Abstract

Protein acetylation has been implicated in the regulation of essential cellular processes in diverse organisms. Previous proteomic analysis from our group revealed differential protein acetylation among the three main Leishmania mexicana stages (procyclic, metacyclic and amastigote), suggesting a central role for this modification during parasite differentiation. Lysine acetylation is regulated by lysine acetyltransferases (KATs), which add acetyl groups to lysines, and lysine deacetylases (KDACs) that remove the acetyl groups. The KDACs are divided into two classes: Zinc-dependent (DACs) and NAD+-dependent or sirtuins. Thus, to expand our knowledge on how changes in protein acetylation affects Leishmania stage differentiation, we decided to characterize the four DACs (DAC1, 3, 4 and 5) of L. mexicana. Using the CRISPR-Cas9 system to obtain DAC null mutant parasites, we found that DAC1 and 3 are essential for survival of the procyclic parasite stage (only heterozygous null mutants generated), while DAC4 and 5 are dispensable. Using fluorescent-tagged DAC-expressing parasites, we demonstrated that DAC1 and 5 are cytoplasmic, while DAC3 and 4 have nuclear localization in the three parasite stages. Phenotype screening assays using the null mutant parasites demonstrated that DAC1, 3 and 5 are involved in procyclic multiplication, while DAC1 and 5 gene knockout impairs differentiation from procyclic to infective metacyclics in vitro. Experimental in vivo infection of Lutzomyia longipalpis vector further confirmed the importance of DAC5 for parasite differentiation by revealing impaired metacyclogenesis. In addition, we found that the absence of DAC5 significantly affects procyclic differentiation to axenic amastigotes and vice versa, which might be related to morphological alterations observed during time-course differentiation experiments. Finally, we evaluated the impact of DACs in the progression of host infection in vitro and in vivo. We found a significant reduction in intracellular amastigote multiplication of DAC5 null mutants in vitro compared to the parental cell line. A similar scenario was observed in the mouse in vivo infection assays for DAC5 mutants, with no apparent lesion development compared to parental parasites, indicating that DAC5 gene disruption leads to an attenuated parasite. Altogether, these results suggest that regulation of protein acetylation levels is important for Leishmania stage differentiation, opening the opportunity to explore DACs as potential drug targets in the future.