In vitro screening of natural product-based compounds for leishmanicidal activity.

Leishmaniasis is one of the major parasitic diseases, caused by obligate intracellular protozoa Leishmania, having high mortality as well as morbidity rate. As there is no human licensed vaccine available against leishmaniasis, chemotherapy remains the major way of combating this disease. Many disadvantages are known to be associated with the current drug regime including severe side effects and toxicity, long duration and expensive treatment, and the emergence of resistance. An alternative approach is being utilized to search for active molecules using natural sources, rather than relying on synthetic drugs. Many plant-derived secondary metabolites like phenolic compounds, steroids, quinones, etc. are being extensively investigated for their anti-leishmanial potential. One such group of complex phenolic compounds are diarylheptanoids. These compounds have been shown to exhibit anti-inflammatory, anti-parasitic, anti-fungal, and other pharmacological activities. In the present study, a set of sixteen tetrahydropyran derivatives including three natural products were obtained in lyophilized form. These compounds with trans-2,6-disubstituted tetrahydropyrans, Diospongin A, Diospongin B (isolated from Dioscorea spongiosa) and Centrolobine (Centrolobium sclerophyllum) as parent compounds were synthesized by the reaction of 1-phenyl-1-triemthylsiloxyethylene with six-membered cyclic hemiacetals in the presence of iodine as a catalyst. All the sixteen synthesized tetrahydropyran derivatives were used for toxicity analysis against L. donovani promastigotes, amastigotes and THP-1-derived human macrophages. IC50 values and selectivity index were calculated for all the compounds. Out of these sixteen, five compounds showed the best effect in vitro in terms of both leishmanicidal activity and non-toxicity to human macrophages.

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.

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.

Role of two aminoacyl-tRNA synthetase associated proteins (Endothelial Monocyte Activating Polypeptides 1 and 2) of Leishmania donovani in chemotaxis of human monocytes.

Visceral leishmaniasis is caused by the protozoan parasite Leishmania donovani. It is a fatal form of leishmaniasis prevalent in Indian subcontinent. Since there are no human licensed vaccines available for leishmaniasis, chemotherapeutic drugs remain the only means for combating parasitic infections. We have earlier identified a total of 26 amino-acyl tRNA synthetases (aaRS) along with five stand-alone editing domains and two aaRS-associated proteins in Leishmania donovani. In addition to their canonical role of tRNA aminoacylation, aaRS have been involved in novel functions by acquiring novel domains during evolution. The aaRS-associated proteins have been reported to be analogous to a human cytokine, EMAP II, as they possess a modified version of the heptapeptide motif responsible for the cytokine activity. In this manuscript, we report the characterization of two L. donovani aminoacyl-tRNA synthetase associated proteins which showed a human chemokine like activity. Both the proteins, L. donovani EMAP-1 and EMAP-2, possess a modified form of the heptapeptide motif, which is responsible for cytokine activity in human EMAP-2. LdEMAP-1 and LdEMAP-2 were cloned, expressed, and purified. Both LdEMAP-1 and LdEMAP-2 proteins in the promastigote stage were found to be localized in cytoplasm as confirmed by immunofluorescence. In case of L. donovani infected human THP-1 derived macrophages, secretion of LdEMAP-1 and LdEMAP-2 proteins in the cytosol of the macrophages was observed. The role of LdEMAP-1 and LdEMAP-2 in the aminoacylation of rLdTyrRS was also tested and LdEMAP-2 but not LdEMAP-1 increased the rate of aminoacylation of tyrosyl tRNA synthetase (rLdTyrRS). L. donovani EMAP-1 and EMAP-2 proteins managed to exhibit the capability of attracting human origin cells as determined by chemotaxis assay, and also were able to induce the secretion of cytokines from macrophages like their human counterpart (EMAP II). Our working hypothesis is that both of these proteins might be involved in helping the parasite to establish the infection within the host.

Sensing Host Arginine Is Essential for Leishmania Parasites' Intracellular Development.

