Searching for Inositol Phosphorylceramide Synthase in Leishmania major

Timothy Notton, Kai Zhang, Stephen M. Beverley, Department of Molecular Microbiology, Washington University Medical School

Protozoan parasites of Leishmania are responsible for Leishmaniasis, a zoonotic disease that affects millions globally, with at least 2 million new cases reported annually. There is no vaccine for Leishmaniasis and standard drug treatment is expensive and outdated. Therefore, the need to develop new drug targets in Leishmania is of paramount importance.  

Previous studies with L. major have demonstrated the importance of inositol phosphorylceramide (IPC), a highly abundant lipid molecule on the parasite surface.    Furthermore, IPC is absent from mammals, a fact that is of particular chemotherapeutic interest. Although IPC synthase (IPCS), the enzyme responsible for IPC synthesis, has been well characterized in Saccharomyces cerevisiae and other fungi, its gene remains unknown in L. major. Here we attempt to uncover the gene for IPCS in L. major through a genomic approach.

IPCS is an essential enzyme in S. cerevisiae. Aureobasidin A (AbA), an antifungal antibiotic, inhibits IPCS, and therefore growth. This vulnerability was exploited when conducting a genetic screen for the L. major IPCS.

A library of the L. major Friedlin VI strain was constructed by the insertion of 4-6 kbp random genomic DNA fragments into the pYES2 yeast expression vector.  The library was transformed into a S. cerevisiae strain deficient in uracil synthesis. Transformants were first selected by growing yeasts on uracil deficient plates. Selection for clones that could contain the L. major IPCS gene in their plasmid was then performed by growing the transformants in the presence of AbA at levels just sufficient to inhibit control cells.  Since pYES2 vectors are maintained as multicopy episomes, transformants expressing the Leishmania IPCS were expected to be more resistant to AbA due to overexpression of the IPCS and/or the relative insensitivity of the Leishmania IPCS activity to AbaA.  The steps above had been carried out previously in collaboration with the laboratory of Maurizio del Poeta at the University of South Carolina.   From screening about 200,000 independent pYES2 transformants, 32 candidate colonies had been recovered.  

In my work I grew up the yeast transformants, made plasmid DNA, and transformed it into E. coli to obtain DNA for sequencing.   End sequencing of these and searches against the Leishmania genome by Blast comparisons were used to deduce the sequence of the complete insert.   Surprisingly all but 1 clone contained different sequences.   This result could arise for the following reasons, singly or in combination:  1) the AbaA selection was not performing as expected, yielding essentially a ‘random’ selection of transformants, possibly arising from mutations occurring in the yeast host itself; 2) the recovered clones encoded ‘supressor’ loci; and 3) that within this collection is a single representative of the desired IPCS, which was not recognizable to us via homology or motif analysis.   The latter possibility is consistent with the finding that as yet IPCS genes have not been identified in protozoal or plant genomes, even though the activity exists.

The next step will be to re-introduce these plasmids back into yeast and confirm their activity (addressing possibility 1 above), first for AbaA resistance and if positive, for their ability to rescue IPCS-null mutants.   Further characterization is dependent upon the results of the re-transfection.