Leishmania protein KMP-11 modulates cholesterol transport and membrane fluidity to facilitate host cell invasion.

The first step of successful infection by any intracellular pathogen relies on its ability to invade its host cell membrane. However, the detailed structural and molecular understanding underlying lipid membrane modification during pathogenic invasion remains unclear. In this study, we show that a specific Leishmania donovani (LD) protein, KMP-11, forms oligomers that bridge LD and host macrophage (MΦ) membranes. This KMP-11 induced interaction between LD and MΦ depends on the variations in cholesterol (CHOL) and ergosterol (ERG) contents in their respective membranes. These variations are crucial for the subsequent steps of invasion, including (a) the initial attachment, (b) CHOL transport from MΦ to LD, and (c) detachment of LD from the initial point of contact through a liquid ordered (Lo) to liquid disordered (Ld) membrane-phase transition. To validate the importance of KMP-11, we generate KMP-11 depleted LD, which failed to attach and invade host MΦ. Through tryptophan-scanning mutagenesis and synthesized peptides, we develop a generalized mathematical model, which demonstrates that the hydrophobic moment and the symmetry sequence code at the membrane interacting protein domain are key factors in facilitating the membrane phase transition and, consequently, the host cell infection process by Leishmania parasites.

Vitexin alters Staphylococcus aureus surface hydrophobicity to obstruct biofilm formation.

Cell Surface hydrophobicity is one of the determinant biophysical parameters of bacterial aggregation for being networked to form a biofilm. Phytoconstituent, like vitexin, has long been in use for their antibacterial effect. The present work demonstrates the role of vitexin in modulating Staphylococcus aureus surface hydrophobicity while aggregating to form biofilm and pathogenesis in a host. In planktonic form, vitexin shows minimum inhibitory concentration at 252 µg/ml against S. aureus. Sub-MIC doses of vitexin and antibiotics (26 µg/ml of vitexin, 55 µg/ml of azithromycin, and 2.5 µg/ml of gentamicin) were selected to treat S. aureus. Dead cell counts after treatment were studied through flow cytometry. As dead cell counts were minimal (<5 %), these doses were considered for all subsequent experiments. While studying aggregating cells, it was observed that vitexin reduces S. aureus surface hydrophobicity and membrane permeability at the sub-MIC dose of 26 µg/ml. The in silico binding analysis showed a higher binding affinity of vitexin with surface proteins (IcaA, DltA, and SasG) of S. aureus. Down-regulation of dltA and icaAB expression, along with the reduction in membrane potential with a sub-MIC dose of vitexin, explains reduced S. aureus surface hydrophobicity. Vitexin was found to interfere with S. aureus biofilm-associated protein biomass, EPS production, and swarming movement. Subsequently, the suppression of proteases production and down-regulation of icaAB and agrAC gene expression with a sub-MIC dose of vitexin explained the inhibition of S. aureus virulence in vitro. Besides, vitexin was also found to potentiate the antibiofilm activity of sub-MIC doses of gentamicin and azithromycin. Treatment with vitexin exhibits a protective response in S. aureus infected macrophages through modulation of expression of cytokines like IL-10 and IL-12p40 at protein and mRNA levels. Furthermore, CFU count and histological examination of infected mouse tissue (liver and spleen) justify the in vivo protective effect of vitexin from S. aureus biofilm-associated infection. From this study, it can be inferred that vitexin can reduce S. aureus surface hydrophobicity, leading to interference with aggregation at the time of biofilm formation and subsequent pathogenesis in a host.

Regulatory role of Transcription factor-EB (TFEB) in parasite control through alteration of antigen presentation in visceral leishmaniasis.

