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.

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.

Maternal and severe anaemia in delivering women is associated with risk of preterm and low birth weight: A cross sectional study from Jharkhand, India.

BACKGROUND AND OBJECTIVES: Haemoglobin content is the well accepted indicator for anaemia assessment. The high prevalence of anaemia, maternal health care issues and adverse delivery outcome in Jharkhand, we investigated whether delivering women with anaemia would present a modifiable risk of preterm (PTB) and low birth weight (LBW). METHODS: A facility-based cross-sectional study involving pregnant women, with screening for pregnancy endpoints and haemoglobin assay, were conducted. Anaemia was classified according to World Health Organization's definition of anaemia in pregnancy. Confounding variables were adjusted in a logistic model. The adjusted odds ratios (AORs) with 95% confidence intervals (CIs) were used for analyzing the association among maternal anaemia, PTB and LBW. RESULTS: We observed a high prevalence of anaemia (78.45%) in delivering women, whereas high prevalence of preterm birth (34.75%) and LBW (32.81%) in delivering women overall. In the adjusted analysis, overall anaemia in pregnancy was strongly associated with preterm birth (OR, 3.42; 95% CI, 1.98-5.88; P ≤ .0001) as compared to LBW (OR, 1.12; 95% CI, 0.65-1.61; P = .0003). The risk of PTB and LBW were dependent on the stratification of the anaemia group, as the strongest association was observed in severe (OR, 4.86) followed by mild (OR, 3.66) and moderate (OR, 3.18) anaemia in PTB; whereas risk of LBW was found in severe (OR, 2.5) followed by moderate (OR, 1.11) and mild (OR, 0.57) anaemia. The risk of PTB and LBW across six pregnancy haemoglobin groups were compared, haemoglobin of 10-10.9 g/dl (OR, 1.25) and ≤ 8 g/dl (OR, 1.03) have shown association with PTB and LBW, respectively. However, high haemoglobin concentration was not associated with either PTB or LBW. CONCLUSIONS: Anaemia in delivering women was associated with an elevated risk of PTB and LBW and the risk increased with the severity of anaemia in pregnant women.

LdIscU is a [2Fe-2S] scaffold protein which interacts with LdIscS and its expression is modulated by Fe-S proteins in Leishmania donovani.

The pathogenicity of protozoan parasites is frequently attributed to their ability to circumvent the deleterious effects of ROS and Fe-S clusters are among their susceptible targets with paramount importance for parasite survival. The biogenesis of Fe-S clusters is orchestrated by ISC system; the sulfur donor IscS and scaffold protein IscU being its core components. However, among protozoan parasites including Leishmania, no information is available regarding biochemical aspect of IscU, its interaction partners and regulation. Here, we show that Leishmania donovani IscU homolog, LdIscU, readily assembles [2Fe-2S] clusters and, interestingly, follows Michaelis-Menten enzyme kinetics. It is localized in the mitochondria of the parasite and interacts with LdIscS to form a stable complex. Additionally, LdIscU and Fe-S proteins activity is significantly upregulated in resistant isolates and during stationary growth stage indicating an association between them. The differential expression of LdIscU modulated by Fe-S proteins demand suggests its potential role in parasite survival and drug resistance. Thus, our study provides novel insight into the Fe-S scaffold protein of a protozoan parasite.

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.

Role of IL-1ß, IL-6 and TNF-α cytokines and TNF-α promoter variability in Plasmodium vivax infection during pregnancy in endemic population of Jharkhand, India.

