Dendritic cell engineered cTXN as new vaccine prospect against L. donovani.

Dendritic cells (DCs), as antigen-presenting cells, can reportedly be infected withLeishmaniaparasites and hence provide a better option to trigger T-cell primary immune responses and immunological memory. We consistently primed DCs during culture with purified recombinant cytosolic tryparedoxin (rcTXN) and then evaluated the vaccine prospect of presentation of rcTXN against VL in BALB/c mice. We reported earlier the immunogenic properties of cTXN antigen derived fromL. donovani when anti-cTXN antibody was detected in the sera of kala-azar patients. It was observed that cTXN antigen, when used as an immunogen with murine DCs acting as a vehicle, was able to induce complete protection against VL in an infected group of immunized mice. This vaccination triggered splenic macrophages to produce more IL-12 and GM-CSF, and restricted IL-10 release to a minimum in an immunized group of infected animals. Concomitant changes in T-cell responses against cTXN antigen were also noticed, which increased the release of protective cytokine-like IFN-γ under the influence of NF-κß in the indicated vaccinated group of animals. All cTXN-DCs-vaccinated BALB/c mice survived during the experimental period of 120 days. The results obtained in our study suggest that DCs primed with cTXN can be used as a vaccine prospect for the control of visceral leishmaniasis.

Mining the Proteome of Leishmania donovani for the Development of Novel MHC Class I Restricted Epitope for the Control of Visceral Leishmaniasis.

Although, the precise host defence mechanism(s) is not completely understood, T cell-mediated immune responses is believed to play a pivotal role in controlling parasite infection. Here we target the stage dependent over expressed gene. Here, the consensus based computational approach was adopted for the screening of potential major histocompatibility complex class I restricted epitopes. Based on the computational analysis and previously published report, a set 19 antigenic proteins derived from Leishmania donovani were screened for further characterization as vaccine candidates. A total of 49 epitopes were predicted, which revealed a comprehensive binding affinity to the 40 different MHC class I supertypes. Based on the population coverage and HLA cross presentation, nine highly promiscuous epitopes such as LTYDDVWTV (P1), FLFPQRTAL(P2), FLFSNGAVV (P3), YIYNFGIRV (P4), YMTAAFAAL (P5), KLLRPFAPL (P6), FMLGWIVTI (P7), SLFERNKRV (P8), and SVWNRIFTL (P9) which have either a high or an intermediate TAP binding affinity were selected for further analysis. Theoretical population coverage analysis of polytope vaccine (P1-P9) revealed more than 92% population. Stimulation with the cocktail of peptide revealed a proliferative CD8+ T cell response and increased IFN-γ production. An upregulated NF-κB activity is thought to be play a pivotal role in T cell proliferation against the selected peptide. The Th1-type cytokine profile (presence of IFN-γ and absence of IL-10) suggests the potentiality of the cocktail of epitope as a subunit vaccine against leishmaniasis. However, the efficiency of these epitopes to trigger other Th1 cytokines and chemokines in a humanized mice model could explore its plausibility as a vaccine candidate. J. Cell. Biochem. 119: 378-391, 2018. © 2017 Wiley Periodicals, Inc.

Proteomic approaches unravel the intricacy of secreted proteins of Leishmania: An updated review.

Leishmaniasis, a parasitic protozoan disease, is still a worldwide concern due to persistent issues with chemotherapy, rapid emerging drug resistance; and non- availability of approved vaccine for the control of disease. Therefore, the search of parasite specific proteins to identify new anti-leishmanial drug targets and vaccine candidates is an urgent priority. In this context, proteins that are secreted, in vitro during parasite growth under defined conditions, can be explored as potential tool for studying their roles in parasite survival inside host and disease pathogenesis. From the last few years, various approaches have been exploited to identify the proteins secreted out by the parasites under defined conditions at particular stage or time. Due to availability of genomic information on various Leishmania species, proteomics have been emerged as most promising approach for analyzing the complexity of exoproteome of different Leishmania species. Herein, we have summarized various secretion mechanisms used by Leishmania parasites to export the proteins into the extracellular space; followed by the role of proteomics in exoproteome analysis along with special emphasis on various applications to study the exoproteome, which might provide potential targets for drug design or novel antigens for vaccine development.

