Identification of a De Novo Peptide against Palmitoyl Acyltransferase 6 to Block Survivability and Infectivity of Leishmania donovani.

Palmitoylation is an essential post-translational modification in Leishmania donovani, catalyzed by enzymes called palmitoyl acyl transferases (PATs) and has an essential role in virulence. Due to the toxicity and promiscuity of known PAT inhibitors, identification of new molecules is needed. Herein, we identified a specific novel de novo peptide inhibitor, PS1, against the PAT6 Leishmania donovani palmitoyl acyl transferase (LdPAT6). To demonstrate specific inhibition of LdPAT6 by PS1, we employed a bacterial orthologue system and metabolic labeling-coupled click chemistry where both LdPAT6 and PS1 were coexpressed and displayed palmitoylation suppression. Furthermore, strong binding of the LdPAT6-DHHC domain with PS1 was observed through analysis using microscale thermophoresis, ELISA, and dot blot assay. PS1 specific to LdPAT6 showed significant growth inhibition in promastigotes and amastigotes by expressing low cytokines levels and invasion. This study reveals discovery of a novel de novo peptide against LdPAT6-DHHC which has potential to block survivability and infectivity of L. donovani.

Screening of traditional medicinal plant extracts and compounds identifies a potent anti-leishmanial diarylheptanoid from Siphonochilus aethiopicus.

Available anti-leishmanial drugs are associated with toxic side effects, necessitating the search for safe and effective alternatives. This study is focused on identifying traditional medicinal plant natural products for anti-leishmanial potential and possible mechanism of action. Compounds S and T. cordifolia residual fraction (TC-5) presented the best anti-leishmanial activity (IC50: 0.446 and 1.028 mg/ml) against promastigotes at 48 h and less cytotoxicity to THP-1 macrophages. These test agents elicited increased expression of pro-inflammatory cytokines; TNFα and IL-12. In infected untreated macrophages, NO release was suppressed but was significantly (p < 0.05) increased in infected cells treated with compound S. Importantly, Compound S was found to interact with LdTopoIIdimer in silico, resulting in a likely reduced ability of nucleic acid (dsDNA)-remodelling and, as a result, parasite proliferation in vitro. Thereby, Compound S possesses anti-leishmanial activity and this effect occurs via a Th1-mediated pro-inflammatory response. An increase in NO release and its inhibitory effect on LdTopoII may also contribute to the anti-leishmanial effect of compound S. These results show the potential of this compound as a potential starting point for the discovery of novel anti-leishmanial leads.Communicated by Ramaswamy H. Sarma.

Targeting an essential Plasmodium cold shock protein to block growth and transmission of malaria parasite.

Cold shock proteins are characterized by the presence of one or more cold shock domains that bestow them with nucleic acid binding ability. Although cold shock proteins are well characterized in bacteria, plants and humans, there is no information on their existence and role in malaria parasite. Here, we have identified and delineated the function of a cold shock protein of Plasmodium falciparum (Pf) 'PfCoSP'. We demonstrate that PfCoSP exhibits nucleic acid binding properties and regulates gene expression. PfCoSP promotes microtubule assembly by interacting with Pf α/ß tubulin. We identified a human cold shock protein LIN28A inhibitor 'LI71' as a binding partner of PfCoSP which inhibited PfCoSP-DNA and α/ß tubulin interactions and, also inhibited the development of asexual blood stages and gametocyte stage of malaria parasite. Because PfCoSP is essential for parasite survival, characterization of its interacting partners may form the basis for development of future anti-malarials.

Cuscuta reflexa Possess Potent Inhibitory Activity Against Human Malaria Parasite: An In Vitro and In Vivo Study.

