Genome deletions to overcome the directed loss of gene function in Leishmania

Alpizar-Sosa, E. A., Kumordzi, Y., Wei, W., Whitfield, P. D. , Barrett, M. P. and Denny, P. W. (2022) Genome deletions to overcome the directed loss of gene function in Leishmania. Frontiers in Cellular and Infection Microbiology, 12, 988688. (doi: 10.3389/fcimb.2022.988688) (PMID:36211960) (PMCID:PMC9539739)

Abstract

With the global reach of the Neglected Tropical Disease leishmaniasis increasing, coupled with a tiny armory of therapeutics which all have problems with resistance, cost, toxicity and/or administration, the validation of new drug targets in the causative insect vector borne protozoa Leishmania spp is more important than ever. Before the introduction of CRISPR Cas9 technology in 2015 genetic validation of new targets was carried out largely by targeted gene knockout through homologous recombination, with the majority of genes targeted (~70%) deemed non-essential. In this study we exploit the ready availability of whole genome sequencing technology to reanalyze one of these historic cell lines, a L. major knockout in the catalytic subunit of serine palmitoyltransferase (LCB2), which causes a complete loss of sphingolipid biosynthesis but remains viable and infective. This revealed a number of Single Nucleotide Polymorphisms, but also the complete loss of several coding regions including a gene encoding a putative ABC3A orthologue, a putative sterol transporter. Hypothesizing that the loss of such a transporter may have facilitated the directed knockout of the catalytic subunit of LCB2 and the complete loss of de novo sphingolipid biosynthesis, we re-examined LCB2 in a L. mexicana line engineered for straightforward CRISPR Cas9 directed manipulation. Strikingly, LCB2 could not be knocked out indicating essentiality. However, simultaneous deletion of LCB2 and the putative ABC3A was possible. This indicated that the loss of the putative ABC3A facilitated the loss of sphingolipid biosynthesis in Leishmania, and suggested that we should re-examine the many other Leishmania knockout lines where genes were deemed non-essential.

• Item Type: Articles
• Additional Information: Funding: This work was supported by MRC Confidence in Concept MC-PC-17157 (PD); the UKRI - Global Challenges Research Fund, ‘A Global Network for Neglected Tropical Diseases’ MR/ P027989/1 (PD; https://www.ukri.org); and an MRC Newton grant Bridging epigenetics, metabolism and cell cycle in pathogenic trypanosomatids, MR/S019650/1 (MB).
• Keywords: Cellular and Infection Microbiology, Leishmania, genome, knockout, plasticity, lipids
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Whitfield, Mr Phil and Barrett, Professor Michael
• Authors: Alpizar-Sosa, E. A., Kumordzi, Y., Wei, W., Whitfield, P. D., Barrett, M. P., and Denny, P. W.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Frontiers in Cellular and Infection Microbiology
• Publisher: Frontiers Media
• ISSN: 2235-2988
• ISSN (Online): 2235-2988
• Copyright Holders: Copyright © 2022 Alpizar-Sosa, Kumordzi, Wei, Whitfield, Barrett and Denny
• First Published: First published in Frontiers in Cellular and Infection Microbiology 12: 988688
• Publisher Policy: Reproduced under a Creative Commons License

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
304784		Bridging epigenetic, metabolism and cell cycle in pathogenic trypanosomatids	Michael Barrett	Medical Research Council (MRC)	MR/S019650/1	III - Parasitology