Cytosolic tryparedoxin of Leishmania donovani modulates host immune response in visceral leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by the unicellular protozoan parasite of genus Leishmania. Tryparedoxin (TXN) is a low molecular mass dithiol protein belonging to oxidoreductases super-family; which function in concert with tryparedoxin peroxidase (TXNPx) as a system in protozoan parasites including Leishmania. Leishmanial hydroperoxides detoxification cascade uses trypanothione as electron donor to reduce hydroperoxide inside the macrophages during infection. However, the mechanism by which tryparedoxin can contribute in progression of visceral leishmaniasis (VL) and its impact on host's cellular immune response during infection in Indian VL patient is unknown. In this study, we purified a ∼17 kDa recombinant cytosolic tryparedoxin (cTXN) protein of Leishmania donovani (rLdcTXN) and investigated its immunological responses in peripheral blood monocytes (PBMC) isolated from VL patients. The protein significantly enhanced the promastigotes count after 96 h of culture showing a direct correlation with parasite growth. Furthermore, stimulation of PBMC isolated from VL patients with rLdcTXN resulted in up-regulation of IL-4 and IL-10 production whereas IL-12 and IFN-γ was significantly down-regulated suggesting a pivotal role of cTXN in provoking the immune suppression during VL. Our study demonstrates the importance of cTXN protein which can potentially modulate the outcome of disease through suppressing host protective Th1 response in VL patients.

Evaluation of Leishmania donovani disulfide isomerase as a potential target of cellular immunity against visceral leishmaniasis.

In Leishmania species, protein disulfide isomerase (PDI) - a redox chaperone is primarily associated with virulence and survival. The precise mechanism, especially in relation to redox changes and its effects on immunological responses in visceral leishmaniasis (VL) is not completely understood as yet. Therefore, we purified a recombinant PDI from Leishmania donovani (r-LdPDI) which was of ∼15 kDa molecular size and examined its effects on immunological responses in peripheral blood (PBMC) of human VL cases. For these studies, alanine was tested as an inhibitor and was used in parallel to all experiments. This protein was identified to have a direct correlation with parasite growth which significantly increased number of promastigotes as well as axenic amastigotes after 96 h of culture. Our experiments examining the immunological response against r-LdPDI also indicate the activation of pro-L. donovani dictated immunological responses in VL. The stimulation of PBMC with r-LdPDI induced lactate dehydrogenase (LDH) activities and up regulated interleukin-10 (IL-10) production but not the HLA-DR expression, Nitric oxide (NO) release and IFN-γ production indicating a pivoted role for r-LdPDI in causing a strong immunosuppression in a susceptible host. Further, we observed that an addition of alanine in L. donovani culture offers a significant inhibition in growth of parasite and helps in reconstitution of protective immune response in VL cases. Therefore, we demonstrate a future cross talk on use of alanine which can reduce the activities of PDI of L. donovani, eliminating the parasite induced immunosuppression and inducing collateral host protective response in VL.

Unveiling the role of serine o-acetyltransferase in drug resistance and oxidative stress tolerance in Leishmania donovani through the regulation of thiol-based redox metabolism.