Drug resistance to practically all antimalarial drugs in use necessitate the development of new chemotherapeutics against malaria. In this aspect, traditionally used plants with folklore reputation are the pillar for drug discovery. Cuscuta reflexa being traditionally used in the treatment of malaria in Odisha, India we aimed to experimentally validate its antimalarial potential. Different solvent extracts of C. reflexa or column fractions from a promising solvent extract were evaluated for in vitro anti-plasmodial activity against Plasmodium falciparum strain Pf3D7. Potent fractions were further evaluated for inhibition of parasite growth against different drug resistant strains. Safety of these fractions was determined by in vitro cyto-toxicity, and therapeutic effectiveness was evaluated by suppression of parasitemia and improvement in survival of experimental mice. Besides, their immunomodulatory effect was investigated in Pf-antigen stimulated RAW cells. GCMS fingerprints of active fractions was determined. Column separation of methanol extract which showed the highest in vitro antiplasmodial activity (IC50 = 14.48 µg/ml) resulted in eleven fractions, three of which (F2, F3, and F4) had anti-plasmodial IC50 ranging from ≤ 10 to 2.2 µg/ml against various P. falciparum strains with no demonstration of in vitro cytotoxicity. F4 displayed the highest in vivo parasite suppression, and had a mean survival time similar to artesunate (19.3 vs. 20.6 days). These fractions significantly modulated expression of inflammatory cytokines in Pf-antigen stimulated RAW cells. The findings of the study confirm the antimalarial potential of C. reflexa. Exploration of phyto-molecules in GCMS fingerprints of active fractions is warranted for possible identification of lead anti-malarial phyto-drugs.

Host-Erythrocytic Sphingosine-1-Phosphate Regulates Plasmodium Histone Deacetylase Activity and Exhibits Epigenetic Control over Cell Death and Differentiation.

The evolution of resistance to practically all antimalarial drugs poses a challenge to the current malaria elimination and eradication efforts. Given that the epigenome of Plasmodium falciparum governs several crucial parasite functions, pharmaceutical interventions with transmission-blocking potential that target epigenetic molecular markers and regulatory mechanisms are likely to encounter drug resistance. In the malaria parasite, histone deacetylases (HDACs) are essential epigenetic modulators that regulate cellular transcriptional rearrangements, notably the molecular mechanisms underlying parasite proliferation and differentiation. We establish "lipid sequestration" as a mechanism by which sphingolipids, specifically Sphingosine-1-Phosphate (S1P) (a metabolic product of Sphingosine Kinase 1 [SphK-1]), regulate epigenetic reprogramming in the parasite by interacting with, and modulating, the histone-deacetylation activity of PfHDAC-1, thereby regulating Plasmodium pathogenesis. Furthermore, we demonstrate that altering host S1P levels with PF-543, a potent and selective Sphk-1 inhibitor, dysregulates PfHDAC-1 activity, resulting in a significant increase in the global histone acetylation signals and, consequently, transcriptional modulation of genes associated with gametocytogenesis, virulence, and proliferation. Our findings point to a hitherto unrecognized functional role for host S1P-mediated sphingolipid signaling in modulating PfHDAC-1's enzymatic activity and, as a result, the parasite's dynamic genome-wide transcriptional patterns. The epigenetic regulation of parasite proliferation and sexual differentiation offers a novel approach for developing host-targeted therapeutics to combat malaria resistance to conventional regimens. IMPORTANCE Sphingolipid is an 18-carbon amino-alcohol-containing lipid with a sphingosine backbone, which when phosphorylated by sphingosine kinase 1 (SphK-1), generates sphingosine-1-phosphate (S1P), an essential lipid signaling molecule. Dysregulation of S1P function has been observed in a variety of pathologies, including severe malaria. The malaria parasite Plasmodium acquires a host S1P pool for its growth and survival. Here, we describe the molecular attuning of histone deacetylase-1 (PfHDAC-1), a crucial epigenetic modulator that contributes to the establishment of epigenetic chromatin states and parasite survival, in response to S1P binding. Our findings highlight the host lipid-mediated epigenetic regulation of malaria parasite key genes.

Rapid diagnosis of Plasmodium falciparum malaria using a point-of-care loop-mediated isothermal amplification device.

