Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes.

Abstract

Trypanosomatid protozoan parasites cause several important tropical diseases and have been a fertile ground for the discovery of molecular paradigms such as trans-splicing and RNA editing. Transfection-based methods for the study of these organisms have recently been developed, and we have now designed an expression vector, pX, which contains only 2.3 kilobases of Leishmania DNA and can be stably transfected with high efficiency. Genes encoding Escherichia coli ,B-galactosidase or a Leishmania amazonensis protective membrane glycoprotein (GP46A/M-2) were inserted into the pX expression site and transfected into Leishmania major, where they directed the synthesis of high levels of mRNAs formed by 5' and 3' processing events occurring predominantly at the sites used by the normal transcripts. Colony assays and immunoblot analysis showed that both proteins were produced; enzymatically active f8-galactosidase comprised =1% of total protein. Sizes of the GP46A protein synthesized in transfected L. major or L. amazonensis were similar and differed from the predominant L. amazonensis GP46, suggesting that the GP46A gene may encode a variant GP46 family member. Because these vectors function efficiently in pathogenic species of Leishmania, pX will facilitate the genetic analyses of parasite proteins crucial for infectivity as well as the identification of cis-acting elements mediating transcription and replication.

Arginase Plays a Pivotal Role in Polyamine Precursor Metabolism in Leishmania

“…The resulting expression plasmid was designated pXG-GFPϩ2Ј-arginase. This plasmid was then transfected into wild type L. mexicana using standard electroporation conditions (17,18). Transfected parasites were selected in 20 g/ml G418 and used for localization studies.…”

“…pX63-HYG-⌬arg and pX63-PHLEO⌬arg were digested with HindIII and BglII to liberate linear fragments, designated HYG-⌬arg and PHLEO-⌬arg, respectively, containing the drug resistance marker and the arginase flanking regions. HYG-⌬arg and PHLEO-⌬arg were isolated from agarose gels and then transfected into parasites using standard electroporation conditions for transfection of Leishmania promastigotes (17,18). First, HYG-⌬arg was transfected into wild type L. mexicana to create the arginase/arg heterozygote, and colonies were isolated by selection on plates of semi-solid DME-L-CS medium containing 50 g/ml hygromycin.…”

Infection and Activation of Human Neutrophils with Fluorescent Leishmania infantum

“…The gene encoding either mCherry or dTomato [28] was cloned into Leishmania expression vector pIR1SAT (kindly provided by Stephen M. Beverly, Ph.D., Washington University at St. Louis). Wild-type parasite cultures were transfected with gel-purified integrating expression constructs by electroporation, and clones were selected from semisolid agar plates [29,30]. Transfectants, called mCherry-Li or dTomato-Li respectively, were grown to stationary phase in HOMEM.…”

Manipulación genética y el estudio del parásito protozoario Leishmania.

“…Por ejemplo, la fosfatasa ácida de secreción (SAP) y la glicoproteína de membrana Gp46/M2 de L. mexicana han sido expresadas por células de L. major (108). Además, se ha observado que Leishmania puede expresar genes flanqueados con secuencias intergénicas de T. cruzi, y se han construido vectores que pueden expresar productos génicos en ambos parásitos.…”

“…El uso de la tecnología de transfección de ADN en Leishmania comenzó con la expresión transitoria de genes luego de la electroporación de parásitos con vectores circulares (5), y se ha desarrollado hasta incluir un espectro amplio de métodos para el análisis funcional de genes que han permitido estudiar la expresión y la regulación génica del parásito (6-27), la identificación de genes esenciales para supervivencia (28-49), la investigación de los mecanismos de resistencia a drogas y de virulencia (50-92), la producción de proteínas foráneas (91)(92)(93)(94)(95)(96)(97)(98)(99)(100)(101)(102)(103)(104)(105)(106)(107), la identificación y la expresión de antígenos de superficie (108). En la presente revisión se divulgan los avances y aplicaciones más recientes y notorias en el campo de la manipulación genética en Leishmania.…”