Glyoxalase I Gene Deletion Mutants of Leishmania donovani Exhibit Reduced Methylglyoxal Detoxification

Background

Glyoxalase I is a metalloenzyme of the glyoxalase pathway that plays a central role in eliminating the toxic metabolite methyglyoxal. The protozoan parasite Leishmania donovani possesses a unique trypanothione dependent glyoxalase system.

Principal Findings

Analysis of the L. donovani GLOI sequence predicted a mitochondrial targeting sequence, suggesting that the enzyme is likely to be targeted to the mitochondria. In order to determine definitively the intracellular localization of GLOI in L. donovani, a full-length GLOI gene was fused to green fluorescent protein (GFP) gene to generate a chimeric construct. Confocal microscopy of L. donovani promastigotes carrying this chimeric construct and immunofluorescence microscopy using anti-GLOI antibodies demonstrated that GLOI is localized in the kinetoplast of the parasite apart from the cytosol. To study the physiological role of GLOI in Leishmania, we first created promastigote mutants heterozygous for GLOI by targeted gene replacement using either hygromycin or neomycin phosphotransferases as selectable markers. Heterozygous mutants of L. donovani display a slower growth rate, have lower glyoxalase I activity and have reduced ability to detoxify methylglyoxal in comparison to the wild-type parasites. Complementation of the heterozygous mutant with an episomal GLOI construct showed the restoration of heterozygous mutant phenotype nearly fully to that of the wild-type. Null mutants were obtained only after GLOI was expressed from an episome in heterozygous mutants.

Conclusions

We for the first time report localization of GLOI in L. donovani in the kinetoplast. To study the physiological role of GLOI in Leishmania, we have generated GLOI attenuated strains by targeted gene replacement and report that GLOI is likely to be an important gene since GLOI mutants in L. donovani showed altered phenotype. The present data supports that the GLOI plays an essential role in the survival of this pathogenic organism and that inhibition of the enzyme potentiates the toxicity of methylglyoxal.     

Linking In Vitro and In Vivo Survival of Clinical Leishmania donovani Strains

Background

Leishmania donovani is an intracellular protozoan parasite that causes a lethal systemic disease, visceral leishmaniasis (VL), and is transmitted between mammalian hosts by phlebotomine sandflies. Leishmania expertly survives in these ‘hostile’ environments with a unique redox system protecting against oxidative damage, and host manipulation skills suppressing oxidative outbursts of the mammalian host. Treating patients imposes an additional stress on the parasite and sodium stibogluconate (SSG) was used for over 70 years in the Indian subcontinent.

Methodology/Principal Findings

We evaluated whether the survival capacity of clinical L. donovani isolates varies significantly at different stages of their life cycle by comparing proliferation, oxidative stress tolerance and infection capacity of 3 Nepalese L. donovani strains in several in vitro and in vivo models. In general, the two strains that were resistant to SSG, a stress encountered in patients, attained stationary phase at a higher parasite density, contained a higher amount of metacyclic parasites and had a greater capacity to cause in vivo infection in mice compared to the SSG-sensitive strain.

Conclusions/Significance

The 2 SSG-resistant strains had superior survival skills as promastigotes and as amastigotes compared to the SSG-sensitive strain. These results could indicate that Leishmania parasites adapting successfully to antimonial drug pressure acquire an overall increased fitness, which stands in contrast to what is found for other organisms, where drug resistance is usually linked to a fitness cost. Further validation experiments are under way to verify this hypothesis.     

Role of Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase in Visceral Organ Infection by Leishmania donovani

The initial 7 steps of the glycolytic pathway from glucose to 3-phosphoglycerate are localized in the glycosomes in Leishmania, including step 6, catalyzed by the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In L. donovani and L. mexicana, there exists a second GAPDH enzyme present in the cytosol that is absent in L. braziliensis and that has become a pseudogene in L. major. To investigate the role of the cytosolic GAPDH (cGAPDH), an L. donovani cGAPDH-null mutant was generated, and conversely, the functional L. donovani cGAPDH was introduced into L. major and the resulting engineered parasites were characterized. The L. donovani cGAPDH-null mutant was able to proliferate at the same rate as the wild-type parasite in glucose-deficient medium. However, in the presence of glucose, the L. donovani cGAPDH-null mutant consumed less glucose and proliferated more slowly than the wild-type parasite and displayed reduced infectivity in visceral organs of experimentally infected mice. This demonstrates that cGAPDH is functional in L. donovani and is required for survival in visceral organs. Restoration of cGAPDH activity in L. major, in contrast, had an adverse effect on L. major proliferation in glucose-containing medium, providing a possible explanation of why it has evolved into a pseudogene in L. major. This study indicates that there is a difference in glucose metabolism between L. donovani and L. major, and this may represent an important factor in the ability of L. donovani to cause visceral disease.     

