Plasmodium palmitoylation machinery engineered in <i>E. coli</i> for high‐throughput screening of palmitoyl acyl‐transferase inhibitors

Abstract

Lipid-based palmitoylation is a post-translation modification (PTM) which acts as a biological rheostat in life cycle progression of a deadly human malaria parasite, <i>Plasmodium falciparum</i>. <i>P. falciparum</i> palmitoylation is catalyzed by 12 putative palmitoyl acyl-transferase enzymes containing the conserved DHHC-CRD (DHHC motif within a cysteine-rich domain) which can serve as a druggable target. However, the paucity of high-throughput assays has impeded the design of drugs targeting palmitoylation. We have developed a novel strategy which involves engineering of <i>Escherichia coli</i>, a PTM-null system, to enforce ectopic expression of palmitoyl acyl-transferase in order to study <i>Plasmodium</i>-specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic <i>E. coli</i> strains expressing <i>Plasmodium</i>-specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl-transferase activity via acyl-biotin exchange (ABE) and clickable chemistry methods. <i>E. coli</i> proteome was found to be palmitoylated in PfDHHC-expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of <i>E. coli</i> proteins. Upon treatment with generic inhibitor 2-bromopalmitate (2-BMP), a predominant reduction in palmitic acid incorporation is detected. Overall, these findings suggest that synthetic <i>E. coli</i> strains expressing PfDHHCs can enforce global palmitoylation in the <i>E. coli</i> proteome. Interestingly, this finding was corroborated by our <i>in silico</i> palmitoylome profiling, which revealed that out of the total <i>E. coli</i> proteome, 108 proteins were predicted to be palmitoylated as represented by the presence of three cysteine consensus motifs (cluster type I, II, III). In summary, our study reports a proof of concept for screening of chemotherapeutics targeting the palmitoylation machinery using a high-throughput screening platform.