Investigating the 'Leishmania donovani sacp' gene and its role in macrophage infection and survival in mice

The protozoan parasite 'Leishmania donovani' is a causative agent of the neglected tropical disease known as visceral leishmaniasis, which can be lethal when untreated. Studying Leishmania viru-lence factors is crucial in determining how the parasite causes disease and identifying new targets for treatment. One potential virulence factor is 'L. donovani''s abundantly secreted protein: secreted acid phosphatase (SAcP). Whole-genome analysis revealed that the sacp gene was present in three copies in wild type 'L. donovani'. Using CRISPR-Cas9 gene editing; we generated a sacp gene knockout termed Ld SAcP, which demonstrated a loss of both the SAcP protein and an associated reduction in secreted acid phosphatase activity. Genome sequencing confirmed the precise dele-tion of the sacp gene in LdDSAcP and identified several changes in the genome. LdDSAcP demonstrated no significant changes in promastigote proliferation or its ability to infect and survive in macrophages compared to the wildtype strain. LdDSAcP also demonstrated no change in murine liver infection; however, survival was impaired in the spleen. Taken together these results show that SAcP is not necessary for the survival of promastigotes in culture but may support long-term survival in the spleen. These observations also show that the use of CRISPR gene editing and WGS together are effective to investigate the function and phenotype of complex potential drug targets such as multicopy genes.