Targeting Ergosterol Biosynthesis in Leishmania donovani: Essentiality of Sterol 14alpha-demethylase

Leishmania protozoan parasites (Trypanosomatidae family) are the causative agents of cutaneous, mucocutaneous and visceral leishmaniasis worldwide. While these diseases are associated with significant morbidity and mortality, there are few adequate treatments available. Sterol 14alpha-demethylase (CYP51) in the parasite sterol biosynthesis pathway has been the focus of considerable interest as a novel drug target in Leishmania. However, its essentiality in Leishmania donovani has yet to be determined. Here, we use a dual biological and pharmacological approach to demonstrate that CYP51 is indispensable in L. donovani. We show via a facilitated knockout approach that chromosomal CYP51 genes can only be knocked out in the presence of episomal complementation and that this episome cannot be lost from the parasite even under negative selection. In addition, we treated wild-type L. donovani and CYP51-deficient strains with 4-aminopyridyl-based inhibitors designed specifically for Trypanosoma cruzi CYP51. While potency was lower than in T. cruzi, these inhibitors had increased efficacy in parasites lacking a CYP51 allele compared to complemented parasites, indicating inhibition of parasite growth via a CYP51-specific mechanism and confirming essentiality of CYP51 in L. donovani. Overall, these results provide support for further development of CYP51 inhibitors for the treatment of visceral leishmaniasis.     

Hsp90 inhibitors radicicol and geldanamycin have opposing effects on <Emphasis Type="Italic">Leishmania</Emphasis> Aha1-dependent proliferation

Hsp90 and its co-chaperones are essential for the medically important parasite Leishmania donovani, facilitating life cycle control and intracellular survival. Activity of Hsp90 is regulated by co-chaperones of the Aha1 and P23 families. In this paper, we studied the expression of L. donovani Aha1 in two life cycle stages, its interaction with Hsp90 and the phenotype of Aha1 null mutants during the insect stage and inside infected macrophages. This study provides a detailed in vitro analysis of the function of Aha1 in Leishmania parasites and the first instance of a reverse genetic analysis of Aha1 in a protozoan parasite. While Aha1 is non-essential under standard growth conditions and at elevated temperature, Aha1 protects against ethanol stress. However, both overexpression and lack of Aha1 affected parasite growth in the presence of the Hsp90 inhibitors radicicol (RAD) and geldanamycin (GA). Under RAD pressure, P23 and Aha1 act in an antagonistic way. By contrast, expression levels of both co-chaperones have similar effects under GA treatment, indicating different inhibition mechanisms by the two compounds. Aha1 is also secreted in virulence-enhancing exosomes. This may explain why the loss of Aha1 reduces the infectivity of L. donovani in ex vivo mouse macrophages, indicating a role during the intracellular mammalian stage.     

A tyrosyl DNA phosphodiesterase 1 from kinetoplastid parasite Leishmania donovani (LdTdp1) capable of removing topo I–DNA covalent complexes

Tyrosyl DNA phosphodiesterase 1 (Tdp1) is a member of phospholipase D superfamily, which cleaves a broad range of 3′‐DNA adducts, the best characterized of which is the phosphodiester bond formed between DNA and topoisomerase IB. This study describes cloning and functional characterization of the enzyme, termed as LdTdp1 in the kinetoplastid parasite Leishmania donovani. Sequence analysis confirmed conservation of the active site motifs typical for all Tdp1 proteins. LdTdp1 activity was detected in the parasite nucleus as well as in the kinetoplast. The enzyme harbours a nuclear localization signal at its C‐terminus. Overexpression of the active enzyme protected the parasites against topoisomerase IB inhibitor camptothecin (CPT) and oxidative agent H₂O₂‐mediated cytotoxicity and its downregulation rendered the parasites hypersensitive to CPT. Trapping of mutant LdTdp1 on DNA takes place following CPT treatment in L. donovani cells. The expression level and associated activity of LdTdp1 were found to be higher in CPT‐resistant L. donovani parasites. Altogether, this is the first report of Tdp1 from the kinetoplastid parasite L. donovani, which actively participates in topoisomerase I‐mediated DNA damage repair process and thereby counteracts the cytotoxic effect of topoisomerase I inhibitors.     

