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

Alpizar-Sosa, E.A.; Kumordzi, Y.; Wei, W.; Whitfield, P.; Barrett, M.P.; Denny, P.W.

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Authors

Y. Kumordzi

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Dr Wenbin Wei wenbin.wei2@durham.ac.uk
Chief Experimental Officer (Bioinformatics)

P. Whitfield

M.P. Barrett



Abstract

With the global reach of the Neglected Tropical Disease leishmanaisis increasing, coupled with a tiny armoury 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 reanalyse 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. Hypothesising 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.

Citation

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

Journal Article Type Article
Acceptance Date Sep 6, 2022
Online Publication Date Sep 23, 2022
Publication Date 2022
Deposit Date Jul 11, 2022
Publicly Available Date Mar 21, 2023
Journal Frontiers in Cellular and Infection Microbiology, section Parasite and Host
Publisher Frontiers Media
Peer Reviewed Peer Reviewed
Volume 12
DOI https://doi.org/10.3389/fcimb.2022.988688

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http://creativecommons.org/licenses/by/4.0/

Copyright Statement
© 2022 Alpizar-Sosa, Kumordzi, Wei, Whitfield, Barrett and Denny. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.






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