DNA microarrays for comparative genomics and analysis of gene expression in Trypanosoma cruzi

Abstract

Trypanosoma cruzi presents high genetic diversity and parasite isolates show remarkable differences in biological parameters. In this study, we evaluated whether DNA microarrays containing CL Brener cDNAs can be used for comparative genomics and for the analysis of gene expression in T. cruzi. We constructed a prototype microarray with 710 expression sequence tags of CL Brener and 20 sequences of T. cruzi strains. These probes represent 665 unique genes. Results from four hybridisations with genomic DNA of Silvio (T. cruzi I) and CL Brener (hybrid genotype) identified 9.3% of the probes (68/730) differentially represented in the two genomes. Data from eight hybridisations with cDNA obtained from three independent parasite harvests of Silvio and CL Brener disclosed 84 sequences of 730 (11.5%) that showed statistical significant (P ≤ 0.01) changes in expression (1.6–6.5-fold). Some of the array-identified sequences were confirmed by Southern and Northern blot analysis. Only 20% of the probes with increased expression in Silvio or CL Brener presented higher hybridisation with genomic DNA of either strain. Approximately 2.5% (18/730) and 9.0% (65/730) of the probes were differentially expressed (P ≤ 0.01), respectively, in epimastigotes and metacyclic trypomastigotes of two T. cruzi II strains isolated from chronic chagasic patients. Microarrays identified several sequences for which differences in gene copy number and/or in the levels of RNA transcripts were previously demonstrated by different approaches. The data indicate that DNA microarrays are a useful tool for comparative studies between strains and provide further evidence for a high level of post-transcriptional regulation of RNA abundance in T. cruzi.

Introduction

The protozoon Trypanosoma cruzi is the etiological agent of Chagas disease, which affects 16–18 million people in South and Central America. T. cruzi life cycle alternates between vertebrates and triatomine insects, with different developmental stages in each host: epimastigotes and metacyclic trypomastigotes in the insect vector and intracellular amastigotes and bloodstream trypomastigotes in the mammalian host. T. cruzi is diploid, its mode of replication is predominantly asexual and, therefore, parasite strains represent independent clonal lineages [1]. The strains, also named as stocks or isolates, were shown to be divergent for various biological characteristics such as in vitro growth and differentiation, course of experimental infection, tissue tropism, and susceptibility to drugs. In addition, the diversity of symptoms of Chagas disease: indeterminate, cardiac and digestive forms have been attributed to the interplay between the genetic characteristics of the parasite and the human host [2]. In the last years, evidence provided by independent DNA markers such as ribosomal RNA (rRNA) and mini-exon genes [3], isoenzyme phenotypes [4] and phylogenies of the 18S rRNA gene [5] indicate that T. cruzi clonal genotypes cluster into two major lineages, that were named as T. cruzi I and II [6]. Epidemiological studies showed that T. cruzi I strains circulate in the sylvatic cycle of the parasite transmission, whereas T. cruzi II strains predominate in the domestic cycle where Chagas disease is more severe [7], [8]. Recently, genetic analysis points to the presence of hybrid genotypes in natural populations of T. cruzi [9], [10], [11], [12]. CL Brener, the reference organism of the T. cruzi genome project [13], is one example of these putative hybrid strains.

Although DNA microarrays have proven to be a useful tool to discover new genes and to identify coordinated expression of families of genes in apicomplexan parasites, few laboratories have employed this technology for expression profiling of genes in trypanosomatids (revised in [14]). These studies revealed stage-specific gene expression in bloodstream and procyclic forms of Trypanosoma brucei [15] and changes in RNA abundance between different stages of Leishmania major [16], [17]. At present, a single paper has been published that describes the application of a microarray containing genomic clones and open reading frames of T. cruzi to identify genes up-regulated during trypomastigote to amastigote transformation in Brazil strain [18]. Because T. cruzi isolates present high genetic diversity, in the present study we aimed at evaluating whether DNA microarrays bearing CL Brener expressed sequence tags (ESTs) can be used for comparative genomics and to investigate differential gene expression in parasite strains. For this purpose, we have produced a T. cruzi prototype DNA microarray slide containing 710 ESTs and 20 well-characterised sequences of various T. cruzi isolates. The microarray was hybridised with DNA or cDNA from two pairs of isolates: CL Brener (hybrid) and Silvio X10 cl1 (T. cruzi I) and two T. cruzi II strains isolated, respectively, from an asymptomatic individual and a patient with cardiac and digestive disorders. Some differentially recognised DNA probes were confirmed by Southern and Northern blot experiments. The data indicate that DNA microarrays can be used for comparative studies of parasite strains and provide evidence for a high level of post-transcriptional regulation of RNA abundance in T. cruzi.