Understanding the unique metabolic pathway of L. donovani is crucial for comprehending its biology under oxidative stress conditions. The de novo cysteine biosynthetic pathway of L. donovani is absent in humans and its product, cysteine regulates the downstream components of trypanothione-based thiol metabolism, important for maintaining cellular redox homeostasis. The role of serine o-acetyl transferase (SAT), the first enzyme of this pathway remains unexplored. In order to investigate the role of SAT protein, we cloned SAT gene into pXG-GFP+ vector for episomal expression of SAT in Amphotericin B sensitive L. donovani promastigotes. The SAT overexpression was confirmed by SAT enzymatic assay, GFP fluorescence, immunoblotting and PCR. Our study unveiled an upregulated expression of both LdSAT and LdCS of cysteine biosynthetic pathway and other downstream thiol pathway proteins in LdSAT-OE promastigotes. Additionally, there was an increase in enzymatic activities of LdSAT and LdCS proteins in LdSAT-OE, which was found similar to the Amp B resistant parasites, indicating a potential role of SAT protein in modulating drug resistance. We observed that the overexpression of SAT in Amp B sensitive parasites increases tolerance to drug pressure and oxidative stress via trypanothione-dependent antioxidant mechanism. Moreover, the in vitro J774A.1 macrophage infectivity assessment showed that SAT overexpression augments parasite infectivity. In LdSAT-OE promastigotes, antioxidant enzyme activities like APx and SOD were upregulated, intracellular reactive oxygen species were reduced with a corresponding increase in thiol level, emphasizing SAT's role in stress tolerance and enhanced infectivity. Additionally, the ROS mediated upregulation in the expression of LdSAT, LdCS, LdTryS and LdcTXNPx proteins reveals an essential cross talk between SAT and proteins of thiol metabolism in combating oxidative stress and maintaining redox homeostasis. Taken together, our results provide the first insight into the role of SAT protein in parasite infectivity and survival under drug pressure and oxidative stress.

Interaction between Cfd1 and Nbp35 proteins involved in cytosolic FeS cluster assembly machinery deciphers a stable complexation in Leishmania donovani.

Leishmania donovani is the causative unicellular parasite for visceral leishmaniasis (VL); and FeS proteins are likely to be very essential for their survival and viability. Cytosolic FeS cluster assembly (CIA) machinery is one of the four systems for the biosynthesis and transfer of FeS clusters among eukaryotes; Cfd1 and Nbp35 are the scaffold components for cytosolic FeS cluster biogenesis. We investigated the role of CIA machinery components and purified Cfd1 and Nbp35 proteins of L. donovani. We also investigated the interactive nature between LdCfd1 and LdNbp35 proteins by in silico analysis, in vitro co-purification, pull down assays along with in vivo immuno-precipitation; which inferred that both LdCfd1 and LdNbp35 proteins are interacting with each other. Thus, our collective data revealed the interaction between these two proteins which forms a stable complex that can be attributed to the cellular process of FeS clusters biogenesis, and transfer to target apo-proteins of L. donovani. The expression of Cfd1 and Nbp35 proteins in Amp B resistant parasites is up-regulated leading to increased amount of FeS proteins. Hence, it favors increased tolerance towards ROS level, which helps parasites survival under drug pressure contributing in Amphotericin B resistance.

Role of conserved active site tryptophan-101 in functional activity and stability of phosphoserine aminotransferase from an enteric human parasite.

Site-directed mutagenesis study was performed to elucidate the role of conserved tryptophan-101 present at the active site of phosphoserine aminotransferase from an enteric human parasite Entamoeba histolytica. Fluorescence resonance energy transfer and molecular dynamic simulation show that the indole ring of Trp101 stacks with the cofactor PLP. Loss of enzymatic activity and PLP polarization values suggest that Trp101 plays a major role in maintaining a defined PLP microenvironment essentially required for optimal enzymatic activity. Studies on W101F, W101H and W101A mutants show that only the indole ring of the conserved Trp101 forms most favorable stacking interaction with the pyridine ring of the cofactor PLP. Protein stability was compromised on substitution of Trp101 with Phe/His/Ala amino acids. A difference in conformational free energy of 1.65 kcal mol(-1) was observed between WT-protein and W101A mutant.

Unique thiol metabolism in trypanosomatids: Redox homeostasis and drug resistance.