LAMP diagnosis of malaria is simple and cost-effective with acceptable sensitivity and specificity as compared to standard diagnostic modules such as microscopy, RDTs and nested PCR, and thus its deployment for onsite screening of malaria in resource-limited regions is under consideration. However, the requirement of an electricity-operated dry bath and bulky read-out unit is still a major concern. In an effort to simplify this limitation, we have developed a portable LAMP device and fluorescence readout unit which can be used in the rapid point-of-care diagnosis of malaria. We have developed a point-of-care diagnostic LAMP device that is easy to operate by a mobile application, and the results can be quantified with a fluorescent readout unit. The diagnostic performance of the device was evaluated in 90 P. falciparum-infected clinical isolates stored at 4°C for 6-7 years and 10 freshly collected isolates from healthy volunteers. The LOD and quantitative ability of LAMP in estimating parasitemia levels were revealed with laboratory-grown P. falciparum strain (3D7). The LAMP assay performed in our device was exclusive for P. falciparum detection with sensitivity and specificity determined to be 98.89% and 100%, respectively, in clinical isolates. The LOD was documented to be 1 parasite/µl at the cut-off ADC value of 20. Parasite density estimated from ADC values showed concordance with microscopically determined parasite density of the cultured P. falciparum 3D7 strain. The LAMP assay performed in our device provides a possible portable platform for its deployment in the point-of-care diagnosis of malaria. Further validation of the quantitative ability of the assay with freshly collected or properly stored clinical samples of known parasitemia is necessary for field applicability.

Host SUMOylation Pathway Negatively Regulates Protective Immune Responses and Promotes Leishmania donovani Survival.

SUMOylation is one of the post-translational modifications that have recently been described as a key regulator of various cellular, nuclear, metabolic, and immunological processes. The process of SUMOylation involves the modification of one or more lysine residues of target proteins by conjugation of a ubiquitin-like, small polypeptide known as SUMO for their degradation, stability, transcriptional regulation, cellular localization, and transport. Herein, for the first time, we report the involvement of the host SUMOylation pathway in the process of infection of Leishmania donovani, a causative agent of visceral leishmaniasis. Our data revealed that infection of L. donovani to the host macrophages leads to upregulation of SUMOylation pathway genes and downregulation of a deSUMOylating gene, SENP1. Further, to confirm the effect of the host SUMOylation on the growth of Leishmania, the genes associated with the SUMOylation pathway were silenced and parasite load was analyzed. The knockdown of the SUMOylation pathway led to a reduction in parasitic load, suggesting the role of the host SUMOylation pathway in the disease progression and parasite survival. Owing to the effect of the SUMOylation pathway in autophagy, we further investigated the status of host autophagy to gain mechanistic insights into how SUMOylation mediates the regulation of growth of L. donovani. Knockdown of genes of host SUMOylation pathway led to the reduction of the expression levels of host autophagy markers while promoting autophagosome-lysosome fusion, suggesting SUMOylation-mediated autophagy in terms of autophagy initiation and autophagy maturation during parasite survival. The levels of reactive oxygen species (ROS) generation, nitric oxide (NO) production, and pro-inflammatory cytokines were also elevated upon the knockdown of genes of the host SUMOylation pathway during L. donovani infection. This indicates the involvement of the SUMOylation pathway in the modulation of protective immune responses and thus favoring parasite survival. Taken together, the results of this study indicate the hijacking of the host SUMOylation pathway by L. donovani toward the suppression of host immune responses and facilitation of host autophagy to potentially facilitate its survival. Targeting of SUMOylation pathway can provide a starting point for the design and development of novel therapeutic interventions to combat leishmaniasis.

Chromatin-Remodeling Factor BRG1 Is a Negative Modulator of L. donovani in IFNγ Stimulated and Infected THP-1 Cells.

