Sigrid C. Roberts

An Effect of Parasite-Encoded Arginase on the Outcome of Murine Cutaneous Leishmaniasis

Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Δarg), as well as wild-type and complemented Δarg controls (Δarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p < 0.05); 2) poorer survival of Δarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Δarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Δarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Δarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Δarg parasites produced more IFN-γ and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.

Ornithine Decarboxylase Gene Deletion Mutants of Leishmania donovani

A knockout strain of Leishmania donovani lacking both ornithine decarboxylase (ODC) alleles has been created by targeted gene replacement. Growth of Δodccells in polyamine-deficient medium resulted in a rapid and profound depletion of cellular putrescine pools, although levels of spermidine were relatively unaffected. Concentrations of trypanothione, a spermidine conjugate, were also reduced, whereas glutathione concentrations were augmented. The Δodc L. donovaniexhibited an auxotrophy for polyamines that could be circumvented by the addition of the naturally occurring polyamines, putrescine or spermidine, to the culture medium. Whereas putrescine supplementation restored intracellular pools of both putrescine and spermidine, exogenous spermidine was not converted back to putrescine, indicating that spermidine alone is sufficient to meet the polyamine requirement, and that L. donovani does not express the enzymatic machinery for polyamine degradation. The lack of a polyamine catabolic pathway in intact parasites was confirmed radiometrically. In addition, the Δodc strain could grow in medium supplemented with either 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), but polyamine auxotrophy could not be overcome by other aliphatic diamines or spermine. These data establish genetically that ODC is an essential gene in L. donovani, define the polyamine requirements of the parasite, and reveal the absence of a polyamine-degradative pathway.

Genetic analysis of spermidine synthase from Leishmania donovani.

The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Deltaspdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Deltaspdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3-4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.

S-adenosylmethionine decarboxylase from Leishmania donovani: Molecular, genetic, and biochemical characterization of null mutants and overproducers

The polyamine biosynthetic enzyme, S-adenosylmethionine decarboxylase (ADOMETDC) has been advanced as a potential target for antiparasitic chemotherapy. To investigate the importance of this protein in a model parasite, the gene encoding ADOMETDC has been cloned and sequenced fromLeishmania donovani. The Δadometdc null mutants were created in the insect vector form of the parasite by double targeted gene replacement. The Δadometdc strains were incapable of growth in medium without polyamines; however, auxotrophy could be rescued by spermidine but not by putrescine, spermine, or methylthioadenosine. Incubation of Δadometdcparasites in medium lacking polyamines resulted in a drastic increase of putrescine and glutathione levels with a concomitant decrease in the amounts of spermidine and the spermidine-containing thiol trypanothione. Parasites transfected with an episomalADOMETDC construct were created in both wild type and Δadometdc parasites. ADOMETDC overexpression abrogated polyamine auxotrophy in the Δadometdc L. donovani. In addition, ADOMETDC overproduction in wild type parasites alleviated the toxic effects of 5′-(((Z)-4-amino-2-butenyl)methylamino)-5′-deoxyadenosine (MDL 73811), but not pentamidine, berenil, or methylglyoxyl bis(guanylhydrazone), all inhibitors of ADOMETDC activities in vitro. The molecular, biochemical, and genetic characterization of ADOMETDC establishes that it is essential in L. donovani promastigotes and a potential target for therapeutic validation.

Arginine Homeostasis and Transport in the Human Pathogen Leishmania donovani

Arginine is an essential amino acid for the human pathogen Leishmania but not to its host. Thus, the mechanism by which this protozoan parasite regulates cellular homeostasis of arginine is critical for its survival and virulence. In a previous study, we cloned and functionally characterized a high affinity arginine-specific transporter, LdAAP3, from Leishmania donovani. In this investigation, we have characterized the relationship between arginine transport via LdAAP3 and amino acid availability. Starving promastigotes for amino acids decreased the cellular level of most amino acids including arginine but also increased the abundance of both LdAAP3 mRNA and protein and up-regulated arginine transport activity. Genetic obliteration of the polyamine biosynthesis pathway for which arginine is the sole precursor caused a significant decrease in the rate of arginine transport. Cumulatively, we established that LdAAP3 expression and activity changed whenever the cellular level of arginine changed. Our findings have led to the hypothesis that L. donovani promastigotes have a signaling pathway that senses cellular concentrations of arginine and subsequently activates a mechanism that regulates LdAAP3 expression and activity. Interestingly, this response of LdAAP3 to amino acid availability in L. donovani is identical to that of the mammalian cation amino acid transporter 1. Thus, we conjecture that Leishmania mimics the host response to amino acid availability to improve virulence.