Leishmania donovani, an obligate intracellular parasite, the causative agent of visceral leishmaniasis is known to subvert the host immune system for its own survival. Although the precise mechanism is still unknown, emerging evidences indicate that L. donovani efficiently suppress MHC I mediated antigen presentation, rendering inadequate CD8+T cell activation and weakening host defense against parasite. The role of transcription factor EB (TFEB) was recognized in modulating antigen presentation besides its role in lysosomal biogenesis and function. Here, we investigated the regulatory role of TFEB in the modulation of presentation of Leishmania antigen in host tissue. Our results showed an increased expression of TFEB after Leishmania infection both in vitro and in vivo and there was a decrease in the expression of Th-1 cytokine IFNγ along with MHC class I and CD8+T cells indicating attenuation of cell mediated immunity and possibly MHC I restricted antigen presentation. Silencing of TFEB resulted in increased expression of IFNγ and MHC I along with increased CD8+T cells population without any significant change in CD4+T cell number. We also observed a decreased parasite burden in TFEB silenced condition which indicates enhanced parasite clearance by alteration of immunological response possibly through induction of presentation of Leishmania antigen through MHC I. The present study explains the role of TFEB silencing in parasite clearance through regulating the antigen presentation of Leishmania antigen thereby promises to formulate a potential therapeutic strategy against visceral leishmaniasis.

Study of epidemiological behaviour of malaria and its control in the Purulia district of West Bengal, India (2016-2020).

Purulia is a malaria-prone district in West Bengal, India, with approximately half of the blocks defined as malaria endemic. We analyzed the malaria case in each block of the Purulia district from January 1, 2016, to December 31, 2020. As per the API, 20 blocks of Purulia were assigned to four different categories (0-3) and mapped using ArcGIS software. An exponential decay model was fitted to forecast the trend of malaria cases for each block of Purulia (2021-2025). There was a sharp decrease in total malaria cases and API from 2016 to 2020 due to the mass distribution of LLINs. The majority of cases (72.63%) were found in ≥ 15-year age group. Males were more prone to malaria (60.09%). Malaria was highly prevalent among Scheduled Tribes (48.44%). Six blocks were reported in Category 3 (high risk) and none in Category 0 (no risk) in 2016, while no blocks were determined to be in Category 3, and three blocks were in Category 0 in 2020. The exponential decay model prediction is oriented towards gaining malaria-free status in thirteen blocks of Purulia by 2025. This study will incite the government to uphold and strengthen the current efforts to meet the malaria elimination goals.

Oncotherapeutic Application of Resveratrol-based Inorganic Nanoparticles.

BACKGROUND: Potential therapeutic benefits of natural phytoconstituents and the emergence of nano-structured drug delivery systems have expanded the scope of enhanced chemotherapy with minimal adverse effects. Various in vivo and in vitro studies have revealed Resveratrol to be a potent anti-carcinogenic agent. Researchers are currently applying the concept of nano-science for enhancing the delivery of phyto-drugs like resveratrol, in order to carry the drug to the affected tissues and organs of cancer patients with much ease and efficiency. METHODS: The current review emphasizes the use of inorganic nanoparticles for enhancing the delivery and efficacy of resveratrol into otherwise inaccessible tumorigenic tissues. CONCLUSION: The present review work summarizes a comprehensive update on the mechanism of actions of the resveratrol-based inorganic nanocomposite particles that are currently being studied against various cancer models. This work may be significant in laying the foundation for the future of metallic nanoparticles-based delivery and efficacy of phytochemicals in general and resveratrol in specific against non-invasive metastatic cancer.

Lupeol and amphotericin B mediate synergistic anti-leishmanial immunomodulatory effects in Leishmania donovani-infected BALB/c mice.

