Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene.

Abstract

Leishmania protozoans are the causative agents of human leishmaniasis, which includes a spectrum of diseases ranging from self-healing skin ulcers to fatal visceral infections. Leishmania donovani causes visceral leishmaniasis, also known as kala-azar, which has a high mortality rate if not treated. The Leishmania protozoans exist as extracellular flagellated promastigotes in the alimentary tract of the sand fly and are transmitted to the mammalian hosts through the bite of the insect. Once injected through the skin, the promastigotes are taken up by macrophages, rapidly differentiate into amastigotes, and start to multiply within the phagolysosomal compartment. As the infected cells rupture, amastigotes subsequently infect other macrophages, giving rise to the various symptoms associated with leishmaniasis (23).While in the midgut of the insect, newly transformed promastigotes, functionally avirulent, progressively acquire capacity for infection and migrate to the mouthparts (30, 32). This process, termed metacyclogenesis, is concurrent with the differential expression of major surface glycoconjugates which mediate the migration of promastigotes in the alimentary tract and prepare the organism for the serum environment (27,28,31,33). In comparison, the promastigote-to-amastigote cytodifferentiation is a profound morphological and physiological transformation. During differentiation, the parasite loses its flagellum, rounds up, changes its glycoconjugate coat (13,21,22,39), and starts to express a set of metabolic enzymes optimally active at low pHs (14,25 functions of its host cell, including oxygen metabolite-mediated killing and the capacity of the macrophage to act as an efficient antigen-presenting cell (5, 20). Glycoconjugates that coat the promastigote have been shown to play a critical role in the mechanism of entry of the parasite into its host cell and in the modulation of the host immune response (reviewed in references 24 and 38).Because of difficulties in obtaining a sufficient number of pure and viable amastigotes, the biology of this form of the parasite has not been well characterized. The importance of the promastigote-amastigote cytodifferentiation for the establishment of infection in the mammalian host has prompted us to identify molecular events involved in this process. Our approach was to identify Leishmania genes developmentally expressed during the amastigote stage. Our assumption was that genes expressed only by amastigotes were likely to be essential for the survival of the parasite in its human host.In this study, we have characterized an amastigote-stagespecific gene (A2 gene) and determined the physiological conditions required to induce its expression. We also demonstrate that the A2 gene product is recognized by serum from a kala-azar patient and has identity with a major antigen developmentally expressed by the pathologic forms of the malariaassociated protozoan Plasmodium falciparum. These results suggest that the Leishmania and Plasmodium parasites, phylogenetically distant par...

Putting theLeishmaniagenome to work: functional genomics by transposon trapping and expression profiling

“…A number of workers have carried out methods searching for changes in gene expression amongst Leishmania stages, using methods such as differential or subtractive hybridization and differential display (Coulson & Smith 1990;Charest & Matlashewski 1994;Pogue et al 1995;Heard et al 1996;Liu et al 2000;Wu et al 2000). While a number of genes showing significant regulation by transcript abundance were found, most workers remarked that they were able to identify only a relatively small number of differentially regulated genes, in agreement with our preliminary microarray results.…”

Heat Shock Protein 90 Homeostasis Controls Stage Differentiation in Leishmania donovani

“…Promastigote-to-amastigote differentiation can be induced in vitro by the application of a heat shock for 24 h and subsequent acidification of the culture medium. The synthesis of a set of closely related proteins, the A2 protein family (Charest and Matlashewski, 1994;Charest et al, 1996), is a hallmark for this stage differentiation process. These proteins with so far unknown functions are important for the virulence of the parasites and can be detected both in axenically cultured and in tissue-derived amastigotes, but not in promastigotes.…”

“…Moreover, species such as L. mexicana, L. pifanoi, and L. donovani will, during heat stress and acidification of the medium, show a morphological transition toward the mammalian stage, the amastigote (Zilberstein and Shapira, 1994;Saar et al, 1998). 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.…”

Comparison of the A2 Gene Locus in Leishmania donovani and Leishmania major and Its Control over Cutaneous Infection

“…The A2 gene family was first identified in L. donovani by virtue of its expression, which is specific to the amastigote stage of the life cycle (5), and it has been established that the A2 protein is among the few widely accepted amastigote-specific molecular markers identified to date (reviewed in Ref. 6).…”