Trypanosomatids are mainly responsible for heterogeneous parasitic diseases: Leishmaniasis, Sleeping sickness, and Chagas disease and control of these diseases implicates serious challenges due to the emergence of drug resistance. Redox-active biomolecules are the endogenous substances in organisms, which play important role in the regulation of redox homeostasis. The redox-active substances like glutathione, trypanothione, cysteine, cysteine persulfides, etc., and other inorganic intermediates (hydrogen peroxide, nitric oxide) are very useful as defence mechanism. In the present review, the suitability of trypanothione and other essential thiol molecules of trypanosomatids as drug targets are described in Leishmania and Trypanosoma. We have explored the role of tryparedoxin, tryparedoxin peroxidase, ascorbate peroxidase, superoxide dismutase, and glutaredoxins in the anti-oxidant mechanism and drug resistance. Up-regulation of some proteins in trypanothione metabolism helps the parasites in survival against drug pressure (sodium stibogluconate, Amphotericin B, etc.) and oxidative stress. These molecules accept electrons from the reduced trypanothione and donate their electrons to other proteins, and these proteins reduce toxic molecules, neutralize reactive oxygen, or nitrogen species; and help parasites to cope with oxidative stress. Thus, a better understanding of the role of these molecules in drug resistance and redox homeostasis will help to target metabolic pathway proteins to combat Leishmaniasis and trypanosomiases.

Two atypical L-cysteine-regulated NADPH-dependent oxidoreductases involved in redox maintenance, L-cystine and iron reduction, and metronidazole activation in the enteric protozoan Entamoeba histolytica.

We discovered novel catalytic activities of two atypical NADPH-dependent oxidoreductases (EhNO1/2) from the enteric protozoan parasite Entamoeba histolytica. EhNO1/2 were previously annotated as the small subunit of glutamate synthase (glutamine:2-oxoglutarate amidotransferase) based on similarity to authentic bacterial homologs. As E. histolytica lacks the large subunit of glutamate synthase, EhNO1/2 were presumed to play an unknown role other than glutamine/glutamate conversion. Transcriptomic and quantitative reverse PCR analyses revealed that supplementation or deprivation of extracellular L-cysteine caused dramatic up- or down-regulation, respectively, of EhNO2, but not EhNO1 expression. Biochemical analysis showed that these FAD- and 2[4Fe-4S]-containing enzymes do not act as glutamate synthases, a conclusion which was supported by phylogenetic analyses. Rather, they catalyze the NADPH-dependent reduction of oxygen to hydrogen peroxide and L-cystine to L-cysteine and also function as ferric and ferredoxin-NADP(+) reductases. EhNO1/2 showed notable differences in substrate specificity and catalytic efficiency; EhNO1 had lower K(m) and higher k(cat)/K(m) values for ferric ion and ferredoxin than EhNO2, whereas EhNO2 preferred L-cystine as a substrate. In accordance with these properties, only EhNO1 was observed to physically interact with intrinsic ferredoxin. Interestingly, EhNO1/2 also reduced metronidazole, and E. histolytica transformants overexpressing either of these proteins were more sensitive to metronidazole, suggesting that EhNO1/2 are targets of this anti-amebic drug. To date, this is the first report to demonstrate that small subunit-like proteins of glutamate synthase could play an important role in redox maintenance, L-cysteine/L-cystine homeostasis, iron reduction, and the activation of metronidazole.

Metabolome analysis revealed increase in S-methylcysteine and phosphatidylisopropanolamine synthesis upon L-cysteine deprivation in the anaerobic protozoan parasite Entamoeba histolytica.

