Timothy Notton

REVERSE GENETIC ANALYSIS OF LAG1 IN LEISHMANIA MAJOR. Timothy Notton¹, Kai Zhang², Stephen Beverley², Department of Biomedical Engineering, Washington University, St. Louis, MO¹; Department of Molecular Microbiology, Washington University Medical School, St. Louis, MO.²

Trypanosomatid protozoans of the genus Leishmania are the etiological agents of leishmaniasis, a debilitating disease that affects more than 10 million globally. In the human host, parasites reside within the phagolysosome of macrophages, normally toxic to microbes. However, Leishmania elaborates several mechanisms that enable it to survive and cause disease from this site.

Previous studies have demonstrated the importance of inositol phosphorylceramide (IPC), a ubiquitous surface sphingolipid in Leishmania major, although its exact role is unclear. We aim to elucidate the function of IPC in sphingolipid metabolism by preventing IPC synthesis. Although the gene responsible for IPC synthesis (IPCS) is uncharacterized in L. major, a deletion of LAG1, which functions immediately upstream of IPCS in the SL biosynthetic pathway, would presumably have the same phenotype as an IPCS deletion. Leishmania LAG1 was identified in database searches and its ceramide synthase activity confirmed in collaboration with Anthony Futerman’s laboratory in Israel.

LAG1 null mutants were sought using homologous recombination and replacement. LAG1 exists as a single copy on the tetrasomatic chromosome 31, therefore four antibiotic resistance markers were needed to delete this gene, one for each round of replacement, since Leishmania is asexual in culture (and probably in nature). In each round, parasites were transfected with a suitable DNA construct where the LAG1 ORF was replaced with an ORF encoding a drug resistance marker, and plated on selective media supplemented with appropriate antibiotics. Southern blotting of surviving colonies with a LAG1 probe confirmed the success of the first three rounds of transfection, which proceeded in a typical manner for Leishmania transfections.

After a final round of transfection, cells were cultured using two selection methods expected to facilitate growth of a LAG1 null mutant. In the first, parasites were plated on selective media containing all four antibiotics and supplemented with ethanolamine (EtN) and C₁₈ ceramide (Cer). The second method involved the liquid selection for null mutants in media containing all components of the plate selection media with the addition of myriocin, a potent serine palmitoyltransferase (SPT) inhibitor. Myriocin was added to prevent the buildup of sphingoid bases, which are toxic at high concentrations.

The genotypes of 106 clones were determined by PCR. WT LAG1 alleles were identified by the synthesis of a 1.4 kb PCR product with primers complementary to sequences on either end of LAG1. As a positive control we used primers for an unrelated ORF.

All of the 106 screened colonies tested positive for both LAG1 and the positive control. Thus, we were unable to generate a LAG1 null mutant in this study. Since knockouts of non-essential Leishmania genes are readily generated on a routine basis in many laboratories, this potentially suggests that LAG1 null mutants are lethal to Leishmania under the conditions tested here. This conclusion is surprising since SPT null mutants, which completely lack sphingolipids, are viable and grow relatively normally. Potentially, the conditions used do not completely rescue LAG1 deficiency or LAG1 carries out novel roles in Leishmania.