Sadri Ertem (1898-1943)

Well characterised molecular markers for specific T. cruzi life-cycle stages are still scarce. Qualitative and quantitative differences between proteins expressed in the various life-cycle stages have been demonstrated on ID and 2D protein gels and Western blots (Contreras et al., 1985a; Lanar and Manning, 1984; Rangel-Aldao et al., 1986). However the identity of most of these molecules remains to be deter­

mined. With the technological advances that have been made since those early studies were conducted 2 0 years ago, and the advent of high throughput functional genomics approaches (section 1.7.2), it is now feasible to identify such stage-specific gene products on a large scale. A brief overview of the current knowledge of mo­

lecular events associated with metacyclogenesis is presented below.

Expression o f metacyclic-specific surface glycoproteins gp82 and gp90. The best characterised stage-specific T. cruzi genes code for surface proteins, including the epimastigote-specific small mucin genes TcSMUG (Di Noia et al., 2000), the amastigote-specific amastin (Teixeira et al., 1994) and proteins of the gp85/sialidase multi-gene family expressed in cell-derived trypomastigotes. The latter belong to the trans-sialidase gene superfamily (Cross and Takle, 1993; Schenkman et al., 1994).

Two proteins that belong to this family, gp82 and gp90, have been identified as the major surface glycoproteins specific to metacyclics (Araya et al., 1994; Ruiz et al., 1998). As yet, gp82 represents the only well characterised protein marker for meta­

cyclics in a wide variety of T. cruzi strains. Expression of gp90 on the other hand is strain-specific (Beard et al., 1988; Ruiz et al., 1998). The genes coding for gp82 and gp90 have been cloned and shown to belong to multi-gene families, dispersed throughout the T. cruzi genome. The protein sequences are 40-60% identical to those of the gp85/sialidase family. Steady state levels of gp82 and gp90 mRNA are developmental^ regulated. Transcript levels are high in metacyclics, very low in

33

epimastigotes, and absent from other life-cycle stages (Araya et al., 1994; Carmo et al., 1999; Franco et al., 1993).

Gp82 has been implicated in the process of host cell invasion and in the initiation of signalling cascades, both in the parasite and in the target cell. It functions as a para­

site surface receptor that binds to an unidentified target cell molecule. This initiates a signalling cascade in the parasite cell, that involves tyrosine phosphorylation of a 175 kDa protein, and leads to invasion of the target cell (Favoreto et al., 1998). The strains o f T. cruzi revealed an inverse correlation between the infectivity o f a T. cruzi strain and the level of gp90 expression. While gp90 was found to be one of the major surface components in the poorly invasive G strain, the antibody failed to detect the protein in the highly invasive CL and Y strains (Ruiz et al., 1998).

Changes in chromatin. A general decrease in transcription rates (Elias et al., 2001b), and phosphorylation of histone HI (Marques Porto et al., 2002) occurs concomi­

tantly with extensive nuclear reorganisation when replicating forms differentiate to the non-replicating and infective forms (including metacyclics). This is detailed in section 1.2.2.

Differential gene expression during metacyclogenesis. Prior to in vitro differentiation in TAU, epimastigotes of the Dm28c strain adhere to the culture flask, and these cpimastigotes express specific surface antigens (Bonaldo et al., 1988). Recent dis­

coveries may provide the first clue as to the molecules and control mechanisms involved in this process. To identify genes expressed transiently during metacyclo­

genesis, a method has been devised recently (Krieger et al., 1999; Krieger and Goldenberg, 1998), called representation of differential expression (RDE). RDE is based on PCR amplification of cDNA sequences unique to a cell population (tester) after subtractive hybridisation with DNA sequences of a related cell population

(driver). Using as driver the polysomal RNA fractions from replicating epimas- tigotes. and as tester the polysomal RNA fractions from epimastigotes induced to differentiate in TAU, several transcripts have been shown to be specific to adhered (and by implication, differentiating) cells. Interestingly, these studies suggest that mRNA mobilisation to the polysomes may be one of the post-transcriptional mecha­

nisms for regulating gene expression in trypanosomes.

The RDE method has so far led to the identification of two types of surface proteins thought to be involved in the differentiation process. This will be discussed in the following two sections. In addition, several novel upregulated transcripts of unknown function await further characterisation.

Chitin-binding-like protein (CBLP). During metacyclogenesis in TAU, transcripts were shown to be mobilised to the polysomal fraction that code for a family of small cysteine-rich proteins with sequence similarity to the chitin-binding domain of wheat germ agglutinin. Maximal transcript levels were detected 24 hours after initiation of differentiation. Consistent with this, the levels o f a 7 kDa protein detected by a poly­

clonal antiserum raised against recombinant CBLP1, are highest in adhered epimas­

tigotes after 24 hours (Dallagiovanna et al., 2001).

Metacyclogenin. A second putative surface protein specifically expressed in differ­

entiating epimastigotes is metacyclogenin (Avila et al., 2001). Metacyclogenin mRNA is associated with the polysomes of 24 hours adhered epimastigotes. The T. cruzi genome contains at least 3 copies of the gene arranged in a tandem repeat with an 'associated' gene (AG) and a cytosolic tryparedoxin peroxidase gene (CPX).

The AG gene shows an expression pattern similar to metacyclogenin (Avila et al., 2001). The 13 kDa metacyclogenin protein is detected exclusively in adhered epimastigotes and it appears to be associated with the parasite cell membrane, as determined by immunocytochemical electron microscopy. The function of metacyc­

logenin is unknown.

35

Using reverse genetic approaches it should be feasible to determine the role that these proteins play in differentiation and how commitment to differentiation and attachment of the parasite are linked.

1.5 DISSECTING SIGNAL TRANSDUCTION