Arginine homeostasis in lysosomes is critical for the growth and metabolism of mammalian cells. Phagolysosomes of macrophages are the niche where the parasitic protozoan Leishmania resides and causes human leishmaniasis. During infection, parasites encounter arginine deprivation, which is monitored by a sensor on the parasite cell surface. The sensor promptly activates a mitogen-activated protein kinase 2 (MAPK2)-mediated arginine deprivation response (ADR) pathway, resulting in upregulating the abundance and activity of the Leishmania arginine transporter (AAP3). Significantly, the ADR is also activated during macrophage infection, implying that arginine levels within the host phagolysosome are limiting for growth. We hypothesize that ADR-mediated upregulation of AAP3 activity is necessary to withstand arginine starvation, suggesting that the ADR is essential for parasite intracellular development. CRISPR/Cas9-mediated disruption of the AAP3 locus yielded mutants that retain a basal level of arginine transport but lack the ability to respond to arginine starvation. While these mutants grow normally in culture, they were impaired in their ability to develop inside THP-1 macrophages and were ∼70 to 80% less infective in BALB/c mice. Hence, inside the host macrophage, Leishmania must overcome the arginine "hunger games" by upregulating the transport of arginine via the ADR. We show that the ability to monitor and respond to changes in host metabolite levels is essential for pathogenesis.IMPORTANCE In this study, we report that the ability of the human pathogen Leishmania to sense and monitor the lack of arginine in the phagolysosome of the host macrophage is essential for disease development. Phagolysosomes of macrophages are the niche where Leishmania resides and causes human leishmaniasis. During infection, the arginine concentration in the phagolysosome decreases as part of the host innate immune response. An arginine sensor on the Leishmania cell surface activates an arginine deprivation response pathway that upregulates the expression of a parasite arginine transporter (AAP3). Here, we use CRISPR/Cas9-mediated disruption of the AAP3 locus to show that this response enables Leishmania parasites to successfully compete with the host macrophage in the "hunger games" for arginine.

Deciphering the interaction of benzoxaborole inhibitor AN2690 with connective polypeptide 1 (CP1) editing domain of Leishmania donovani leucyl-tRNA synthetase.

Leucyl-tRNA synthetases (LRS) catalyze the linkage of leucine with tRNALeu. A large insertion CP1 domain (Connective Polypeptide 1) in LRS is responsible for post-transfer editing of mis-charged aminoacyl-tRNAs. Here, we characterized the CP1 domain of Leishmania donovani, a protozoan parasite, and its role in editing activity and interaction with broad spectrum anti-fungal, AN2690. The deletion mutant of LRS, devoid of CP1 domain (LRS-CP1Δ) was constructed, followed by determination of its role in editing and aminoacylation. Binding of AN2690 and different amino acids with CP1 deletion mutant and full length LRS was evaluated using isothermal titration calorimetry (ITC) and molecular dynamics simulations. The recombinant LRS-CP1Δ protein did not catalyze the aminoacylation and the editing reaction when compared to full-length LRS. Thus, indicating that CP1 domain was imperative for both aminoacylation and editing activities of LRS. Binding studies with different amino acids indicated selectivity of isoleucine by CP1 domain over other amino acids. These studies also indicated high affinity of AN2690 with the editing domain. Molecular docking studies indicated that AN2690-CP1 domain complex was stabilized by hydrogen bonding and hydrophobic interactions resulting in high binding affinity between the two. Our data suggests CP1 is crucial for the function of L.donovani LRS.

Epigenetic regulation of defense genes by histone deacetylase1 in human cell line-derived macrophages promotes intracellular survival of Leishmania donovani.

