Functional characterization of the first lipoyl-relay pathway from a parasitic protozoan.

Lipoic acid (LA) is a sulfur-containing cofactor covalently attached to key enzymes of central metabolism in prokaryotes and eukaryotes. LA can be acquired by scavenging, mediated by a lipoate ligase, or de novo synthesized by a pathway requiring an octanoyltransferase and a lipoate synthase. A more complex pathway, referred to as "lipoyl-relay", requires two additional proteins, GcvH, the glycine cleavage system H subunit, and an amidotransferase. This route was described so far in Bacillus subtilis and related Gram-positive bacteria, Saccharomyces cerevisiae, Homo sapiens, and Caenorhabditis elegans. Using collections of S. cerevisiae and B. subtilis mutants, defective in LA metabolism, we gathered evidence that allows us to propose for the first time that lipoyl-relay pathways are also present in parasitic protozoa. By a reverse genetic approach, we assigned octanoyltransferase and amidotransferase activity to the products of Tb927.11.9390 (TblipT) and Tb927.8.630 (TblipL) genes of Trypanosoma brucei, respectively. The B. subtilis model allowed us to identify the parasite amidotransferase as the target of lipoate analogs like 8-bromo-octanoic acid, explaining the complete loss of protein lipoylation and growth impairment caused by this compound in T. cruzi. This model could be instrumental for the screening of selective and more efficient chemotherapies against trypanosomiases.

Lipoic acid metabolism in Trypanosoma cruzi as putative target for chemotherapy.

Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC50 values one order of magnitude lower (62-66 µM). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [14C]-octanoic acid was taken up 12 fold less efficiently than [14C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments.

Zinc(II)-Dipicolylamine Coordination Complexes as Targeting and Chemotherapeutic Agents for Leishmania major.

Cutaneous leishmaniasis is a neglected tropical disease that causes painful lesions and severe disfigurement. Modern treatment relies on a few chemotherapeutics with serious limitations, and there is a need for more effective alternatives. This study describes the selective targeting of zinc(II)-dipicolylamine (ZnDPA) coordination complexes toward Leishmania major, one of the species responsible for cutaneous leishmaniasis. Fluorescence microscopy of L. major promastigotes treated with a fluorescently labeled ZnDPA probe indicated rapid accumulation of the probe within the axenic promastigote cytosol. The antileishmanial activities of eight ZnDPA complexes were measured using an in vitro assay. All tested complexes exhibited selective toxicity against L. major axenic promastigotes, with 50% effective concentration values in the range of 12.7 to 0.3 µM. Similar toxicity was observed against intracellular amastigotes, but there was almost no effect on the viability of mammalian cells, including mouse peritoneal macrophages. In vivo treatment efficacy studies used fluorescence imaging to noninvasively monitor changes in the red fluorescence produced by an infection of mCherry-L. major in a mouse model. A ZnDPA treatment regimen reduced the parasite burden nearly as well as the reference care agent, potassium antimony(III) tartrate, and with less necrosis in the local host tissue. The results demonstrate that ZnDPA coordination complexes are a promising new class of antileishmanial agents with potential for clinical translation.

Fitness and Phenotypic Characterization of Miltefosine-Resistant Leishmania major.

Trypanosomatid parasites of the genus Leishmania are the causative agents of leishmaniasis, a neglected tropical disease with several clinical manifestations. Leishmania major is the causative agent of cutaneous leishmaniasis (CL), which is largely characterized by ulcerative lesions appearing on the skin. Current treatments of leishmaniasis include pentavalent antimonials and amphotericin B, however, the toxic side effects of these drugs and difficulty with distribution makes these options less than ideal. Miltefosine (MIL) is the first oral treatment available for leishmaniasis. Originally developed for cancer chemotherapy, the mechanism of action of MIL in Leishmania spp. is largely unknown. While treatment with MIL has proven effective, higher tolerance to the drug has been observed, and resistance is easily developed in an in vitro environment. Utilizing stepwise selection we generated MIL-resistant cultures of L. major and characterized the fitness of MIL-resistant L. major. Resistant parasites proliferate at a comparable rate to the wild-type (WT) and exhibit similar apoptotic responses. As expected, MIL-resistant parasites demonstrate decreased susceptibility to MIL, which reduces after the drug is withdrawn from culture. Our data demonstrate metacyclogenesis is elevated in MIL-resistant L. major, albeit these parasites display attenuated in vitro and in vivo virulence and standard survival rates in the natural sandfly vector, indicating that development of experimental resistance to miltefosine does not lead to an increased competitive fitness in L. major.

Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major.

Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs) and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5) in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention.

LmaPA2G4, a homolog of human Ebp1, is an essential gene and inhibits cell proliferation in L. major.

We have identified LmaPA2G4, a homolog of the human proliferation-associated 2G4 protein (also termed Ebp1), in a phosphoproteomic screening. Multiple sequence alignment and cluster analysis revealed that LmaPA2G4 is a non-peptidase member of the M24 family of metallopeptidases. This pseudoenzyme is structurally related to methionine aminopeptidases. A null mutant system based on negative selection allowed us to demonstrate that LmaPA2G4 is an essential gene in Leishmania major. Over-expression of LmaPA2G4 did not alter cell morphology or the ability to differentiate into metacyclic and amastigote stages. Interestingly, the over-expression affected cell proliferation and virulence in mouse footpad analysis. LmaPA2G4 binds a synthetic double-stranded RNA polyriboinosinic polyribocytidylic acid [poly(I∶C)] as shown in an electrophoretic mobility shift assay (EMSA). Quantitative proteomics revealed that the over-expression of LmaPA2G4 led to accumulation of factors involved in translation initiation and elongation. Significantly, we found a strong reduction of de novo protein biosynthesis in transgenic parasites using a non-radioactive metabolic labeling assay. In conclusion, LmaPA2G4 is an essential gene and is potentially implicated in fundamental biological mechanisms, such as translation, making it an attractive target for therapeutic intervention.

In vitro screening test using Leishmania promastigotes stably expressing mCherry protein.

Transgenic Leishmania major and Leishmania donovani axenic promastigotes constitutively expressing mCherry were used for in vitro antileishmanial drug screening. This method requires minimal sample manipulation and can be easily adapted to automatic drug tests, allowing primary high-throughput screenings without the need for expensive and sophisticated instruments.

Characterization of bifunctional sphingolipid Δ4-desaturases/C4-hydroxylases of trypanosomatids by liquid chromatography-electrospray tandem mass spectrometry.

Six genes encoding putative sphingolipid desaturases have been identified in trypanosomatid genomes: one in Trypanosoma brucei (TbSLdes protein), one in Trypanosoma cruzi (TcSLdes) and four in Leishmania major (LmSLdes1-4), tandemly arrayed on chromosome 26. The six amino acid sequences showed the three characteristic histidine boxes, with a long spacer between the first and second box, as in fungal desaturases and bifunctional desaturases/hydroxylases, to which they are phylogenetically related. We functionally characterized the trypanosomatid enzymes by their expression in Saccharomyces cerevisiae sur2Δ mutant, which lacks C4-hydroxylase activity. The sphingoid base profile (dinitrophenyl derivatives) of each yeast mutant transformed with each one of the different parasite genes was analyzed by HPLC, using a sur2Δ mutant expressing the Schyzosaccharomyces pombe sphingolipid desaturase (SpSLdes) as positive control. TbSLdes was capable of desaturating endogenous sphingolipids at levels comparable to those found in SpSLdes. By contrast, L. major and T. cruzi enzymes showed either no or negligible activities. Using the HPLC system coupled to electrospray tandem quadrupole/time of flight mass spectrometry we were able to detect significant levels of desaturated and hydroxylated sphingoid bases in extracts of all transformed yeast mutants, except for those transformed with the empty vector. These results indicate that S. pombe, T. brucei, T. cruzi and L. major enzymes are all bifunctional. Using the same methodology, desaturated and hydroxylated sphingoid bases were detected in T. cruzi epimastigotes and L. major promastigote cells, as described previously, and in T. brucei procyclic and bloodstream forms for the first time.