Plasmodium palmitoylation machinery engineered in E. coli for high-throughput screening of palmitoyl acyl-transferase inhibitors

FEBS Open Bio. 2019 Jan 10;9(2):248-264. doi: 10.1002/2211-5463.12564. eCollection 2019 Feb.


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, Plasmodium falciparum. P. falciparum 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 Escherichia coli, a PTM-null system, to enforce ectopic expression of palmitoyl acyl-transferase in order to study Plasmodium-specific palmitoylation and screening of inhibitors. In this study, we have developed three synthetic E. coli strains expressing Plasmodium-specific DHHC proteins (PfDHHC7/8/9). These cells were used for validating acyl-transferase activity via acyl-biotin exchange (ABE) and clickable chemistry methods. E. coli proteome was found to be palmitoylated in PfDHHC-expressing clones, suggesting that plasmodium DHHC can catalyze palmitoylation of E. coli 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 E. coli strains expressing PfDHHCs can enforce global palmitoylation in the E. coli proteome. Interestingly, this finding was corroborated by our in silico palmitoylome profiling, which revealed that out of the total E. coli 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.

Keywords: E. coli; PfDHHCs; acyl‐biotin exchange; click chemistry; drug screening; palmitoylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / antagonists & inhibitors*
  • Acyltransferases / metabolism
  • Biocatalysis
  • Click Chemistry
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • Escherichia coli / drug effects*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Genetic Engineering*
  • High-Throughput Screening Assays*
  • Molecular Docking Simulation
  • Palmitic Acid / antagonists & inhibitors*
  • Palmitic Acid / metabolism
  • Plasmodium falciparum / drug effects*
  • Plasmodium falciparum / growth & development
  • Plasmodium falciparum / metabolism


  • Enzyme Inhibitors
  • Palmitic Acid
  • Acyltransferases