Sylvia Krobitsch

Leishmania major Hsp100 is required chiefly in the mammalian stage of the parasite.

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.

Leishmania donovani Heat Shock Protein 100 CHARACTERIZATION AND FUNCTION IN AMASTIGOTE STAGE DIFFERENTIATION

We report the cloning and molecular analysis of the Leishmania donovani clpB gene. The protein-coding region is highly conserved compared with its L. major homologue, while 5′- and 3′-flanking DNA sequences display considerable divergence. The encoded mRNA has an unusually long 5′-leader sequence typical for RNAs, which are translated preferentially under heat stress. The gene product, a 100-kDa heat shock protein, Hsp100, becomes abundant only during sustained heat stress, but not under common chemical stresses. Hsp100 associates into trimeric complexes and is found mostly in a cytoplasmic, possibly membrane-associated, localization as determined by immune electron microscopy. Hsp100 shows immediate early expression kinetics during axenic amastigote development. In its absence, expression of at least one amastigote stage-specific protein family is impaired.

A novel role for 100 kD heat shock proteins in the parasite Leishmania donovani.

Heat shock proteins of the 100 kD family have been known to confer general stress tolerance in yeast and plants. Several protozoan parasites possess genes for Hsp100 proteins. In Leishmania species the protein is expressed under heat stress and during the mammalian stage, the amastigote. We show here that replacement of the clpB gene which encodes Hsp100 does not affect thermotolerance or general viability in Leishmania donovani insect stages (promastigotes) nor in axenically cultured mammalian stages (amastigotes). However, its expression is required for normal development of the parasite inside mammalian host cells. Hsp100 appears to function as an antagonist of amastigote-to-promastigote differentiation and a promoter of full amastigote development.

Heat shock protein 100 and the amastigote stage-specific A2 proteins of Leishmania donovani

Abstract. HSP100 protein in Leishmania spp. plays an important role for the survival and integrity of intracellular amastigotes. The A2 proteins of L. donovani are functionally linked to HSP100. There is evidence for an interdependence between these two proteins, which are both expressed predominantly in the amastigote stage of Leishmania donovani. Mutant strains lacking either of these proteins display very similar phenotypes, i.e. loss of virulence both in vivo and in vitro. Also, both proteins colocalise specifically to small foci within the cytoplasm of amastigotes.

Cross-species homologous recombination in Leishmania donovani reveals the sites of integration.

The targeted replacement of genes in kinetoplastid protozoa was initially described by Cruz and Beverley [1] and its usefulness for reverse genetics is well established [2–8]. In order to target genes for replacement their 5%and 3%-flanking non-coding sequences are usually fitted on either side of a resistance marker gene such as neomycin phosphotransferase or hygromycin phosphotransferase and ligated into a plasmid vector. The chimeric gene is linearized and transfected into Leishmania promastigotes by means of electroporation and will recombine with the flanking sequences of an allelic gene copy. This implies that each gene must be targeted by a specifically designed construct. Moreover, the fact that only DNA constructs with identical or near-identical sequences have been successfully used for homologous recombination allows no distinction between acceptor and donor sequences. This fact has so far precluded a mapping of the actual sites at which recombination occurs in Leishmania or Trypanosoma. In Leishmania the flanking DNA sequences must have a certain minimal length (\180 bp) and sequence identity (\89%) for homologous recombination to occur [9]. It was also reported that sequence identities below 92% are not tolerated for homologous recombination in Trypanosoma brucei [10]. However, to test the impact of gene replacements in the various aspects of parasite biology and parasite–host interactions it is often of advantage to obtain null mutants of a gene in more than one species. The clpB genes of Leishmania major and Leishmania dono6ani are highly (96%) conserved in the coding regions [11,12]. Sequence conservation in the flanking DNA sequences, in contrast, is considerably lower at an overall 87% identity; long stretches of almost perfect sequence conservation alternate with inserted or deleted parts. Since some of the highly conserved stretches exceed the minimal length required for a faithful recombination [9] we tested whether the replacement constructs used to replace the L. major clpB gene [7] could be used to replace the L. dono6ani clpB alleles. The success of this strategy also allowed us to map the sites of recombination. * Corresponding author. Tel.: +49-40-42818481; fax: +4940-42818400. E-mail address: clos@bni.uni-hamburg.de (J. Clos) 1 Present address: Howard Hughes Medical Institute, The University of Chicago, 5841 S. Maryland Ave. MC 1028, Chicago, IL 60637, USA.