Leishmania donovani, a protozoan parasite, inflicts the disease Visceral leishmaniasis (VL) Worldwide. The only orally bioavailable drug miltefosine is toxic, whereas liposomal amphotericin B (AmpB) is expensive. Lupeol, a triterpenoid from Sterculia villosa bark, was exhibited immunomodulatory and anti-leishmanial activity in experimental VL. Herein, we evaluated synergism between sub-optimum dose of AmpB and lupeol in anti-leishmanial and immunomodulatory effects in L. donovani-infected BALB/c mice. We observed that a combination of sub-optimum dose of lupeol and AmpB significantly reduced the hepatic and splenic parasitic burden accompanied by enhanced nitric oxide production, robust induction of Th1 cytokines (IL-12 and IFN-γ) but suppressed Th2 cytokine (IL-10 and TGF- ß) production. The treatment with the lupeol-AmpB combination enhanced p38mitogen-activated protein kinase (p38MAPK), but reduced extracellular signal-related kinase (ERK-1/2), phosphorylation and up-regulated pro-inflammatory response. The present work thus indicates a lupeol-AmpB-mediated immunotherapeutic approach for eliminating the parasite-induced immunosuppression.

In vivo experiments demonstrate the potent antileishmanial efficacy of repurposed suramin in visceral leishmaniasis.

BACKGROUND: Treatment failure and resistance to the commonly used drugs remains a major obstacle for successful chemotherapy against visceral leishmaniasis (VL). Since the development of novel therapeutics involves exorbitant costs, the effectiveness of the currently available antitrypanosomatid drug suramin has been investigated as an antileishmanial, specifically for VL,in vitro and in animal model experiments. METHODOLOGY/PRINCIPAL: Leishmania donovani promastigotes were treated with suramin and studies were performed to determine the extent and mode of cell mortality, cell cycle arrest and other in vitro parameters. In addition, L. donovani infected BALB/c mice were administered suramin and a host of immunological parameters determined to estimate the antileishmanial potency of the drug. Finally, isothermal titration calorimetry (ITC) and enzymatic assays were used to probe the interaction of the drug with one of its putative targets namely parasitic phosphoglycerate kinase (LmPGK). FINDINGS: The in vitro studies revealed the potential efficacy of suramin against the Leishmania parasite. This observation was further substantiated in the in vivo murine model, which demonstrated that upon suramin administration, the Leishmania infected BALB/c mice were able to reduce the parasitic burden and also generate the host protective immunological responses. ITC and enzyme assays confirmed the binding and consequent inhibition of LmPGK due to the drug. CONCLUSIONS/SIGNIFICANCE: All experiments affirmed the efficacy of suramin against L. donovani infection, which could possibly lead to its inclusion in the repertoire of drugs against VL.

Conformational Switch Driven Membrane Pore Formation by Mycobacterium Secretory Protein MPT63 Induces Macrophage Cell Death.

Virulent Mycobacterium tuberculosis (MTB) strains cause cell death of macrophages (Mϕ) inside TB granuloma using a mechanism which is not well understood. Many bacterial systems utilize toxins to induce host cell damage, which occurs along with immune evasion. These toxins often use chameleon sequences to generate an environment-sensitive conformational switch, facilitating the process of infection. The presence of toxins is not yet known for MTB. Here, we show that MTB-secreted immunogenic MPT63 protein undergoes a switch from ß-sheet to helix in response to mutational and environmental stresses. MPT63 in its helical form creates pores in both synthetic and Mϕ membranes, while the native ß-sheet protein remains inert toward membrane interactions. Using fluorescence correlation spectroscopy and atomic force microscopy, we show further that the helical form undergoes self-association to produce toxic oligomers of different morphology. Trypan blue and flow cytometry analyses reveal that the helical state can be utilized by MTB for killing Mϕ cells. Collectively, our study emphasizes for the first time a toxin-like behavior of MPT63 induced by an environment-dependent conformational switch, resulting in membrane pore formation by toxic oligomers and Mϕ cell death.

Functional aspects of T cell diversity in visceral leishmaniasis.

