Differential expression profiles in the midgut of Triatoma infestans infected with Trypanosoma cruzi

Abstract

Chagas disease, or American trypanosomiasis, is a parasitic disease caused by the protozoan Trypanosoma cruzi and is transmitted by insects from the Triatominae subfamily. To identify components involved in the protozoan-vector relationship, we constructed and analyzed cDNA libraries from RNA isolated from the midguts of uninfected and T. cruzi-infected Triatoma infestans, which are major vectors of Chagas disease. We generated approximately 440 high-quality Expressed Sequence Tags (ESTs) from each T. infestans midgut cDNA library. The sequences were grouped in 380 clusters, representing an average length of 664.78 base pairs (bp). Many clusters were not classified functionally, representing unknown transcripts. Several transcripts involved in different processes (e.g., detoxification) showed differential expression in response to T. cruzi infection. Lysozyme, cathepsin D, a nitrophorin-like protein and a putative 14 kDa protein were significantly upregulated upon infection, whereas thioredoxin reductase was downregulated. In addition, we identified several transcripts related to metabolic processes or immunity with unchanged expressions, including infestin, lipocalins and defensins. We also detected ESTs encoding juvenile hormone binding protein (JHBP), which seems to be involved in insect development and could be a target in control strategies for the vector. This work demonstrates differential gene expression upon T. cruzi infection in the midgut of T. infestans. These data expand the current knowledge regarding vector-parasite interactions for Chagas disease.

Figure 1. Length distribution of T. infestans clusters.

Figure 2. A pie chart showing the species distribution of BLASTx hits of the T. infestans clusters for several organisms.

Figure 3. qRT-PCR of Thioredoxin reductase upon T. cruzi infection.

The levels of mRNA from Thioredoxin reductase were obtained by relative quantification. Adult insects infected with T. cruzi and uninfected T. infestans were used for analysis (three biological samples were used for both the uninfected and infected groups). All data were normalized to 18S ribosomal RNA, representing the mean (n = 3) of identical triplicates ± standard deviation. An unpaired t test was performed for statistical analysis, and differences were considered significant at P<0.05. Asterisks represent significant differences (*** P<0.001).

Figure 4. qRT-PCR of four transcripts upregulated upon T. cruzi infection.

Amounts of mRNA from nitrophorin-like protein (A), 14 kDa protein (B), lysozyme (C) and cathepsin D (D) were obtained by relative quantification. Adult insects infected with T. cruzi and uninfected T. infestans were used for analysis (three biological samples were used for both the uninfected and infected groups). All data were normalized to 18S ribosomal RNA, representing the mean (n = 3) of identical triplicates ± standard deviation. An unpaired t test was performed for statistical analysis, and differences were considered significant at P<0.05. Asterisks represent significant differences (** P<0.01; *** P<0.001).

Figure 5. qRT-PCR of infestin.

Adult insects infected with T. cruzi and uninfected T. infestans were used for analysis (three biological samples were used for both the uninfected and infected groups). All data were normalized to 18S ribosomal RNA, representing the mean (n = 3) of identical triplicates ± standard deviation. An unpaired t test was performed for statistical analysis.