Leishmania donovani, an intracellular protozoan parasite upon infection, encounters a range of antimicrobial factors within the host cells. Consequently, the parasite has evolved mechanisms to evade this hostile defense system through inhibition of macrophage activation that, in turn, enables parasite replication and survival. There is growing evidence that epigenetic down-regulation of the host genome by intracellular pathogens leads to acute infection. Epigenetic modification is mediated by chromatin remodeling, histone modifications, or DNA methylation. Histone deacetylases (HDACs) removes acetyl groups from lysine residues on histones, thereby leading to chromatin remodeling and gene silencing. Here, using L. donovani infected macrophages differentiated from THP-1 human monocytic cells, we report a link between host chromatin modifications, transcription of defense genes and intracellular infection with L. donovani. Infection with L. donovani led to the silencing of host defense gene expression. Histone deacetylase 1 (HDAC1) transcript levels, protein expression, and enzyme activity showed a significant increase upon infection. HDAC1 occupancy at the promoters of the defense genes significantly increased upon infection, which in turn resulted in decreased histone H3 acetylation in infected cells, resulting in the down-regulation of mRNA expression of host defense genes. Small molecule mediated inhibition and siRNA mediated down-regulation of HDAC1 increased the expression levels of host defense genes. Interestingly, in this study, we demonstrate that the silencing of HDAC1 by both siRNA and pharmacological inhibitors resulted in decreased intracellular parasite survival. The present data not only demonstrate that up-regulation of HDAC1 and epigenetic silencing of host cell defense genes is essential for L. donovani infection but also provides novel therapeutic strategies against leishmaniasis.

Stigmasterol as a potential biomarker for amphotericin B resistance in Leishmania donovani.

BACKGROUND: Leishmania donovani, a protozoan parasite, is the primary causative agent for visceral leishmaniasis. Toxicity and increased resistance to existing drugs have led to an urgent need for identifying new drugs and drug targets. Understanding the risks and mechanisms of resistance is of great importance. Amphotericin B (AmB) is a polyene antimicrobial, the mainstay therapy for visceral leishmaniasis in several parts of India. OBJECTIVES: In the present study, we established a line of AmB-resistant L. donovani promastigotes in vitro and demonstrated the molecular basis of resistance to AmB. METHODS: AmB-resistant promastigotes were generated and characterized to evaluate the mechanism of resistance to AmB. We examined the sterol composition of the promastigotes and the axenic amastigotes derived from the WT and AmB-resistant promastigotes. The role of the plant-like C-22 desaturase responsible for stigmasterol production was also evaluated in the AmB-resistant strain. RESULTS: The IC50 for resistant cells was four times higher than for the WT. AmB-resistant promastigotes showed an increase in the conversion of ß-sitosterol into stigmasterol. The presence of higher amounts of stigmasterol in resistant promastigotes, as well as in axenic amastigotes, signifies its role in AmB resistance in Leishmania. The resistant strain showed reduced infectivity in vitro. CONCLUSIONS: We have elucidated the mode of action and resistance mechanisms to the drug. However, further work is required to validate the potential role of stigmasterol in resistance and to help develop a diagnostic kit that can assist in diagnosing potentially resistant lines in the field.

A Plant like Cytochrome P450 Subfamily CYP710C1 Gene in Leishmania donovani Encodes Sterol C-22 Desaturase and its Over-expression Leads to Resistance to Amphotericin B.

BACKGROUND: Leishmania donovani is a protozoan parasite, a primary causative agent of visceral leishmaniasis. Sterol produced via the mevalonate pathway, show differences in composition across biological kingdoms. The specific occurrence of Δ22-unsaturated sterols, containing a double bond at the C-22 position in the side chain occurs in fungi as ergosterol and as stigmasterol in plants. In the present study, we report the identification and functional characterization of a plant-like Cytochrome P450 subfamily CYP710C1 in L. donovani as the Leishmania C-22 desaturase. METHODOLOGY: In silico analysis predicted the presence of a plant like CYP710C1 gene that encodes a sterol C-22 desaturase, a key enzyme in stigmasterol biosynthesis. The enzymatic function of recombinant CYP710C1 as C-22 desaturase was determined. To further study the physiological role of CYP710C1 in Leishmania, we developed and characterized an overexpressing strain and a gene deletion mutant. C-22 desaturase activity and stigmasterol levels were estimated in the wild-type, overexpressing promastigotes and heterozygous mutants. CONCLUSION: We for the first time report the presence of a CYP710C1 gene that encodes a plant like sterol C-22 desaturase leading to stigmasterol biosynthesis in Leishmania. The recombinant CYP710C1 exhibited C-22 desaturase activity by converting ß-sitosterol to stigmasterol. Axenic amastigotes showed higher expression of CYP710C1 mRNA, protein and stigmasterol levels compared to the promastigotes. Sterol profiling of CYP710C1 overexpressing L. donovani and heterozygous mutant parasites demonstrated that CYP710C1 was responsible for stigmasterol production. Most importantly, we demonstrate that these CYP710C1 overexpressing promastigotes are resistant to amphotericin B, a drug of choice for use against leishmaniasis. We report that Leishmania sterol biosynthesis pathway has a chimeric organisation with characteristics of both plant and fungal pathways.