Co-ordination between innate and adaptive immunity is a foremost crucial immunological interactions. The interaction is beneficial for the survival of the host against infectious agent and also detrimental for the pathogen during their future encounter. Major cellular components to bridge the gap of innate and adaptive immune system include B cells, varieties of T cell subsets and their interaction with antigen presenting cells. T cells are the components of immune system which recognise antigen that are specifically presented with the different class of MHC molecules like MHCI and MHCII marking the diversity of exogenous and endogenous nature of antigen. T cells further differentiate in varieties of morphological and immunological forms like CD4+, CD8+ T cells, Th-17, Treg and γδ-T cells based on the nature of antigen, interaction and polarizing factors. Therefore the evolutionary selections of these diversities have a different functional aspect which is not only dependent upon their percentage presence but more promisingly dependent upon their physiological state and local environment. Thus this review is highlighting the major contributions of T cells subsets using an infectious disease model of visceral leishmaniasis and also helpful in explaining the reason for the non-responsiveness of the T cells subsets during the onset and progression of infection.

Free radical stress induces DNA damage response in RAW264.7 macrophages during Mycobacterium smegmatis infection.

Genomic instability resulting from oxidative stress responses may be traced to chromosomal aberration. Oxidative stress suggests an imbalance between the systemic manifestation of reactive free radicals and biological system's ability to repair resulting DNA damage and chromosomal aberration. Bacterial infection associated insult is considered as one of the major factors leading to such stress conditions. To study free radical responses by host cells, RAW 264.7 macrophages were infected with non-pathogenic M. smegmatis mc2155 at different time points. The infection process was followed up with an assessment of free radical stress, cytokine, toll-like receptors (TLRs) and the resulting DNA damage profiles. Results of CFU count showed that maximum infection in macrophages was achieved after 9 h of infection. Host responses to the infection across different time periods were validated from nitric oxide quantification and expression of iNOS and were plotted at regular intervals. IL-10 and TNF-α expression profile at protein and mRNA level showed a heightened pro-inflammatory response by host macrophages to combat M. smegmatis infection. The expression of TLR4, a receptor for recognition of mycobacteria, in infected macrophages reached the highest level at 9 h of infection. Furthermore, comet tail length, micronuclei and γ-H2AX foci recorded the highest level at 9 h of infection, pointing to the fact that breakage in DNA double strands in macrophage reaches its peak at 9 h of infection. In contrast, treatment with ROS inhibitor N-acetyl-L-cysteine (NAC) prevented host cell death through reduction in oxidative stress and DNA damage response during M. smegmatis infection. Therefore, it can be concluded that enhanced oxidative stress response in M. smegmatis infected macrophages might be correlated with DNA damage response.

Immunomodulatory effect of Arabinosylated lipoarabinomannan restrict the progression of visceral leishmaniasis through NOD2 inflammatory pathway: Functional regulation of T cell subsets.

NOD like receptors (NLR) are essential pathogen associated molecular pattern receptors of cytoplasmic origin. During several intracellular parasitic infections NLR played vital role for host protective immune response against the pathogen. Amongst various classes of NLR, NOD1 and NOD2 had been extensively studied and were found to be the most active member of the NLR family. Therefore, we wanted to study the role of NOD1/NOD2 during Leishmania donovani infection and the mechanism behind the utilization of this pathway as a therapeutic approach. Using the infected model of macrophage and BALB/c mice the expression of NOD1 and NOD2 were analysed. Our study showed that NOD2 but not NOD1 has been exploited during experimental VL, leading to the imbalance between Th-1/Th-2 cytokines profile. Over-expression of NOD2 and stimulation with its ligand muramyl dipeptide leads to successful clearance of parasite. During in vivo experiments we found that arabinosylated lipoarabinomannan helps in the restoration of NOD2 and with MDP in combination leads to effective clearance of parasite which rescued host protective immunity and comparatively more effective than Mw and MDP combination resulting in increase T cell response. Consequently, our study highlighted the significance of NOD2 during infection the immune-modulations of which can be used as a therapeutic target.

Nanoparticle Induced Conformational Switch Between α-Helix and ß-Sheet Attenuates Immunogenic Response of MPT63.