L-cysteine is ubiquitous in all living organisms and is involved in a variety of functions, including the synthesis of iron-sulfur clusters and glutathione and the regulation of the structure, stability, and catalysis of proteins. In the protozoan parasite Entamoeba histolytica, the causative agent of amebiasis, L-cysteine plays an essential role in proliferation, adherence, and defense against oxidative stress; however, the essentiality of this amino acid in the pathways it regulates is not well understood. In the present study, we applied capillary electrophoresis time-of-flight mass spectrometry to quantitate charged metabolites modulated in response to L-cysteine deprivation in E. histolytica, which was selected as a model for examining the biological roles of L-cysteine. L-cysteine deprivation had profound effects on glycolysis, amino acid, and phospholipid metabolism, with sharp decreases in the levels of L-cysteine, L-cystine, and S-adenosylmethionine and a dramatic accumulation of O-acetylserine and S-methylcysteine. We further demonstrated that S-methylcysteine is synthesized from methanethiol and O-acetylserine by cysteine synthase, which was previously considered to be involved in sulfur-assimilatory L-cysteine biosynthesis. In addition, L-cysteine depletion repressed glycolysis and energy generation, as it reduced acetyl-CoA, ethanol, and the major nucleotide di- and triphosphates, and led to the accumulation of glycolytic intermediates. Interestingly, L-cysteine depletion increased the synthesis of isopropanolamine and phosphatidylisopropanolamine, and it was confirmed that their increment was not a result of oxidative stress but was a specific response to L-cysteine depletion. We also identified a pathway in which isopropanolamine is synthesized from methylglyoxal via aminoacetone. To date, this study represents the first case where L-cysteine deprivation leads to drastic changes in core metabolic pathways, including energy, amino acid, and phospholipid metabolism.

Bacterial-type oxygen detoxification and iron-sulfur cluster assembly in amoebal relict mitochondria.

The assembly of vital reactive iron-sulfur (Fe-S) cofactors in eukaryotes is mediated by proteins inherited from the original mitochondrial endosymbiont. Uniquely among eukaryotes, however, Entamoeba and Mastigamoeba lack such mitochondrial-type Fe-S cluster assembly proteins and possess instead an analogous bacterial-type system acquired by lateral gene transfer. Here we demonstrate, using immunomicroscopy and biochemical methods, that beyond their predicted cytosolic distribution the bacterial-type Fe-S cluster assembly proteins NifS and NifU have been recruited to function within the relict mitochondrial organelles (mitosomes) of Entamoeba histolytica. Both Nif proteins are 10-fold more concentrated within mitosomes compared with their cytosolic distribution suggesting that active Fe-S protein maturation occurs in these organelles. Quantitative immunoelectron microscopy showed that amoebal mitosomes are minute but highly abundant cellular structures that occupy up to 2% of the total cell volume. In addition, protein colocalization studies allowed identification of the amoebal hydroperoxide detoxification enzyme rubrerythrin as a mitosomal protein. This protein contains functional Fe-S centres and exhibits peroxidase activity in vitro. Our findings demonstrate the role of analogous protein replacement in mitochondrial organelle evolution and suggest that the relict mitochondrial organelles of Entamoeba are important sites of metabolic activity that function in Fe-S protein-mediated oxygen detoxification.

Biophysical characterization of Entamoeba histolytica phosphoserine aminotransferase (EhPSAT): role of cofactor and domains in stability and subunit assembly.

We investigated the role of the cofactor PLP and its binding domain in stability and subunit assembly of phosphoserine aminotransferase (EhPSAT) from an enteric human parasite Entamoeba histolytica. Presence of cofactor influences the tertiary structure of EhPSAT because of the significant differences in the tryptophan microenvironment and proteolytic pattern of holo- and apo-enzyme. However, the cofactor does not influence the secondary structure of the enzyme. Stability of the protein is significantly affected by the cofactor as holo-enzyme shows higher T(m) and C(m) values for thermal and GdnHCl-induced denaturation, respectively, when compared to the apo-enzyme. The cofactor also influences the unfolding pathway of the enzyme. Although urea-dependent unfolding of both holo- and apo-EhPSAT is a three-state process, the intermediates stabilized during unfolding are significantly different. For holo-EhPSAT a dimeric holo-intermediate was stabilized, whereas for apo-EhPSAT, a monomeric intermediate was stabilized. This is the first report on stabilization of a holo-dimeric intermediate for any aminotransferase. The isolated PLP-binding domain is stabilized as a monomer, thus suggesting that either the N-terminal tail or the C-terminal domain of EhPSAT is required for stabilization of dimeric configuration of the wild-type enzyme. To the best of our knowledge, this is a first report investigating the role of PLP and various protein domains in structural and functional organization of a member of subgroup IV of the aminotransferases.

