Screen of Leishmania mexicana transporter mutants in mice
For the protozoan parasite Leishmania, successful completion of their life cycle requires sequential adaptation of cellular morphology, physiology and nutrient scavenging mechanisms to the different environments of a sand fly alimentary tract and the acidic host cell phagolysosome. We carried out a systematic gene-deletion screen covering the entire predicted set of 312 L. mexicana membrane transporters and 57 candidate proteins associated with the amastigote flagellum to discover, which genes are vital for promastigote or amastigote fitness. Each cell line was tagged with a unique 17-nt barcode. Pooled gene deletion mutants were assessed for survival and growth as promastigotes in laboratory cultures, and as amastigotes in macrophages and in mice. For barcode quantification, DNA was extracted at different study time points, for PCR-amplification of the barcode region, Illumina sequencing of the amplicons (bar-seq). Changes in barcode proportions over time, compared to the starting pool, were used as a measure of cell line fitness. This screen identified genes that were dispensable under the tested conditions, genes that were refractory to deletion in the promastigote form and genes whose deletion caused significant loss of fitness in the promastigote and intracellular amastigote forms. Data submission includes genomic sequence mutants and bar-seq samples, used to calculate mutant fitness in vitro and in vivo.