Although significant efforts have been devoted to develop nanoparticle-based biopharmaceuticals, it is not understood how protein conformation and nanoparticle surface modulate each other in optimizing the activity and/or toxicity of the biological molecules. This is particularly important for a protein, which can adopt different conformational states separated by a relatively small energy barrier. In this paper, we have studied nanoparticle binding-induced conformational switch from ß-sheet to α-helix of MPT63, a small major secreted protein from Mycobacterium tuberculosis and a drug target against Tuberculosis. The binding of magnetite nanoparticles to MPT63 results in a ß-sheet to α-helix switch near the sequence stretch between the 19th and 30th amino acids. As a consequence, the immunogenic response of the protein becomes compromised, which could be restored by protein engineering. This study emphasizes that conformational stability toward NP surface binding may require optimization involving genetic engineering for development of a nanoparticle conjugated pharmaceutical.

TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner.

Ceramide is one of the important cellular components involved in cancer regulation and exerts its pleiotropic role in the protective immune response without exhibiting any adverse effects during malignant neoplasm. Although, the PKCδ-ceramide axis in cancer cells has been an effective target in reduction of cancer, involvement of PKCδ in inducing nephrotoxicity have become a major questionnaire. In the present study, we have elucidated the mechanism by which cisplatin exploits the ceramide to render cancer cell apoptosis leading to the abrogation of malignancy in a PKCδ independent pathway with lesser toxicity. Our study revealed that cisplatin treatment in PKCδ silenced melanoma cells induces ceramide mediated apoptosis. Moreover, cisplatin induced upregulation of the transcription factor IRF1 leading to the induction of the transcriptional activity of the TNFα promoter was evident from the pharmacological inhibition and RNA interference studies. Increased cellular expression of TNFα resulted in an elevated ceramide generation by stimulating acid-sphingomyelinase and cPLA2. Furthermore, reciprocity in the regulation of sphingosine kinase 1 (Sphk1) and sphingosine kinase 2 (Sphk2) during PKCδ independent ceramide generation was also observed during cisplatin treatment. PKCδ inhibited murine melanoma model showed reduction in nephrotoxicity along with tumor regression by ceramide generation. Altogether, the current study emphasized the unexplored signaling cascade of ceramide generation by cisplatin during PKCδ silenced condition, which is associated with increased TNFα generation. Our findings enlightened the detailed mechanistic insight of ceramide mediated signaling by chemotherapeutic drugs in cancer therapy exploring a new range of targets for cancer treatment strategies.

Immunomodulation of dual specificity phosphatase 4 during visceral leishmaniasis.

DUSP4, an inducible protein has a substrate specificity toward ERK1/2, a component of MAP kinase which is enhanced during Leishmania infection. The DUSP4-/- mice show increased susceptibility towards the infection caused by Toxoplasma gondii and Leishmania mexicana. These observations emphatically established the fact that unlike DUSP1, DUSP4 has host protective role. In our study, it has been Leishmania donovani, the causative agent of visceral leishmaniasis (VL) significantly reduced the expression of DUSP4 during infection. In order to find out the host protective role of DUSP4 in macrophages during VL, we silenced DUSP4 prior to infection and the parasite number within macrophage was counted. Under DUSP4 knock-down condition, phosphorylation of p38 MAPK and generation of pro-inflammatory response like IL-12, TNF-α, and iNOS was decreased significantly. Silencing DUSP4 promoted the phosphorylation of ERK1/2 and the generation of anti-inflammatory response like- IL-10, TGF-ß with increased Arginase-1 and Cox-2 activity. Glycyrrhizic Acid (GA), an immunomodulator, already known to suppress L. donovani infection, found to up-regulate DUSP4 expression during L. donovani infection. On the other hand, GA failed to increase Th1 cytokine production and decrease Th2 response during DUSP4 knock-down condition suggesting the key role of DUSP4 while providing protection during L. donovani infection.

Antileishmanial and immunomodulatory activities of lupeol, a triterpene compound isolated from Sterculia villosa.

