations in purine-pyrimidine, amino-acid, nicotinate-nicotinamide, and energy metabolism pathways. Besides these, differential levels of intermediary metabolites involved in α-glucan biosynthesis pathway were observed. We also detected high levels of organic osmolytes, methylamine, and betaine during nutrient starvation and oxidative stress. Further, tracing the differential levels of these osmolytes through computational search tools, gene expression studies (using reverse transcription-PCR [RT-PCR]), and enzyme assays, we detected the presence of a putative pathway of biosynthesis of betaine, methylamine, and dimethylamine previously unreported in Mycobacterium smegmatisIMPORTANCE Alterations in metabolite levels provide fast and direct means to regulate enzymatic reactions and, therefore, metabolic pathways. This study documents, for the first time, the metabolic changes that occur in Mycobacterium smegmatis as a response to three stresses, namely, acidic stress, oxidative stress, and nutrient starvation. These stresses are also faced by intracellular mycobacteria during infection and therefore may be extended to frame therapeutic interventions for pathogenic mycobacteria. In addition to the purine-pyrimidine, amino acid, nicotinate-nicotinamide, and energy metabolism pathways that were found to be affected in response to different stresses, a novel putative methylamine biosynthesis pathway was identified to be present in Mycobacterium smegmatis.