The arginine sensing and transport binding sites are distinct in the human pathogen Leishmania.

The intracellular protozoan parasite Leishmania donovani causes human visceral leishmaniasis. Intracellular L. donovani that proliferate inside macrophage phagolysosomes compete with the host for arginine, creating a situation that endangers parasite survival. Parasites have a sensor that upon arginine deficiency activates an Arginine Deprivation Response (ADR). L. donovani transport arginine via a high-affinity transporter (LdAAP3) that is rapidly up-regulated by ADR in intracellular amastigotes. To date, the sensor and its ligand have not been identified. Here, we show that the conserved amidino group at the distal cap of the arginine side chain is the ligand that activates ADR, in both promastigotes and intracellular amastigotes, and that arginine sensing and transport binding sites are distinct in L. donovani. Finally, upon addition of arginine and analogues to deprived cells, the amidino ligand activates rapid degradation of LdAAP3. This study provides the first identification of an intra-molecular ligand of a sensor that acts during infection.

Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase.

Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.

Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target.

BACKGROUND: Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis. METHODOLOGY: Recombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human. CONCLUSIONS: Recombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 µM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 µM). Borrelidin inhibited the promastigotes (IC50: 21 µM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts.

Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity.

Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC50], 4.19 µM) and intracellular amastigotes (IC50, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LdLysRS-1 as a potential drug target.

The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity.

BACKGROUND: Leishmania donovani, a protozoan parasite is the major causative agent of visceral leishmaniasis. Increased toxicity and resistance to the existing repertoire of drugs has been reported. Hence, an urgent need exists for identifying newer drugs and drug targets. Previous reports have shown sirtuins (Silent Information Regulator) from kinetoplastids as promising drug targets. Leishmania species code for three SIR2 (Silent Information Regulator) related proteins. Here, we for the first time report the functional characterization of SIR2 related protein 2 (SIR2RP2) of L. donovani. METHODOLOGY: Recombinant L. donovani SIR2RP2 was expressed in E. coli and purified. The enzymatic functions of SIR2RP2 were determined. The subcellular localization of LdSIR2RP2 was done by constructing C-terminal GFP-tagged full-length LdSIR2RP2. Deletion mutants of LdSIR2RP2 were generated in Leishmania by double targeted gene replacement methodology. These null mutants were tested for their proliferation, virulence, cell cycle defects, mitochondrial functioning and sensitivity to known SIR2 inhibitors. CONCLUSION: Our data suggests that LdSIR2RP2 possesses NAD+-dependent ADP-ribosyltransferase activity. However, NAD+-dependent deacetylase and desuccinylase activities were not detected. The protein localises to the mitochondrion of the promastigotes. Gene deletion studies showed that ΔLdSIR2RP2 null mutants had restrictive growth phenotype associated with accumulation of cells in the G2/M phase and compromised mitochondrial functioning. The null mutants had attenuated infectivity. Deletion of LdSIR2RP2 resulted in increased sensitivity of the parasites to the known SIR2 inhibitors. The sirtuin inhibitors inhibited the ADP-ribosyltransferase activity of recombinant LdSIR2RP2. In conclusion, sirtuins could be used as potential new drug targets for visceral leishmaniasis.