Visceral leishmaniasis (VL) is one of the most severe forms of leishmaniasis, caused by the protozoan parasite Leishmania donovani. Nowadays there is a growing interest in the therapeutic use of natural products to treat parasitic diseases. Sterculia villosa is an ethnomedicinally important plant. A triterpenoid was isolated from this plant and was screened for its antileishmanial and immunomodulatory activities in vitro and in vivo. Biochemical colour test and spectroscopic data confirmed that the isolated pure compound was lupeol. Lupeol exhibited significant antileishmanial activity, with IC50 values of 65 ± 0.41 µg/mL and 15 ± 0.45 µg/mL against promastigote and amastigote forms, respectively. Lupeol caused maximum cytoplasmic membrane damage of L. donovani promastigote at its IC50 dose. It is well known that during infection the Leishmania parasite exerts its pathogenicity in the host by suppressing nitric oxide (NO) production and inhibiting pro-inflammatory responses. It was observed that lupeol induces NO generation in L. donovani-infected macrophages, followed by upregulation of pro-inflammatory cytokines and downregulation of anti-inflammatory cytokines. Lupeol was also found to reduce the hepatic and splenic parasite burden through upregulation of the pro-inflammatory response in L. donovani-infected BALB/c mice. Strong binding affinity of lupeol was observed for four major potential drug targets, namely pteridine reductase 1, adenine phosphoribosyltransferase, lipophosphoglycan biosynthetic protein and glycoprotein 63 of L. donovani, which also supported its antileishmanial and immunomodulatory activities. Therefore, the present study highlights the antileishmanial and immunomodulatory activities of lupeol in an in vitro and in vivo model of VL.

Mycobacterium indicus pranii (Mw)-mediated protection against visceral leishmaniasis by reciprocal regulation of host dual-specificity phosphatases.

Leishmania donovani resides within the host macrophages by dampening host defence mechanisms and thereby it modulates the host cell functions for its survival. Multiple host cell factors compete during the interplay between the host and the parasite. Roles for dual-specificity phosphatases (DUSPs) are implicated in various pathological conditions. However, the reciprocity of these DUSPs was unknown in L. donovani infection in a susceptible model. Here, we show that Mycobacterium indicus pranii (Mw), an immunomodulator, reciprocally regulates DUSP1 and DUSP6 through the TLR4 pathway. Association of PKC-ß with DUSP6 increases after Mw treatment resulting in decreased IL-10, phosphorylation of ERK1/2 and Arginase-1, whereas Mw treatment decreases the association between PKC-ε and DUSP1 resulting in increased IL-12, phosphorylation of p38 and inducible nitric oxide synthase expression. Silencing of DUSP1 or over-expression of DUSP6 in L. donovani-infected BALB/c mice decreases the parasite burden by inducing IL-12 and reducing IL-10 production. Therefore, we identify DUSP1 and DUSP6 as therapeutic targets, functions of which could be favourably modulated by Mw during L. donovani infection.

SLA-PGN-primed dendritic cell-based vaccination induces Th17-mediated protective immunity against experimental visceral leishmaniasis: a crucial role of PKCß.

Emergence of drug resistance during visceral leishmaniasis (VL) is a major obstacle imposed during successful therapy. An effective vaccine strategy against this disease is therefore necessary. Our present study exploited the SLA (soluble leishmanial antigen) and PGN (peptidoglycan) stimulated bone marrow-derived dendritic cells (DCs) as a suitable vaccine candidate during experimental VL. SLA-PGN-stimulated DCs showed a significant decrease in hepatic and splenic parasite burden, which were associated with increased production of nitric oxide and pro-inflammatory cytokines such as IL-12, IFN-γ and IL-17. Elevated level of IL-17 was accompanied with the generation of more Th17 cells. Further studies on DC provided the evidence that these SLA-PGN-stimulated DCs played an important role in providing necessary cytokines such as IL-6, IL-23 and TGF-ß for the generation of Th17 cells. Interestingly, inhibition of protein kinase C-ß (PKCß) in DCs led to decreased production of Th17 polarizing cytokines, causing reduction of the Th17 population size. Altogether, our finding highlighted the important role of DC-based PKCß in regulation of the function and generation of Th17 cells.