Figure 6.

Genetic deletion of the Leishmania mexicana inositol phosphorylceramide synthase (LmxIPCS) leads to loss of IPC synthesis. (A) Cell lines were generated by deleting (KO; LmxIPCS–/−) and restoring (AB; LmxIPCS–/–:LmxIPCS) the LmxIPCS utilizing CRISPR/Cas9 technology. Two KO clones (KO1 and KO2) were tested to ensure complete removal of the target gene before the restoration of LmxIPCS (AB1 and AB2). The success of transgenic generation was confirmed through PCR, using primer sets that anneal to the open reading frame or resistance markers. The parental line (P) was used as a positive control, and a negative control (C−) was included with no DNA added. (B) RT-qPCR was performed to assess the expression level of the gene in the samples from P (LmxT7:Cas9), KO (KO1 LmxIPCS–/−) and AB (AB1 LmxIPCS–/–:LmxIPCS), with the β-tubulin gene used as reference. (C) LC-MS analyses confirmed the loss and return of IPC in KO (KO1) and AB (AB1), and the associated loss and return of ceramide species. Differences between samples (P vs AB; P vs KO; KO vs AB) were evaluated using paired t tests in Prism software version 9.3.0. Statistically significant values are shown with stars: *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. Changes in cardiolipin species (Figure S9) are also provided.