BACKGROUND: The combinatorial effects of Plasmodium infection, perturbation of inflammatory responses and the dichotomic role of TNF promoter polymorphism has potential clinical and physiological relevance during pregnancy. OBJECTIVE AND METHODS: This coordinated orchestration instigated us to investigate the circulating level of inflammatory cytokines (IL-1ß, TNF-α and IL-6) employing ELISA in a stratified group of samples and the plausible genetic association of TNF-α -308 G/A using PCR-RFLP/sequencing during Plasmodium vivax infection in pregnancy. RESULTS: We observed significantly elevated concentrations of IL-1ß were observed, followed by IL-6 and TNF-α in women with malaria (WWM) and in malaria in pregnancy (MIP). Further, elevated IL-1ß, followed by TNF-α and IL-6 were detected in the non-infected pregnancy group. The differential dynamics of inflammatory cytokine concentration during each trimester of pregnancy with and without P. vivax infection were detected. For the first time, a high level of IL-6 was observed in the first trimester of MIP and high IL-1ß in healthy pregnancies. In the second trimester, however, we observed a high level of IL-1ß in the MIP group compared to a sustained high level of IL-1ß in the healthy pregnancy group. In the third trimester, high IL-1ß was sustained in the MIP group and healthy pregnancies acquired a high TNF-α level. The genotypic distribution for the TNF-α promoter -308 G/A position was observed to be nonsignificant and mildly associated during MIP (OR = 1.4) and in WWM (OR = 1.2). Moreover, based on genotypic distribution, we observed a well-correlated and significantly elevated TNF-α concentration in the mutant homozygote genotype (AA; p = 0.001) followed by heterozygotes (GA; p = 0.0001) and ancestral genotypes (GG; p = 0.0001) in both MIP and WWM subjects. CONCLUSION: The observation of elevated IL-1ß and IL-6 in MIP and TNF-α in WWM may be regarded as a prognostic inflammatory marker of infection and pregnancy. Most particularly, the TNF-α concentration and its polymorphic variability in the promoter region may indicate genetic susceptibility and mildly influence the risk for P. vivax infection during pregnancy and in women with malaria.

iNOS polymorphism modulates iNOS/NO expression via impaired antioxidant and ROS content in P. vivax and P. falciparum infection.

Nitric oxide (NO) has dicotomic influence on modulating host-parasite interplay, synchronizing physiological orchestrations and diagnostic potential; instigated us to investigate the plausible association and genetic regulation among NO level, components of oxidative stress, iNOS polymorphisms and risk of malaria. Here, we experimentally elucidate that iNOS promoter polymorphisms are associated with risk of malaria; employing mutation specific genotyping, functional interplay using western blot and RT-PCR, quantitative estimation of NO, total antioxidant content (TAC) and reactive oxygen species (ROS). Genotyping revealed significantly associated risk of P. vivax (adjusted OR = 1.92 and 1.72) and P. falciparum (adjusted OR = 1.68 and 1.75) infection with SNP at iNOS-954G/C and iNOS-1173C/T positions, respectively; though vivax showed higher risk of infection. Intriguingly, mutation and infection specific differential upregulation of iNOS expression/NO level was observed and found to be significantly associated with mutant genotypes. Moreover, P. vivax showed pronounced iNOS protein (2.4 fold) and mRNA (2.5 fold) expression relative to healthy subjects. Furthermore, TAC and ROS were significantly decreased in infection; and differentially decreased in mutant genotypes. Our findings endorse polymorphic regulation of iNOS expression, altered oxidant-antioxidant components and evidences of risk association as the hallmark of malaria pathogenesis. iNOS/NO may serve as potential diagnostic marker in assessing clinical malaria.

Intracellular zinc flux causes reactive oxygen species mediated mitochondrial dysfunction leading to cell death in Leishmania donovani.

Leishmaniasis caused by Leishmania parasite is a global threat to public health and one of the most neglected tropical diseases. Therefore, the discovery of novel drug targets and effective drug is a major challenge and an important goal. Leishmania is an obligate intracellular parasite that alternates between sand fly and human host. To survive and establish infections, Leishmania parasites scavenge and internalize nutrients from the host. Nevertheless, host cells presents mechanism like nutrient restriction to inhibit microbial growth and control infection. Zinc is crucial for cellular growth and disruption in its homeostasis hinders growth and survival in many cells. However, little is known about the role of zinc in Leishmania growth and survival. In this study, the effect of zinc on the growth and survival of L.donovani was analyzed by both Zinc-depletion and Zinc-supplementation using Zinc-specific chelator N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) and Zinc Sulfate (ZnSO4). Treatment of parasites with TPEN rather than ZnSO4 had significantly affected the growth in a dose- and time-dependent manner. The pre-treatment of promastigotes with TPEN resulted into reduced host-parasite interaction as indicated by decreased association index. Zn depletion resulted into flux in intracellular labile Zn pool and increased in ROS generation correlated with decreased intracellular total thiol and retention of plasma membrane integrity without phosphatidylserine exposure in TPEN treated promastigotes. We also observed that TPEN-induced Zn depletion resulted into collapse of mitochondrial membrane potential which is associated with increase in cytosolic calcium and cytochrome-c. DNA fragmentation analysis showed increased DNA fragments in Zn-depleted cells. In summary, intracellular Zn depletion in the L. donovani promastigotes led to ROS-mediated caspase-independent mitochondrial dysfunction resulting into apoptosis-like cell death. Therefore, cellular zinc homeostasis in Leishmania can be explored for new drug targets and chemotherapeutics to control Leishmanial growth and disease progression.

