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.

Genome deletions to overcome the directed loss of gene function in Leishmania.

With the global reach of the Neglected Tropical Disease leishmaniasis increasing, coupled with a tiny armory of therapeutics which all have problems with resistance, cost, toxicity and/or administration, the validation of new drug targets in the causative insect vector borne protozoa Leishmania spp is more important than ever. Before the introduction of CRISPR Cas9 technology in 2015 genetic validation of new targets was carried out largely by targeted gene knockout through homologous recombination, with the majority of genes targeted (~70%) deemed non-essential. In this study we exploit the ready availability of whole genome sequencing technology to reanalyze one of these historic cell lines, a L. major knockout in the catalytic subunit of serine palmitoyltransferase (LCB2), which causes a complete loss of sphingolipid biosynthesis but remains viable and infective. This revealed a number of Single Nucleotide Polymorphisms, but also the complete loss of several coding regions including a gene encoding a putative ABC3A orthologue, a putative sterol transporter. Hypothesizing that the loss of such a transporter may have facilitated the directed knockout of the catalytic subunit of LCB2 and the complete loss of de novo sphingolipid biosynthesis, we re-examined LCB2 in a L. mexicana line engineered for straightforward CRISPR Cas9 directed manipulation. Strikingly, LCB2 could not be knocked out indicating essentiality. However, simultaneous deletion of LCB2 and the putative ABC3A was possible. This indicated that the loss of the putative ABC3A facilitated the loss of sphingolipid biosynthesis in Leishmania, and suggested that we should re-examine the many other Leishmania knockout lines where genes were deemed non-essential.

Amphotericin B resistance in Leishmania mexicana: Alterations to sterol metabolism and oxidative stress response.

Amphotericin B is increasingly used in treatment of leishmaniasis. Here, fourteen independent lines of Leishmania mexicana and one L. infantum line were selected for resistance to either amphotericin B or the related polyene antimicrobial, nystatin. Sterol profiling revealed that, in each resistant line, the predominant wild-type sterol, ergosta-5,7,24-trienol, was replaced by other sterol intermediates. Broadly, two different profiles emerged among the resistant lines. Whole genome sequencing then showed that these distinct profiles were due either to mutations in the sterol methyl transferase (C24SMT) gene locus or the sterol C5 desaturase (C5DS) gene. In three lines an additional deletion of the miltefosine transporter gene was found. Differences in sensitivity to amphotericin B were apparent, depending on whether cells were grown in HOMEM, supplemented with foetal bovine serum, or a serum free defined medium (DM). Metabolomic analysis after exposure to AmB showed that a large increase in glucose flux via the pentose phosphate pathway preceded cell death in cells sustained in HOMEM but not DM, indicating the oxidative stress was more significantly induced under HOMEM conditions. Several of the lines were tested for their ability to infect macrophages and replicate as amastigote forms, alongside their ability to establish infections in mice. While several AmB resistant lines showed reduced virulence, at least two lines displayed heightened virulence in mice whilst retaining their resistance phenotype, emphasising the risks of resistance emerging to this critical drug.

Non-invasive visualisation and identification of fluorescent Leishmania tarentolae in infected sand flies.

Background: The leishmaniases are neglected diseases that affect some of the most vulnerable populations in the tropical and sub-tropical world. The parasites are transmitted by sand flies and novel strategies to control this neglected vector-borne disease are needed. Blocking transmission by targeting the parasite inside the phlebotomine vector offers potential in this regard. Some experimental approaches can be best performed by longitudinal study of parasites within flies, for which non-destructive methods to identify infected flies and to follow parasite population changes are required. Methods: Lutzomyia longipalpis were reared under standard insectary conditions at the Wellcome Centre for Molecular Parasitology. Flies were artificially infected with L. tarentolae expressing green fluorescent protein (GFP. Parasite counts were carried out 5 days post-infection and the percentage of infected flies and survival of infected females was established up to days 5 post-infection. Whole living females were visualised using an epifluorescence inverted microscope to detect the presence parasites inferred by a localised green fluorescent region in the upper thorax. Confirmation of infection was performed by localised-fluorescence of dissected flies and estimates of the parasite population. Results : Leishmania tarentolae was successfully transfected and expressed GFP in vitro. L. tarentolae-GFP Infected flies showed similar parasite populations when compared to non-transfected parasites ( L. tarentolae-WT). Survival of non-infected females was higher than L. tarentolae-infected groups, (Log-rank (Mantel-Cox) test, p<0.05). L. tarentolae-GFP infected females displayed an intense localised fluorescence in the thorax while other specimens from the same infected group did not. Localised fluorescent flies were dissected and showed higher parasite populations compared to those that did not demonstrate high concentrations in this region (t-test, p<0.005). Conclusion : These results demonstrate the feasibility of establishing a safe non-human infectious fluorescent Leishmania-sand fly infection model by allowing non-destructive imaging to signal the establishment of Leishmania infections in living sand flies.