Leishmania donovani Ornithine Decarboxylase Is Indispensable for Parasite Survival in the Mammalian Host

ABSTRACT Mutations within the polyamine biosynthetic pathway of Leishmania donovani, the etiological agent of visceral leishmaniasis, confer polyamine auxotrophy to the insect vector or promastigote form of the parasite. However, whether the infectious or amastigote form of the parasite requires an intact polyamine pathway has remained an open question. To address this issue, conditionally lethal Δodc mutants lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, were created by double targeted gene replacement within a virulent strain of L. donovani. ODC-deficient promastigotes and axenic amastigotes were auxotrophic for polyamines and capable of robust growth only when exogenous putrescine was supplied in the culture medium, confirming that polyamine biosynthesis is an essential nutritional pathway for L. donovani promastigotes. To assess whether the Δodc lesion also affected the ability of amastigotes to sustain a robust infection, macrophage and mouse infectivity experiments were performed. Parasite loads in murine macrophages infected with each of two independent Δodc knockout lines were decreased ∼80% compared to their wild-type counterpart. Furthermore, α-difluoromethylornithine, a suicide inhibitor of ODC, inhibited growth of wild-type L. donovani amastigotes and effectively cured macrophages of parasites, thereby preventing host cell destruction. Strikingly, however, parasitemias of both Δodc null mutants were reduced by 6 and 3 orders of magnitude, respectively, in livers and spleens of BALB/c mice. The compromised infectivity phenotypes of the Δodc knockouts in both macrophages and mice were rescued by episomal complementation of the genetic lesion. These genetic and pharmacological studies strongly implicate ODC as an essential cellular determinant that is necessary for the viability and growth of both L. donovani promastigotes and amastigotes and intimate that pharmacological inhibition of ODC is a promising therapeutic paradigm for the treatment of visceral and perhaps other forms of leishmaniasis.

Identification and characterization of a novel deoxyhypusine synthase in Leishmania donovani.

Deoxyhypusine synthase, an NAD+-dependent enzyme, catalyzes the first step in the post-translational synthesis of an unusual amino acid, hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine), in the eukaryotic initiation factor 5A precursor protein. Two putative deoxyhypusine synthase (DHS) sequences have been identified in the Leishmania donovani genome, which are present on chromosomes 20: DHSL20 (DHS-like gene from chromosome 20) and DHS34 (DHS from chromosome 34). Although both sequences exhibit an overall conservation of key residues, DHSL20 protein lacks a critical lysine residue, and the recombinant protein showed no DHS activity in vitro. However, DHS34 contains the critical lysine residue, and the recombinant DHS34 effectively catalyzed deoxyhypusine synthesis. Furthermore, in vivo labeling confirmed that hypusination of eukaryotic initiation factor 5A occurs in intact Leishmania parasites. Interestingly, the DHS34 is much longer, with 601 amino acids, compared with the human DHS enzyme (369 amino acids) and contains several unique insertions. To study the physiological role of DHS34 in Leishmania, gene deletion mutations were attempted via targeted gene replacement. However, chromosomal null mutants of DHS34 could only be obtained in the presence of a DHS34-containing episome. The present data provide evidence that DHS34 is essential for L. donovani and that structural differences in the human and leishmanial DHS enzyme may be exploited for designing selective inhibitors against the parasite.

Leishmania donovani polyamine biosynthetic enzyme overproducers as tools to investigate the mode of action of cytotoxic polyamine analogs.