Leishmania and its quest for iron: An update and overview.

Parasites of genus Leishmania are the causative agents of complex neglected diseases called leishmaniasis and continue to be a significant health concern globally. Iron is a vital nutritional requirement for virtually all organisms, including pathogenic trypanosomatid parasites, and plays a crucial role in many facets of cellular metabolism as a cofactor of several enzymes. Iron acquisition is essential for the survival of parasites. Yet parasites are also vulnerable to the toxicity of iron and reactive oxygen species. The aim of this review is to provide an update on the current knowledge about iron acquisition and usage by Leishmania species. We have also discussed about host strategy to modulate iron availability and the strategies deployed by Leishmania parasites to overcome iron withholding defences and thus favour parasite growth within host macrophages. Since iron plays central roles in the host's response and parasite metabolism, a comprehensive understanding of the iron metabolism is beneficial to identify potential viable therapeutic opportunities against leishmaniasis.

Interaction between cysteine synthase and serine O-acetyltransferase proteins and their stage specific expression in Leishmania donovani.

Leishmania possess a unique trypanothione redox metabolism with undebated roles in protection from oxidative damage and drug resistance. The biosynthesis of trypanothione depends on l-cysteine bioavailability which is regulated by cysteine biosynthesis pathway. The de novo cysteine biosynthesis pathway is comprised of serine O-acetyltransferase (SAT) and cysteine synthase (CS) enzymes which sequentially mediate two consecutive steps of cysteine biosynthesis, and is absent in mammalian host. However, despite the apparent dependency of redox metabolism on cysteine biosynthesis pathway, the role of SAT and CS in redox homeostasis has been unexplored in Leishmania parasites. Herein, we have characterized CS and SAT to investigate their interaction and relative abundance of these proteins in promastigote vs. amastigote growth stages of L. donovani. CS and SAT genes of L. donovani (LdCS and LdSAT) were cloned, expressed, and fusion proteins purified to homogeneity with affinity column chromatography. Purified LdCS contains PLP as cofactor and showed optimum enzymatic activity at pH 7.5. Enzyme kinetics showed that LdCS catalyses the synthesis of cysteine using O-acetylserine and sulfide with a Km of 15.86 mM and 0.17 mM, respectively. Digitonin fractionation and indirect immunofluorescence microscopy showed that LdCS and LdSAT are localized in the cytoplasm of promastigotes. Size exclusion chromatography, co-purification, pull down and immuno-precipitation assays demonstrated a stable complex formation between LdCS and LdSAT proteins. Furthermore, LdCS and LdSAT proteins expression/activity was upregulated in amastigote growth stage of the parasite. Thus, the stage specific differential expression of LdCS and LdSAT suggests that it may have a role in the redox homeostasis of Leishmania.

Up-regulation of cytosolic tryparedoxin in Amp B resistant isolates of Leishmania donovani and its interaction with cytosolic tryparedoxin peroxidase.