Amplification and Characterization of DDT Metabolizing Delta Class GST in Sand Fly, Phlebotomus argentipes (Diptera: Psychodidae) From Bihar, India.

Phlebotomus argentipes is an established vector for Visceral leishmaniasis prevalent in the Indian subcontinent. Insect Glutathione S-transferases (GST) enzyme plays a pivotal role in the metabolism of xenobiotics and chemical insecticides. We report herein the identification and characterization of a delta class GST from the sandfly, P. argentipes. The resulting clone (rParg-GSTδ) is successfully sequenced, which revealed 76.43% and 66.32% gene identity with GST from Phlebotomus papatasi (Scopoli; Diptera: Psychodidae) and Lutzomiya longipalpis (Lutz and Neiva; Diptera: Psychodidae), respectively. The identified rParg-GST amino acid Blast results revealed 82.6% homology to delta class GST of Phlebotomus papatasi and more than 50% homology to Lepidoptera which comprises butterflies and moths. The Phylogenetic analysis of Parg-GST with different classes of Insect GSTs further supported its classification as delta class. A functional recombinant Parg-GSTδ protein (rParg-GSTδ) was expressed in Escherichia coli (Migula; Enterobacterales: Enterobacteriaceae) cells in a soluble form, purified to homogeneity and found to be active against a substrate 1-chloro-2,4-dintrobenzene (CDNB) and lipid peroxidation by-product 4-Hydrxynonenal (4-HNE). Interestingly, rParg-GSTδ demonstrates high dehydrochlorination activity against dichlorodiphenyltrichloroethane (DDT) i.e., 16.27 nM/µg in high performance liquid chromatography (HPLC) assay. These results provide evidence of direct DDT metabolism property exhibited by P. argentipes GST and set the foundation to decipher the metabolic resistance mechanism in P. argentipes against insecticides.

A randomized, open-label study to evaluate the efficacy and safety of liposomal amphotericin B (AmBisome) versus miltefosine in patients with post-kala-azar dermal leishmaniasis.

BACKGROUND: Treatment of post-kala-azar dermal leishmaniasis cases is of paramount importance for kala-azar elimination; however, limited treatment regimens are available as of now. AIM: To compare the effectiveness of liposomal amphotericin B vs miltefosine in post-kala-azar dermal leishmaniasis patients. METHODOLOGY: This was a randomized, open-label, parallel-group study. A total of 100 patients of post kala azar dermal leishmaniasis, aged between 5 and 65 years were recruited, 50 patients in each group A (liposomal amphotericin B) and B (miltefosine). Patients were randomized to receive either liposomal amphotericin B (30 mg/kg), six doses each 5 mg/kg, biweekly for 3 weeks or miltefosine 2.5 mg/kg or 100 mg/day for 12 weeks. All the patients were followed at 3rd, 6th and 12th months after the end of the treatment. RESULTS: In the liposomal amphotericin B group, two patients were lost to follow-up, whereas four patients were lost to follow-up in the miltefosine group. The initial cure rate by "intention to treat analysis" was 98% and 100% in liposomal amphotericin B and miltefosine group, respectively. The final cure rate by "per protocol analysis" was 74.5% and 86.9% in liposomal amphotericin B and miltefosine, respectively. Twelve patients (25.5%) in the liposomal amphotericin B group and six patients (13%) in the miltefosine group relapsed. None of the patients in either group developed any serious adverse events. LIMITATIONS: Quantitative polymerase chain reaction was not performed at all the follow-up visits and sample sizes. CONCLUSION: Efficacy of miltefosine was found to be better than liposomal amphotericin B, hence, the use of miltefosine as first-line therapy for post-kala-azar dermal leishmaniasis needs to be continued. However, liposomal amphotericin B could be considered as one of the treatment options for the elimination of kala-azar from the Indian subcontinent.