ABSTRACT A number of anticancer and antiparasitic drugs are postulated to target the polyamine biosynthetic pathway and polyamine function, but the exact mode of action of these compounds is still being elucidated. To establish whether polyamine analogs specifically target enzymes of the polyamine pathway, a model was developed using strains of the protozoan parasite Leishmania donovani that overproduce each of the polyamine biosynthetic enzymes. Promastigotes overexpressing episomal constructs encoding ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase (ADOMETDC), or spermidine synthase (SPDSYN) revealed robust overproduction of the corresponding polyamine biosynthetic enzyme. Polyamine pools, however, were either unchanged or only marginally affected, implying that regulatory mechanisms must exist. The ODC, ADOMETDC, and SPDSYN overproducer strains exhibited a high level of resistance to difluoromethylornithine, 5′-{[(Z)-4-amino-2-butenyl]methylamino}-5′-deoxyadenosine, and n-butylamine, respectively, confirming previous observations that these agents specifically target polyamine enzymes. Conversely, augmented levels of polyamine biosynthetic enzymes did not affect the sensitivity of L. donovani promastigotes to pentamidine, berenil, and mitoguazone, drugs that were postulated to target the polyamine pathway, implying alternative and/or additional targets for these agents. The sensitivities of wild-type and overproducing parasites to a variety of polyamine analogs were also tested. The polyamine enzyme-overproducing lines offer a rapid cell-based screen for assessing whether synthetic polyamine analogs exert their mechanism of action predominantly on the polyamine biosynthetic pathway in L. donovani. Furthermore, the drug resistance engendered by the amplification of target genes and the overproduction of the encoded protein offers a general strategy for evaluating and developing therapeutic agents that target specific proteins in Leishmania.

Spermidine Synthase Is Required for Virulence of Leishmania donovani

ABSTRACT Genetic lesions in the polyamine biosynthetic pathway of Leishmania donovani, the causal agent of visceral leishmaniasis, are conditionally lethal mutations that render the insect vector form of the parasite auxotrophic for polyamines. Recently, we have demonstrated that a Δodc L. donovani null mutant lacking ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was profoundly compromised in its ability to infect mice, indicating that ODC is essential for the infectious mammalian stage of the parasite and further validating the enzyme as a possible drug target. To assess whether other components of the polyamine biosynthetic pathway were also essential for parasite virulence, a cell line deficient in spermidine synthase (SPDSYN), the enzyme that converts putrescine to spermidine, was created by double-targeted gene replacement within a virulent L. donovani background. This Δspdsyn strain was auxotrophic for polyamines, required spermidine for growth in its insect vector form, and was adversely impacted in its ability to infect mice. These findings establish that SPDSYN, like ODC, is essential for maintaining a robust infection in mammals and indicate that pharmacologic inhibition of SPDSYN, and perhaps all components of the polyamine biosynthetic pathway, is a valid therapeutic strategy for the treatment of visceral and, potentially, other forms of leishmaniasis.

Crystal Structure of Arginase from Leishmania mexicana and Implications for the Inhibition of Polyamine Biosynthesis in Parasitic Infections

Arginase from parasitic protozoa belonging to the genus Leishmania is a potential drug target for the treatment of leishmaniasis because this binuclear manganese metalloenzyme catalyzes the first committed step in the biosynthesis of polyamines that enable cell growth and survival. The high resolution X-ray crystal structures of the unliganded form of Leishmania mexicana arginase (LmARG) and four inhibitor complexes are now reported. These complexes include the reactive substrate analogue 2(S)-amino-6-boronohexanoic acid (ABH) and the hydroxylated substrate analogue nor-N(ω)-hydroxy-l-arginine (nor-NOHA), which are the most potent arginase inhibitors known to date. Comparisons of the LmARG structure with that of the archetypal arginase, human arginase I, reveal that all residues important for substrate binding and catalysis are strictly conserved. However, three regions of tertiary structure differ between the parasitic enzyme and the human enzyme corresponding to the G62 - S71, L161 - C172, and I219 - V230 segments of LmARG. Additionally, variations are observed in salt link interactions that stabilize trimer assembly in LmARG. We also report biological studies in which we demonstrate that localization of LmARG to the glycosome, a unique subcellular organelle peculiar to Leishmania and related parasites, is essential for robust pathogenesis.