Leishmania is a unicellular protozoan parasite which causes leishmaniasis, a neglected tropical disease. It possess a unique thiol metabolism comprising of several proteins among which, tryparedoxin (cTXN) and tryparedoxin peroxidase (cTXNPx), function in concert as oxidoreductases, utilizing trypanothione as a source of electrons to reduce the hydroperoxides produced by macrophages during infection. This detoxification pathway is unique and essential for the survival of Leishmania. Herein, we report the functional characterization of Leishmania donovani cTXN and its interaction with cTXNPx. The full length recombinant cTXN and cTXNPx proteins were purified in the native state and biochemical analysis showed that the cTXN-cTXNPx coupled system efficiently degraded hydrogen peroxide and tert-butyl hydroperoxide by transferring reducing equivalents from trypanothione. In silico investigation of the potential interaction between cTXN and cTXNPx proteins showed strong interaction of model structures with amino acids Ile109, Thr132, Glu107, Trp70, Trp39, Cys40 and His129 of Ld-cTXN and Thr54, Lys93, Arg128 and Asn152 of Ld-cTXNPx predicted to be involved in interaction. Moreover, co-purification, pull down assay and immunoprecipitation studies confirmed the interaction between Ld-cTXN and Ld-cTXNPx proteins. In addition, for the first time, we demonstrated at the translational level that Ld-cTXN protein is upregulated in Amp B resistant isolates accompanied by enhanced peroxidase activity, as compared to sensitive strains. Thus, our results show that Ld-cTXN and Ld-cTXNPx proteins acts in concert by physical interaction to form a strong peroxide stress detoxification system in Leishmania and their upregulation in Amp B resistant isolates imparts better stress tolerance, and hence fitter pathogens, as compared to sensitive strains.

Interaction of frataxin, an iron binding protein, with IscU of Fe-S clusters biogenesis pathway and its upregulation in AmpB resistant Leishmania donovani.

Leishmania donovani is a unicellular protozoon parasite that causes visceral leishmaniasis (VL), which is a fatal disease if left untreated. Certain Fe-S proteins of the TCA cycle and respiratory chain have been found in the Leishmania parasite but the precise mechanisms for their biogenesis and the maturation of Fe-S clusters remains unknown. Fe-S clusters are ubiquitous cofactors of proteins that perform critical cellular functions. The clusters are biosynthesized by the mitochondrial Iron-Sulphur Cluster (ISC) machinery with core protein components that include the catalytic cysteine desulphurase IscS, the scaffold proteins IscU and IscA, and frataxin as an iron carrier/donor. However, no information regarding frataxin, its regulation, or its role in drug resistance is available for the Leishmania parasite. In this study, we characterized Ld-frataxin to investigate its role in the ISC machinery of L. donovani. We expressed and purified the recombinant Ld-frataxin protein and observed its interaction with Ld-IscU by co-purification and pull-down assay. Furthermore, we observed that the cysteine desulphurase activity of the purified Ld-IscS protein was stimulated in the presence of Ld-frataxin and Ld-IscU, particularly in the presence of iron; neither Ld-frataxin nor Ld-IscU alone had significant effects on Ld-IscS activity. Interestingly, RT-PCR and western blotting showed that Ld-frataxin is upregulated in AmpB-resistant isolates compared to sensitive strains, which may support higher Fe-S protein activity in AmpB-resistant L. donovani. Additionally, Ld-frataxin was localized in the mitochondria, as revealed by digitonin fractionation and indirect immunofluorescence. Thus, our results suggest the role of Ld-frataxin as an iron binding/carrier protein for Fe-S cluster biogenesis that physically interacts with other core components of the ISC machinery within the mitochondria.

Prevalence of Malaria Infection and Risk Factors Associated with Anaemia among Pregnant Women in Semiurban Community of Hazaribag, Jharkhand, India.

The escalating burden, pathogenesis, and clinical sequel of malaria during pregnancy have combinatorial adverse impact on both mother and foetus that further perplexed the situation of diagnosis, treatment, and prevention. This prompted us to evaluate the status of population at risk of MIP in Hazaribag, Jharkhand, India. Cross-sectional study was conducted over a year at Sadar Hospital, Hazaribag. Malaria was screened using blood smear and/or RDT. Anaemia was defined as haemoglobin concentration. Pretested questionnaires were used to gather sociodemographic, clinical, and obstetrical data. The prevalence of MIP was 5.4% and 4.3% at ANC and DU, and 13.2% malaria was in women without pregnancy. Interestingly, majority were asymptomatically infected with P. vivax (over 85%) at ANC and DU. Peripheral parasitemia was significantly associated with fever within past week, rural origin of subjects, and first/second pregnancies in multivariate analysis, with the highest risk factor associated with fever followed by rural residence. Strikingly in cohort, anaemia was prevalent in 86% at ANC as compared to 72% at DU, whereas severe anaemia was 13.6% and 7.8% at ANC and DU. Even more anaemia prevalence was observed in MIP group (88% and 89% at ANC and DU), whereas severe anaemia was 23% and 21%, respectively. In view of observed impact of anaemia, parasitemia and asymptomatic infection of P. vivax during pregnancy and delivery suggest prompt diagnosis regardless of symptoms and comprehensive drug regime should be offered to pregnant women in association with existing measures in clinical spectrum of MIP, delivery, and its outcome.

