Leishmania Major Hsp100 Is Required Chiefly in the Mammalian Stage of the Parasite

Abstract

In Leishmania major a 100-kDa heat shock protein, Hsp100, is abundant in the intracellular amastigote stage which persists in the mammalian host. A replacement of both clpB alleles which encode Hsp100 does not affect promastigote viability under standard culture conditions but impairs thermotolerance in vitro. In experimental infections of BALB/c inbred mice, the lack of Hsp100 in the gene replacement mutants results in a markedly delayed lesion development compared with that in infections with wild-type L. major. Overexpression of exogenous clpB gene copies can partly restore virulence to the gene replacement mutants. Genetic-selection experiments also reveal a strong pressure for Hsp100 expression in the mammalian stage. This requirement for Hsp100 was also observed in in vitro infection experiments with mouse peritoneal macrophages. These experiments indicated a role for Hsp100 during the development from the promastigote to the amastigote stage. Our results suggest an important role for this parasite heat shock protein during the initial stages of a mammalian infection.

Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

“…We have shown previously that Hsp100 is critical for the expression of some amastigote stage-specific proteins and for overall virulence of the parasites (Hubel et al, 1997;Krobitsch et al, 1998); however, gene replacement mutants lacking Hsp100 still show, induced by elevated temperature, morphological differentiation to viable amastigote-like culture stages, indicating that the induction of morphological differentiation occurs independently or upstream of Hsp100 (Krobitsch et al, 1998;Krobitsch and Clos, 1999).…”

“…Such axenic amastigotes are indistinguishable from true host tissue-derived intracellular amastigotes (Bates, 1993;Pan et al, 1993;Charest and Matlashewski, 1994;Charest et al, 1996;Gupta et al, 1996;Saar et al, 1998). Thus, the rise of temperature encountered during transmission can be viewed not as stress but rather as signal for cellular differentiation.We have shown previously that Hsp100 is critical for the expression of some amastigote stage-specific proteins and for overall virulence of the parasites (Hubel et al, 1997;Krobitsch et al, 1998); however, gene replacement mutants lacking Hsp100 still show, induced by elevated temperature, morphological differentiation to viable amastigote-like culture stages, indicating that the induction of morphological differentiation occurs independently or upstream of Hsp100 (Krobitsch et al, 1998;Krobitsch and Clos, 1999).Another chaperone, Hsp90 (Hsp83), is one of the most abundant proteins in Leishmania, constituting 2.8% of the cellular protein . Hsp90 (Hsp83) is encoded by multiple gene copies and is found in the soluble fraction of the cytoplasm (Shapira and Pinelli, 1989;Brandau et al, 1995;Hubel and Clos, 1996).…”

Manipulación genética y el estudio del parásito protozoario Leishmania.

“…Muchos de los experimentos de eliminación de genes han llevado a la identificación de genes de virulencia importantes para la supervivencia o la patogénesis del parásito dentro del insecto vector o del hospedero mamífero, mas no para su crecimiento en los medios rutinarios de cultivo (73). Por ejemplo, el gen A2 fue esencial para la supervivencia de L. donovani en el macrófago, así como lo fueron la fosfomanomutasa (PMM) para L. mexicana y la proteína de choque térmico HSP100 para L. major (81,84,91).…”

“…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.…”

The co-chaperone SGT of Leishmania donovani is essential for the parasite's viability

“…Promastigote stages of L. donovani 1SR (Rosenzweig et al 2007) were grown at 25°C in supplemented M199 medium (Hubel et al 1997). After electroporation, recombinant parasites were cultivated in supplemented M199 medium with the appropriate selection antibiotics.…”