MicroRNA expression profiling of Leishmania donovani-infected host cells uncovers the regulatory role of MIR30A-3p in host autophagy.

Leishmania is an obligate intracellular parasite that replicates inside phagolysosomes or parasitophorous vacuoles (PV) of the monocyte/macrophage lineage. It reprograms macrophages and produces a metabolic state conducive to successful infection and multiplication. MicroRNAs (miRNAs), a class of small 22 to 24 nucleotide noncoding regulatory RNAs alter the gene expression and consequently proteome output by targeting mRNAs, may play a regulatory role in modulating host cell functions. In the present study, we demonstrate the novel regulatory role of host microRNA, MIR30A-3p in modulation of host cell macroautophagy/autophagy after infection with L. donovani. Our in vitro studies showed that MIR30A-3p expression was significantly enhanced after L. donovani infection in a time-dependent manner. Transient transfection with a MIR30A-3p inhibitor followed by L. donovani infection promoted the autophagic response and decreased the intracellular parasite burden in both THP-1 cells and human monocyte-derived macrophages (HsMDM). BECN1/Beclin 1, the mammalian ortholog of yeast Vps30/Atg6, is a key autophagy-promoting protein that plays a key role in the regulation of cell death and survival. We report BECN1-dependent modulation of host cell autophagy in response to L. donovani infection. Pretreatment of L. donovani-infected macrophages with the MIR30A-3p mimic decreased and with antagomir increased the expression of BECN1 protein. We demonstrate that BECN1 is a potential target of MIR30A-3p and this miRNA negatively regulates BECN1 expression. Our present study reveals for the first time a novel role of MIR30A-3p in regulating autophagy-mediated L. donovani elimination by targeting BECN1. The present study has significant impact for the treatment of visceral leishmaniasis.

Twin Attributes of Tyrosyl-tRNA Synthetase of Leishmania donovani: A HOUSEKEEPING PROTEIN TRANSLATION ENZYME AND A MIMIC OF HOST CHEMOKINE.

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes essential for protein synthesis. Apart from their parent aminoacylation activity, several aaRSs perform non-canonical functions in diverse biological processes. The present study explores the twin attributes of Leishmania tyrosyl-tRNA synthetase (LdTyrRS) namely, aminoacylation, and as a mimic of host CXC chemokine. Leishmania donovani is a protozoan parasite. Its genome encodes a single copy of tyrosyl-tRNA synthetase. We first tested the canonical aminoacylation role of LdTyrRS. The recombinant protein was expressed, and its kinetic parameters were determined by aminoacylation assay. To study the physiological role of LdTyrRS in Leishmania, gene deletion mutations were attempted via targeted gene replacement. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. LdTyrRS appears to be an essential gene as the chromosomal null mutants did not survive. Our data also highlights the non-canonical function of L. donovani tyrosyl-tRNA synthetase. We show that LdTyrRS protein is present in the cytoplasm and exits from the parasite cytoplasm into the extracellular medium. The released LdTyrRS functions as a neutrophil chemoattractant. We further show that LdTyrRS specifically binds to host macrophages with its ELR (Glu-Leu-Arg) peptide motif. The ELR-CXCR2 receptor interaction mediates this binding. This interaction triggers enhanced secretion of the proinflammatory cytokines TNF-α and IL-6 by host macrophages. Our data indicates a possible immunomodulating role of LdTyrRS in Leishmania infection. This study provides a platform to explore LdTyrRS as a potential target for drug development.

Leishmania donovani Encodes a Functional Selenocysteinyl-tRNA Synthase.