Interaction of novel proteins,centrin4 and protein of centriole in Leishmania parasite and their effects on the parasite growth

Centrins are cytoskeletal proteins associated with the centrosomes or basal bodies in the eukaryotes. We previously reported the involvement of Centrin 1–3 proteins in cell division in the protozoan parasites Leishmania donovani and Trypanosoma brucei. Centrin4 and 5, unique to such parasites, had never been characterized in Leishmania parasite. In the current study, we addressed the function of centrin4 (LdCen4) in Leishmania. By dominant-negative study, the episomal expression of C-terminal truncated LdCen4 in the parasite reduced the parasite growth. LdCen4 double allele gene deletion by either homologous recombination or CRISPR-Cas9 was not successful in L. donovani. However, CRISPR-Cas9-based deletion of the homologous gene was possible in L. mexicana, which attenuated the parasite growth in vitro, but not ex vivo in the macrophages. LdCen4 also interacts with endogenous and overexpressed LdPOC protein, a homolog of centrin reacting human POC (protein of centriole) in a calcium sensitive manner. LdCen4 and LdPOC binding has also been confirmed through in silico analysis by protein structural docking and validated by co-immunoprecipitation. By immunofluorescence studies, we found that both the proteins share a common localization at the basal bodies. Thus, for the first time, this article describes novel centrin4 and its binding protein in the protozoan parasites.     

Adenylosuccinate Synthetase and Adenylosuccinate Lyase Deficiencies Trigger Growth and Infectivity Deficits in Leishmania donovani

Leishmania are auxotrophic for purines, and consequently purine acquisition from the host is a requisite nutritional function for the parasite. Both adenylosuccinate synthetase (ADSS) and adenylosuccinate lyase (ASL) have been identified as vital components of purine salvage in Leishmania donovani, and therefore Δadss and Δasl null mutants were constructed to test this hypothesis. Unlike wild type L. donovani, Δadss and Δasl parasites in culture exhibited a profoundly restricted growth phenotype in which the only permissive growth conditions were a 6-aminopurine source in the presence of 2′-deoxycoformycin, an inhibitor of adenine aminohydrolase activity. Although both knock-outs showed a diminished capacity to infect murine peritoneal macrophages, only the Δasl null mutant was profoundly incapacitated in its ability to infect mice. The enormous discrepancy in parasite loads observed in livers and spleens from mice infected with either Δadss or Δasl parasites can be explained by selective accumulation of adenylosuccinate in the Δasl knock-out and consequent starvation for guanylate nucleotides. Genetic complementation of a Δasl lesion in Escherichia coli implied that the L. donovani ASL could also recognize 5-aminoimidazole-(N-succinylocarboxamide) ribotide as a substrate, and purified recombinant ASL displayed an apparent K_m of ∼24 μm for adenylosuccinate. Unlike many components of the purine salvage pathway of L. donovani, both ASL and ADSS are cytosolic enzymes. Overall, these data underscore the paramount importance of ASL to purine salvage by both life cycle stages of L. donovani and authenticate ASL as a potential drug target in Leishmania.     

Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC₅₀ values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.     

Leishmania donovani Argininosuccinate Synthase Is an Active Enzyme Associated with Parasite Pathogenesis

Background

Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo.

Methodology/Principal Findings

Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver.

Conclusion/Significance

Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.     