Intracellular pathogens manipulate the host cell for their own survival by contributing to modifications of host epigenome, and thus, altering expression of genes involved in the pathogenesis. Both ATP-dependent chromatin remodeling complex and histone modifications has been shown to be involved in the activation of IFNγ responsive genes. Leishmania donovani is an intracellular pathogen that causes visceral leishmaniasis. The strategies employed by Leishmania donovani to modulate the host epigenome in order to overcome the host defense for their persistence has been worked out in this study. We show that L. donovani negatively affects BRG1, a catalytic subunit of mammalian SWI/SNF chromatin remodeling complex, to alter IFNγ induced host responses. We observed that L. donovani infection downregulates BRG1 expression both at transcript and protein levels in cells stimulated with IFNγ. We also observed a significant decrease in IFNγ responsive gene, Class II transactivator (CIITA), as well as its downstream genes, MHC-II (HLA-DR and HLA-DM). Also, the occupancy of BRG1 at CIITA promoters I and IV was disrupted. A reversal in CIITA expression and decreased parasite load was observed with BRG1 overexpression, thus, suggesting BRG1 is a potential negative regulator for the survival of intracellular parasites in an early phase of infection. We also observed a decrease in H3 acetylation at the promoters of CIITA, post parasite infection. Silencing of HDAC1, resulted in increased CIITA expression, and further decreased parasite load. Taken together, we suggest that intracellular parasites in an early phase of infection negatively regulates BRG1 by using host HDAC1 for its survival inside the host.

Rapid diagnosis of Leishmania infection with a portable loopmediated isothermal amplification device.

L. donovani is an intracellular protozoan parasite, that causes visceral leishmaniasis (VL), and consequently, post-kala azar dermal leishmaniasis (PKDL). Diagnosis and treatment of leishmaniasis is crucial for decreasing its transmission. Various diagnostic techniques like microscopy, enzyme-linked immunosorbent assays (ELISA) and PCR-based methods are used to detect leishmaniasis infection. More recently, loop-mediated isothermal amplification (LAMP) assay has emerged as an ideal diagnostic measure for leishmaniasis, primarily due to its accuracy, speed and simplicity. However, point-of-care diagnosis is still not been tested with the LAMP assay. We have developed a portable LAMP device for the monitoring of Leishmania infection. The LAMP assay performed using our device can detect and amplify as little as 100 femtograms of L. donovani DNA. In a preliminary study, we have shown that the device can also amplify L. donovani DNA present in VL and PKDL patient samples with high sensitivity (100%), specificity (98%) and accuracy (99%), and can be used both for diagnostic and prognostic analysis. To our knowledge, this is the first report to describe the development and application of a portable LAMP device which has the potential to evolve as a point-of-care diagnostic and prognostic tool for Leishmania infections in future.

FAT1 is a novel upstream regulator of HIF1α and invasion of high grade glioma.

The hypoxic microenvironment is an important contributor of glioblastoma (GBM) aggressiveness via HIF1α, while tumour inflammation is profoundly influenced by FAT Atypical Cadherin (FAT1). This study was designed to explore the functional interaction and significance of FAT1 and HIF1α under severe hypoxia-mimicking tumour microenvironment in primary human tumours. We first identified a positive correlation of FAT1 with HIF1α and its target genes in GBM tumour specimens, revealing the significance of the FAT1-HIF1α axis in glioma cells. We found that severe hypoxia leads to an increased expression of FAT1 and HIF1α in U87MG and U373MG cells. To reveal the relevance of FAT1 under hypoxic conditions, we depleted endogenous FAT1 under hypoxia and found a substantial reduction in the expression of HIF1α and its downstream target genes like CA9, GLUT1, VEGFA, MCT4, HK2, BNIP3 and REDD1, as well as a significant reduction in the invasiveness in GBM cells. At the molecular level, under hypoxia the FAT1 depletion-associated reduction in HIF1α was due to compromised EGFR-Akt signaling as well as increased VHL-dependent proteasomal degradation of HIF1α. In brief, for the first time, these results reveal an upstream master regulatory role of FAT1 in the expression and role of HIF1α under hypoxic conditions and that FAT1-HIF1α axis controls the invasiveness of GBM. Hence, FAT1 represents a novel potential therapeutic target for GBM.