The synthesis of selenocysteine, the 21st amino acid, occurs on its transfer RNA (tRNA), tRNA(Sec). tRNA(Sec) is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase (PSTK) in eukaryotes. The selenium donor, selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase. Selenocysteinyl-tRNA synthase (SepSecS) then uses the O-phosphoseryl-tRNA(Sec) and selenophosphate to form Sec-tRNA(Sec) in eukaryotes. Here, we report the characterization of selenocysteinyl-tRNA synthase from Leishmania donovani. Kinetoplastid SepSecS enzymes are phylogenetically closer to worm SepSecS. LdSepSecS was found to exist as a tetramer. Leishmania SepSecS enzyme was found to be active and able to complement the ΔselA deletion in Escherichia coli JS1 strain only in the presence of archaeal PSTK, indicating the conserved nature of the PSTK-SepSecS pathway. LdSepSecS was found to localize in the cytoplasm of the parasite. Gene deletion studies indicate that Leishmania SepSecS is dispensable for the parasite survival. The parasite was found to encode three selenoproteins, which were only expressed in the presence of SepSecS. Selenoproteins of L. donovani are not required for the growth of the promastigotes. Auranofin, a known inhibitor of selenoprotein synthesis showed the same sensitivity toward the wild-type and null mutants suggesting its effect is not through binding to selenoproteins. The three-dimensional structural comparison indicates that human and Leishmania homologs are structurally highly similar but their association modes leading to tetramerization seem different.

Genetically Engineered Ascorbic acid-deficient Live Mutants of Leishmania donovani induce long lasting Protective Immunity against Visceral Leishmaniasis.

Visceral leishmaniasis caused by Leishmania donovani is the most severe systemic form of the disease. There are still no vaccines available for humans and there are limitations associated with the current therapeutic regimens for leishmaniasis. Recently, we reported functional importance of Arabino-1, 4-lactone oxidase (ALO) enzyme from L. donovani involved in ascorbate biosynthesis pathway. In this study, we have shown that ΔALO parasites do not affect the ability of null mutants to invade visceral organs but severely impair parasite persistence beyond 16 week in BALB/c mice and hence are safe as an immunogen. Both short term (5 week) and long term (20 week) immunization with ΔALO parasites conferred sustained protection against virulent challenge in BALB/c mice, activated splenocytes and resulted in induction of pro-inflammatory cytokine response. Protection in immunized mice after challenge correlated with the stimulation of IFN-γ producing CD4(+) and CD8(+) T cells. Antigen-mediated cell immunity correlated with robust nitrite and superoxide generation, macrophage-derived oxidants critical in controlling Leishmania infection. Our data shows that live attenuated ΔALO parasites are safe, induce protective immunity and can provide sustained protection against Leishmania donovani. We further conclude that the parasites attenuated in their anti-oxidative defence mechanism can be exploited as vaccine candidates.

Proteomic-based approach to gain insight into reprogramming of THP-1 cells exposed to Leishmania donovani over an early temporal window.

Leishmania donovani, a protozoan parasite, is the causative agent of visceral leishmaniasis. It lives and multiplies within the harsh environment of macrophages. In order to investigate how intracellular parasite manipulate the host cell environment, we undertook a quantitative proteomic study of human monocyte-derived macrophages (THP-1) following infection with L. donovani. We used the isobaric tags for relative and absolute quantification (iTRAQ) method and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare expression profiles of noninfected and L. donovani-infected THP-1 cells. We detected modifications of protein expression in key metabolic pathways, including glycolysis and fatty acid oxidation, suggesting a global reprogramming of cell metabolism by the parasite. An increased abundance of proteins involved in gene transcription, RNA splicing (heterogeneous nuclear ribonucleoproteins [hnRNPs]), histones, and DNA repair and replication was observed at 24 h postinfection. Proteins involved in cell survival and signal transduction were more abundant at 24 h postinfection. Several of the differentially expressed proteins had not been previously implicated in response to the parasite, while the others support the previously identified proteins. Selected proteomics results were validated by real-time PCR and immunoblot analyses. Similar changes were observed in L. donovani-infected human monocyte-derived primary macrophages. The effect of RNA interference (RNAi)-mediated gene knockdown of proteins validated the relevance of the host quantitative proteomic screen. Our findings indicate that the host cell proteome is modulated after L. donovani infection, provide evidence for global reprogramming of cell metabolism, and demonstrate the complex relations between the host and parasite at the molecular level.