Leishmania donovani chaperonin TCP1γ subunit protects miltefosine induced oxidative damage.

T-complex protein-1 (TCP1) is a chaperonin protein known to fold various proteins like actin and tubulin. In Leishmania donovani only one subunit of TCP1 that is gamma subunit (LdTCP1γ) has been functionally characterized. It not only performs ATP dependent protein folding but is also essential for survival and virulence. The present work demonstrates that LdTCP1γ also has a role in miltefosine resistance. Overexpression of LdTCP1γ in L. donovani promastigotes results in decreased sensitivity of parasites towards miltefosine, while single-allele replacement mutants exhibited increased sensitivity as compared to wild-type promastigotes. This response was specific to miltefosine with no cross-resistance to other drugs. The LdTCP1γ-mediated drug resistance was directly related to miltefosine-induced apoptotic death of the parasite, as was evidenced by 2 to 3-fold decrease in cell death parameters in overexpressing cells and >2-fold increase in single-allele replacement mutants. Further, deciphering the mechanism revealed that resistance of overexpressing cells was associated with efficient ROS neutralization due to increased levels of thiols and upregulation of cytosolic tryparedoxin peroxidase (cTxnPx). Further, modulation of LdTCP1γ expression in parasite also modulates the levels of proinflammatory cytokine (TNF-α) and anti-inflammatory cytokine (IL-10) of the host macrophages. The study provides evidence for the involvement of a chaperonin protein LdTCP1γ in the protection against miltefosine induced oxidative damage and reveals the fundamental role of LdTCP1γ in parasite biology.

Isoform-dependent feedback regulation of serine O-acetyltransferase isoenzymes involved in L-cysteine biosynthesis of Entamoeba histolytica.

Serine acetyltransferase (SAT; EC 2.3.1.30) catalyzes the CoA-dependent acetylation of the side chain hydroxyl group of l-serine to form O-acetyl serine, in the first step of the L-cysteine biosynthetic pathway. Since this pathway is selectively present in a few parasitic protists and absent in mammals, it represents a reasonable target to develop new chemotherapeutics. Entamoeba histolytica apparently possesses three SAT isotypes (EhSAT1-3) showing 48-73% mutual identity, a calculated molecular mass of 34.4-37.7 kDa, and an isoelectric point of 5.70-6.63. To better understand the role of individual SAT isotypes, we determined kinetic and inhibitory parameters of recombinant SAT isotypes. While the three SAT isotypes showed comparable Km and k(cat) for L-serine and acetyl-CoA, they showed remarkable differences in their sensitivity to inhibition by L-cysteine. The Ki values for L-cysteine varied by 100-fold (4.7-460 microM) among SAT isotypes (EhSAT1

In-silico screening and validation of high-affinity tetra-peptide inhibitor of Leishmania donovani O-acetyl serine sulfhydrylase (OASS).