ADF2 is required for transformation of the ookinete and sporozoite in malaria parasite development

Malaria parasites undergo two rounding-up transformations in their life cycle: the ookinete-to-oocyst transformation in the mosquito midgut, and the sporozoite-to-EEF (exo-erythrocytic form) differentiation in the host hepatocyte. Both events are characterized by the loss of polarity, implying that cytoskeletal reorganization is involved. In other eukaryotes, regulation of the actin skeleton is fundamental to subcellular remodeling. Although filamentous actin is well known to be involved in the motility of apicomplexan parasites, its participation in their morphological regulation is still largely unexplored. Here we describe the fundamental role of Actin depolymerization factor 2 (ADF2), a vector-stage-specific ADF isoform, in morphological changes accompanying the parasite life cycle. Among protozoan parasites, Plasmodium is unique in having two actin and two ADF genes. Disruption of the ADF2 gene in the rodent malaria parasite P. berghei had no effect on ookinete development or its subsequent invasion of the midgut. However, both the ookinete-to-oocyst and sporozoite-to-EEF transformations showed significant defects. These results indicated that Plasmodium ADF2 plays a pivotal role in transformation in the malaria parasite life cycle.     

Gregarina hylobii and Ophryocystis hylobii n. sp. (ophryocystidae,neogregarinida) parasitizing Hylobius abietis (Curculionidae,coleoptera)

Two sporozoan parasites, Gregarina hylobii and Ophryocystis hylobii n. sp., parasitizing the adults of natural populations of Hylobius abietis are described. The gregarine G. hylobii, a parasite of the gut of H. abietis, has been reexamined and its complete life cycle, including the solitary gamonts, gametocysts, and sporoducts is described. The life cycle of the neogregarine O. hylobii infecting the Malpighian tubules of host animals examined at light and electron microscope levels is discussed. Some data on the prevalence of infections and host-parasite relationships are also discussed.     

Leishmania donovani Infection Enhances Lateral Mobility of Macrophage Membrane Protein Which Is Reversed by Liposomal Cholesterol

BackgroundThe protozoan parasite Leishmania donovani (LD) reduces cellular cholesterol of the host possibly for its own benefit. Cholesterol is mostly present in the specialized compartment of the plasma membrane. The relation between mobility of membrane proteins and cholesterol depletion from membrane continues to be an important issue. The notion that leishmania infection alters the mobility of membrane proteins stems from our previous study where we showed that the distance between subunits of IFNγ receptor (R1 and R2) on the cell surface of LD infected cell is increased, but is restored to normal by liposomal cholesterol treatment.Conclusions/SignificancesTo our knowledge this is the first direct demonstration that LD parasites during their intracellular life cycle increases lateral mobility of membrane proteins and decreases F-actin level in infected macrophages. Such defects may contribute to ineffective intracellular signaling and other cellular functions.     

Amplification of Adenine Phosphoribosyltransferase Suppresses the Conditionally Lethal Growth and Virulence Phenotype of Leishmania donovani Mutants Lacking Both Hypoxanthine-guanine and Xanthine Phosphoribosyltransferases

Leishmania donovani cannot synthesize purines de novo and obligatorily scavenge purines from the host. Previously, we described a conditional lethal Δhgprt/Δxprt mutant of L. donovani (Boitz, J. M., and Ullman, B. (2006) J. Biol. Chem. 281, 16084–16089) that establishes that L. donovani salvages purines primarily through hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT). Unlike wild type L. donovani, the Δhgprt/Δxprt knock-out cannot grow on 6-oxypurines and displays an absolute requirement for adenine or adenosine and 2′-deoxycoformycin, an inhibitor of parasite adenine aminohydrolase activity. Here, we demonstrate that the ability of Δhgprt/Δxprt parasites to infect mice was profoundly compromised. Surprisingly, mutant parasites that survived the initial passage through mice partially regained their virulence properties, exhibiting a >10-fold increase in parasite burden in a subsequent mouse infection. To dissect the mechanism by which Δhgprt/Δxprt parasites persisted in vivo, suppressor strains that had regained their capacity to grow under restrictive conditions were cloned from cultured Δhgprt/Δxprt parasites. The ability of these suppressor clones to grow in and metabolize 6-oxypurines could be ascribed to a marked amplification and overexpression of the adenine phosphoribosyltransferase (APRT) gene. Moreover, transfection of Δhgprt/Δxprt cells with an APRT episome recapitulated the suppressor phenotype in vitro and enabled growth on 6-oxypurines. Biochemical studies further showed that hypoxanthine, unexpectedly, was an inefficient substrate for APRT, evidence that could account for the ability of the suppressors to metabolize hypoxanthine. Subsequent analysis implied that APRT amplification was also a potential contributory mechanism by which Δhgprt/Δxprt parasites displayed persistence and increased virulence in mice.     

