Microarray profiling of gene expression during trypomastigote to amastigote transition in Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, remains a significant public health concern throughout South and Central America. Although much is known about immune control of T. cruzi and in particular the importance of recognition of parasite-infected cells, relatively little is known about the target antigens of these protective immune responses. For instance, few of the genes expressed in the intracellular amastigote stage have been identified. To gain insight into the molecular events, at the level of mRNA abundance, involved in this critical point in the parasite life-cycle, we used DNA microarrays of 4400 sequences from T. cruzi ORF-selected and random, genomic sequencing libraries to determine relative mRNA abundances in trypomastigotes and developing amastigotes. Results from six hybridizations using independently generated parasite samples consistently identified 60 probes that detected genes upregulated within 2h after extracellular trypomastigotes were induced, in vitro, to differentiate into amastigotes. Sequence analysis from these 60 probes identified 14 known and 25 novel T. cruzi genes. The general direction of regulation was confirmed by quantitative RT-PCR for seven of the array-identified, amastigote upregulated, known genes. This work demonstrates the feasibility of computational and microarray approaches to gene discovery in T. cruzi, an organism for which a fully assembled and annotated genome sequence is not yet available and in which control of transcription initiation is believed to be absent. Moreover, this work is the first report of amastigote up regulation for 38 genes, thus expanding considerably the pool of genes known to be upregulated in this important yet poorly-studied stage of the T. cruzi life-cycle.