OASS is a specific enzyme that helps Leishmania parasite to survive the oxidative stress condition in human macrophages. SAT C-terminal peptides in several organisms, including Leishmania, were reported to inhibit or reduce the activity of OASS. Small peptide and small molecules mimicking the SAT C-terminal residues are designed and tested for the inhibition of OASS in different organisms. Hence, in this study, all the possible tetra-peptide combinations were designed and screened based on the docking ability with Leishmania donovani OASS (Ld-OASS). The top ranked peptides were further validated for the stability using 50 ns molecular dynamic simulation. In order to identify the better binding capability of the peptides, the top peptides complexed with Ld-OASS were also subjected to molecular dynamic simulation. The docking and simulation results favored the peptide EWSI to possess greater advantage than previously reported peptide (DWSI) in binding with Ld-OASS active site. Also, screening of non-peptide inhibitor of Asinex Biodesign library based on the shape similarity of EWSI and DWSI was performed. The top similar molecules of each peptides were docked on to Ld-OASS active site and subsequently simulated for 20 ns. The results suggested that the ligand that shares high shape similarity with EWSI possess better binding capability than the ligand that shares high shape similarity with DWSI. This study revealed that the tetra-peptide EWSI had marginal advantage over DWSI in binding with Ld-OASS, thereby providing basis for defining a pharmacophoric scaffold for the design of peptidomimetic inhibitors as well as non-peptide inhibitors of Ld-OASS. Communicated by Ramaswamy H. Sarma.

Quantitative secretome analysis unravels new secreted proteins in Amphotericin B resistant Leishmania donovani.

Leishmaniasis is second most neglected disease after malaria and seems to be a worldwide concern because of increased drug resistance and non-availability of approved vaccine. The underlying molecular mechanism of drug resistance (Amp B) in Leishmania parasites still remains elusive. Herein, the present study investigated differentially expressed secreted proteins of Amphotericin B sensitive (S) and resistant (R) isolate of Leishmania donovani by using label free quantitative LC-MS/MS approach. A total of 406 differentially expressed secreted proteins were found between sensitive (S) and resistant (R) isolate. Among 406 proteins, 32 were significantly up regulated (>2.0 fold) while 22 were down regulated (<0.5 fold) in resistant isolate of L. donovani. Further, differentially expressed proteins were classified into 11 various biological processes. Interestingly, identified up regulated proteins in resistant parasites were dominated in carbohydrate metabolism, stress response, transporters and proteolysis. Western blot and enzymatic activity of identified proteins validate our proteomic findings. Finally, our study demonstrated some new secreted proteins associated with Amp B resistance which provides a basis for further investigations to understand the role of proteins in L. donovani. BIOLOGICAL SIGNIFICANCE: Although great advances have been achieved in the diagnosis and treatment of leishmaniasis, still drug resistance is major hurdle in control of disease. Present study will enhance the deeper understanding of altered metabolic pathways involved in Amp B resistance mechanism and provide possible new proteins which can be potential candidate either for exploring as new drug target or vaccine. Protein-protein interactions highlighted the up-regulated metabolic pathways in resistant parasites which further unravel the adaptive mechanism of parasites.

Detection and functional characterization of sigma class GST in Phlebotomus argentipes and its role in stress tolerance and DDT resistance.

Several Glutathione S-transferases (GSTs) enzymes, in insects, have previously been implicated in resistance developed against DDT and other insecticides. The GST enzyme particularly sigma class have important physiological role in detoxification of lipid peroxidation by-products in insects. Phlebotomus argentipes has been intensely exposed to DDT over years due to Indoor Residual Spray (IRS) programme for Kala-azar elimination in Bihar, India. However, in P. argentipes, role of GSTs in DDT resistance have not been elucidated. Here, sigma class GST of P. argentipes (Parg-GSTσ) was successfully cloned, expressed and purified by affinity chromatography. The recombinant Parg-GSTσ was found to be highly active towards cumene hydroperoxide and 4-HNE having specific activity 92.47 & 203.92 µM/min/mg of protein, respectively and exhibited low activity towards universal substrate CDNB i.e., 8.75 µM/min/mg of protein. RT-PCR and immunoblot analysis showed at least 2 and 1.8 fold overexpression of Parg-GSTσ in the single exposed and non exposed DDT resistant P. argentipes as compared to susceptible, implicating Parg-GSTσ also involved in DDT resistance probably by imparting enhanced stress tolerance. The DDT, H2O2 and temperature induction assays demonstrated stress-dependent induction of Parg-GSTσ expression indicating its important role in oxidative stress redressal.