Interaction between Nbp35 and Cfd1 proteins of cytosolic Fe-S cluster assembly reveals a stable complex formation in Entamoeba histolytica.

Iron-Sulfur (Fe-S) proteins are involved in many biological functions such as electron transport, photosynthesis, regulation of gene expression and enzymatic activities. Biosynthesis and transfer of Fe-S clusters depend on Fe-S clusters assembly processes such as ISC, SUF, NIF, and CIA systems. Unlike other eukaryotes which possess ISC and CIA systems, amitochondriate Entamoeba histolytica has retained NIF & CIA systems for Fe-S cluster assembly in the cytosol. In the present study, we have elucidated interaction between two proteins of E. histolytica CIA system, Cytosolic Fe-S cluster deficient 1 (Cfd1) protein and Nucleotide binding protein 35 (Nbp35). In-silico analysis showed that structural regions ranging from amino acid residues (P33-K35, G131-V135 and I147-E151) of Nbp35 and (G5-V6, M34-D39 and G46-A52) of Cfd1 are involved in the formation of protein-protein complex. Furthermore, Molecular dynamic (MD) simulations study suggested that hydrophobic forces surpass over hydrophilic forces between Nbp35 and Cfd1 and Van-der-Waal interaction plays crucial role in the formation of stable complex. Both proteins were separately cloned, expressed as recombinant fusion proteins in E. coli and purified to homogeneity by affinity column chromatography. Physical interaction between Nbp35 and Cfd1 proteins was confirmed in vitro by co-purification of recombinant Nbp35 with thrombin digested Cfd1 and in vivo by pull down assay and immunoprecipitation. The insilico, in vitro as well as in vivo results prove a stable interaction between these two proteins, supporting the possibility of its involvement in Fe-S cluster transfer to target apo-proteins through CIA machinery in E. histolytica. Our study indicates that initial synthesis of a Fe-S precursor in mitochondria is not necessary for the formation of Cfd1-Nbp35 complex. Thus, Cfd1 and Nbp35 with the help of cytosolic NifS and NifU proteins can participate in the maturation of non-mitosomal Fe-S proteins without any apparent assistance of mitosomes.

Stage-dependent expression and up-regulation of trypanothione synthetase in amphotericin B resistant Leishmania donovani.

Kinetoplastids differ from other organisms in their ability to conjugate glutathione and spermidine to form trypanothione which is involved in maintaining redox homeostasis and removal of toxic metabolites. It is also involved in drug resistance, antioxidant mechanism, and defense against cellular oxidants. Trypanothione synthetase (TryS) of thiol metabolic pathway is the sole enzyme responsible for the biosynthesis of trypanothione in Leishmania donovani. In this study, TryS gene of L. donovani (LdTryS) was cloned, expressed, and fusion protein purified with affinity column chromatography. The purified protein showed optimum enzymatic activity at pH 8.0-8.5. The TryS amino acids sequences alignment showed that all amino acids involved in catalytic and ligands binding of L. major are conserved in L. donovani. Subcellular localization using digitonin fractionation and immunoblot analysis showed that LdTryS is localized in the cytoplasm. Furthermore, RT-PCR coupled with immunoblot analysis showed that LdTryS is overexpressed in Amp B resistant and stationary phase promastigotes (∼ 2.0-folds) than in sensitive strain and logarithmic phase, respectively, which suggests its involvement in Amp B resistance. Also, H2O2 treatment upto 150 µM for 8 hrs leads to 2-fold increased expression of LdTryS probably to cope up with oxidative stress generated by H2O2. Therefore, this study demonstrates stage- and Amp B sensitivity-dependent expression of LdTryS in L. donovani and involvement of TryS during oxidative stress to help the parasites survival.