A Leishmania donovani gene that confers accelerated recovery from stationary phase growth arrest

We have isolated a gene, LdGF1, from the protozoan parasite Leishmania donovani. Overexpression of this gene confers a strong selective advantage in liquid culture after stationary phase growth arrest. We could show that recombinant L. donovani or Leishmania major, when overexpressing LdGF1, recover faster from a stationary phase growth arrest than control parasite strains. While no advantage of LdGF1 overexpression could be observed in log phase cultures or after a hydroxyurea-induced S-phase growth arrest, recovery from a cell cycle arrest due to serum deprivation was faster in LdGF1-overexpressing strains. This was found to be due to an accelerated release from a G₁ cell cycle arrest. By contrast, in a BALB/c mouse infection system, overexpression of LdGF1 in L. major resulted in reduced virulence. We conclude that increased levels of LdGF1 are beneficiary during recovery from G₁ cell cycle arrest, but pose a disadvantage inside a mammalian host. These results are discussed in the context of the observed loss of virulence during in vitro passage of Leishmania parasites.     

Over-Expression of 60s Ribosomal L23a Is Associated with Cellular Proliferation in SAG Resistant Clinical Isolates of Leishmania donovani

Background

Sodium antimony gluconate (SAG) unresponsiveness of Leishmania donovani (Ld) had effectively compromised the chemotherapeutic potential of SAG. 60s ribosomal L23a (60sRL23a), identified as one of the over-expressed protein in different resistant strains of L.donovani as observed with differential proteomics studies indicates towards its possible involvement in SAG resistance in L.donovani. In the present study 60sRL23a has been characterized for its probable association with SAG resistance mechanism.

Methodology and principal findings

The expression profile of 60s ribosomal L23a (60sRL23a) was checked in different SAG resistant as well as sensitive strains of L.donovani clinical isolates by real-time PCR and western blotting and was found to be up-regulated in resistant strains. Ld60sRL23a was cloned, expressed in E.coli system and purified for raising antibody in swiss mice and was observed to have cytosolic localization in L.donovani. 60sRL23a was further over-expressed in sensitive strain of L.donovani to check its sensitivity profile against SAG (Sb V and III) and was found to be altered towards the resistant mode.

Conclusion/Significance

This study reports for the first time that the over expression of 60sRL23a in SAG sensitive parasite decreases the sensitivity of the parasite towards SAG, miltefosine and paramomycin. Growth curve of the tranfectants further indicated the proliferative potential of 60sRL23a assisting the parasite survival and reaffirming the extra ribosomal role of 60sRL23a. The study thus indicates towards the role of the protein in lowering and redistributing the drug pressure by increased proliferation of parasites and warrants further longitudinal study to understand the underlying mechanism.     

The Leishmania donovani chaperone cyclophilin 40 is essential for intracellular infection independent of its stage‐specific phosphorylation status

During its life cycle, the protozoan pathogen Leishmania donovani is exposed to contrasting environments inside insect vector and vertebrate host, to which the parasite must adapt for extra‐ and intracellular survival. Combining null mutant analysis with phosphorylation site‐specific mutagenesis and functional complementation we genetically tested the requirement of the L. donovani chaperone cyclophilin 40 (LdCyP40) for infection. Targeted replacement of LdCyP40 had no effect on parasite viability, axenic amastigote differentiation, and resistance to various forms of environmental stress in culture, suggesting important functional redundancy to other parasite chaperones. However, ultrastructural analyses and video microscopy of cyp40?/? promastigotes uncovered important defects in cell shape, organization of the subpellicular tubulin network and motility at stationary growth phase. More importantly, cyp40?/? parasites were unable to establish intracellular infection in murine macrophages and were eliminated during the first 24 h post infection. Surprisingly, cyp40?/? infectivity was restored in complemented parasites expressing a CyP40 mutant of the unique S274 phosphorylation site. Together our data reveal non‐redundant CyP40 functions in parasite cytoskeletal remodelling relevant for the development of infectious parasites in vitro independent of its phosphorylation status, and provide a framework for the genetic analysis of Leishmania‐specific phosphorylation sites and their role in regulating parasite protein function.