Evaluation of the Leishmania Inositol Phosphorylceramide Synthase as a Drug Target Using a Chemical and Genetic Approach.

The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.

Disruption of the inositol phosphorylceramide synthase gene affects Trypanosoma cruzi differentiation and infection capacity.

Sphingolipids (SLs) are essential components of all eukaryotic cellular membranes. In fungi, plants and many protozoa, the primary SL is inositol-phosphorylceramide (IPC). Trypanosoma cruzi is a protozoan parasite that causes Chagas disease (CD), a chronic illness for which no vaccines or effective treatments are available. IPC synthase (IPCS) has been considered an ideal target enzyme for drug development because phosphoinositol-containing SL is absent in mammalian cells and the enzyme activity has been described in all parasite forms of T. cruzi. Furthermore, IPCS is an integral membrane protein conserved amongst other kinetoplastids, including Leishmania major, for which specific inhibitors have been identified. Using a CRISPR-Cas9 protocol, we generated T. cruzi knockout (KO) mutants in which both alleles of the IPCS gene were disrupted. We demonstrated that the lack of IPCS activity does not affect epimastigote proliferation or its susceptibility to compounds that have been identified as inhibitors of the L. major IPCS. However, disruption of the T. cruzi IPCS gene negatively affected epimastigote differentiation into metacyclic trypomastigotes as well as proliferation of intracellular amastigotes and differentiation of amastigotes into tissue culture-derived trypomastigotes. In accordance with previous studies suggesting that IPC is a membrane component essential for parasite survival in the mammalian host, we showed that T. cruzi IPCS null mutants are unable to establish an infection in vivo, even in immune deficient mice.

Chalcones identify cTXNPx as a potential antileishmanial drug target.

With current drug treatments failing due to toxicity, low efficacy and resistance; leishmaniasis is a major global health challenge that desperately needs new validated drug targets. Inspired by activity of the natural chalcone 2',6'-dihydroxy-4'-methoxychalcone (DMC), the nitro-analogue, 3-nitro-2',4',6'- trimethoxychalcone (NAT22, 1c) was identified as potent broad spectrum antileishmanial drug lead. Structural modification provided an alkyne containing chemical probe that labelled a protein within the parasite that was confirmed as cytosolic tryparedoxin peroxidase (cTXNPx). Crucially, labelling is observed in both promastigote and intramacrophage amastigote life forms, with no evidence of host macrophage toxicity. Incubation of the chalcone in the parasite leads to ROS accumulation and parasite death. Deletion of cTXNPx, by CRISPR-Cas9, dramatically impacts upon the parasite phenotype and reduces the antileishmanial activity of the chalcone analogue. Molecular docking studies with a homology model of in-silico cTXNPx suggest that the chalcone is able to bind in the putative active site hindering access to the crucial cysteine residue. Collectively, this work identifies cTXNPx as an important target for antileishmanial chalcones.

Down Modulation of Host Immune Response by Amino Acid Repeats Present in a Trypanosoma cruzi Ribosomal Antigen.

Several antigens from Trypanosoma cruzi, the causative agent of Chagas disease (CD), contain amino acid repeats identified as targets of the host immune response. Ribosomal proteins containing an Ala, Lys, Pro-rich repeat domain are among the T. cruzi antigens that are strongly recognized by antibodies from CD patients. Here we investigated the role of amino acid repeats present in the T. cruzi ribosomal protein L7a, by immunizing mice with recombinant versions of the full-length protein (TcRpL7a), as well as with truncated versions containing only the repetitive (TcRpL7aRep) or the non-repetitive domains (TcRpL7aΔRep). Mice immunized with full-length TcRpL7a produced high levels of IgG antibodies against the complete protein as well as against the repeat domain, whereas mice immunized with TcRpL7aΔRep or TcRpL7aRep produced very low levels or did not produce IgG antibodies against this antigen. Also in contrast to mice immunized with the full-length TcRpL7a, which produced high levels of IFN-γ, only low levels of IFN-γ or no IFN-γ were detected in cultures of splenocytes derived from mice immunized with truncated versions of the protein. After challenging with trypomastigotes, mice immunized with the TcRpL7a were partially protected against the infection whereas immunization with TcRpL7aΔRep did not alter parasitemia levels compared to controls. Strikingly, mice immunized with TcRpL7aRep displayed an exacerbated parasitemia compared to the other groups and 100% mortality after infection. Analyses of antibody production in mice that were immunized with TcRpL7aRep prior to infection showed a reduced humoral response to parasite antigens as well as against an heterologous antigen. In vitro proliferation assays with mice splenocytes incubated with different mitogens in the presence of TcRpL7aRep resulted in a drastic inhibition of B-cell proliferation and antibody production. Taken together, these results indicate that the repeat domain of TcRpL7a acts as an immunosuppressive factor that down regulates the host B-cell response against parasite antigens favoring parasite multiplication in the mammalian host.

Development of a dual reporter system to identify regulatory cis-acting elements in untranslated regions of Trypanosoma cruzi mRNAs.

In trypanosomatids, transcription is polycistronic and gene expression control occurs mainly at the post-transcriptional level. To investigate the role of sequences present in the 3'UTR of stage-specific mRNAs of Trypanosoma cruzi, we generated a new vector, named pTcDUALuc, containing the firefly and Renilla luciferase reporter genes. To test this vector, sequences derived from the 3'UTR plus intergenic regions of the alpha tubulin gene, which is up-regulated in epimastigotes, and amastin, which is up-regulated in amastigotes, were inserted downstream from the firefly reporter gene and luciferase activity was compared in transient and stable transfected parasites. As expected, increased luciferase activity was detected in epimastigotes transiently transfected with pTcDUALuc containing tubulin sequences. Using stable transfected cell lines that were allowed to differentiate into amastigotes, we observed increased luciferase activity and mRNA levels in amastigotes transfected with pTcDUALuc containing amastin sequences. We also showed that the spliced leader sequence and poly-A tail were inserted in the predicted sites of the firefly luciferase mRNA and that deletions in the alpha tubulin 3'UTR resulted in decreased luciferase expression because it affects polyadenylation. In contrast to the constructs containing 3'UTR sequences derived from tubulin and amastin genes, the presence of the 3'UTR from a trans-sialidase gene, whose expression is higher in trypomastigotes, resulted in increased luciferase activity in trypomastigotes without a corresponding increase in luciferase mRNA levels.