Probing druggability and biological function of essential proteins in Leishmania combining facilitated null mutant and plasmid shuffle analyses

Mariko Dacher, Miguel A. Morales, Pascale Pescher, Olivier Leclercq, Najma Rachidi, Eric Prina, Mathieu Cayla, Albert Descoteaux, Gerald F. Späth*

*この研究の対応する著者

研究成果: Article査読

22 被引用数 (Scopus)

抄録

Summary: Leishmania parasites cause important human morbidity and mortality. Essential Leishmania genes escape genetic assessment by loss-of-function analyses due to lethal null mutant phenotypes, even though these genes and their products are biologically most significant and represent validated drug targets. Here we overcome this limitation using a facilitated null mutant approach applied for the functional genetic analysis of the MAP kinase LmaMPK4. This system relies on the episomal expression of the target gene from vector pXNG that expresses the Herpes simplex virus thymidine kinase gene thus rendering transgenic parasites susceptible for negative selection using the antiviral drug ganciclovir. Using this system we establish the genetic proof of LmaMPK4 as essential kinase in promastigotes. LmaMPK4 structure/function analysis by plasmid shuffle allowed us to identify regulatory kinase sequence elements relevant for chemotherapeutic intervention. A partial null mutant, expressing an MPK4 derivative with altered ATP-binding properties, showed defects in metacyclogenesis, establishing a first link of MPK4 function to parasite differentiation. The approaches presented here are broadly applicable to any essential gene in Leishmania thus overcoming major bottlenecks for their functional genetic analysis and their exploitation for structure-informed drug development.

本文言語English
ページ(範囲)146-166
ページ数21
ジャーナルMolecular Microbiology
93
1
DOI
出版ステータスPublished - 2014
外部発表はい

ASJC Scopus subject areas

  • 分子生物学
  • 微生物学
  • 医学(全般)

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