Selective binding of <i>T. cruzi</i> metacyclic trypomastigotes to gastric mucin.

<p>A) Microtiter plates coated with varying amounts of gastric or submaxillary mucin were incubated with metacyclic forms for 1 h and processed for detection of bound parasites by ELISA. Values are the means of triplicates of one representative assay out of three. Variation between triplicates was <5%. B) Microtiter plates coated with gastric (G) or submaxillary (S) mucin (10 µg/well) were processed for ELISA using antibodies specific for gastric or submaxillary mucin at 1∶100 dilution. Values are the means of triplicates (variation <5%). C) Metacyclic forms were added to microtiter plates coated with gastric mucin (10 µg/well) and incubated in absence or in the presence of the indicated amounts of the recombinant protein J18 or GST and processed for ELISA. Values are the means of triplicates. (variation <10%). D) Metacyclic trypomastigotes were added to gastric mucin-coated coverslips and, following the procedure described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0000613#s2" target="_blank">methods</a> section, the Giemsa-stained parasites were visualized under the microscope. E) Transwell filters coated with gastric or submaxillary mucin were placed onto wells containing metacyclic forms. At different time points, samples from the filter chamber were collected and the number of parasites counted. Values represent the means ± standard deviation of three independent experiments. The difference in parasite translocation through gastric and submaxillary mucin layer was significant (*), with P<0.05. F) Gastric or submaxillary mucin was added to HeLa cells before addition of metacyclic trypomastigotes. After 1 h at 37°C, the cells were fixed and Giemsa-stained. The number of internalized parasites was counted in a total of 250 cells. Values correspond to means ± SD of 4 independent experiments. There was a significant difference between invasion in absence and in the presence of submaxillary mucin (*), P<0.01.</p

Determination of <i>T. cruzi</i> gp82 sequences implicated in binding to gastric mucin.

<p>A) Amino acid composition of peptides corresponding to gp82 central domain. Shown are the 20-mer peptides with an overlap of 10 residues. B) Effect of peptides shown in (A) on J18 binding to gastric mucin. Values are the means of triplicates of representative assays (variation between triplicates <10%).</p

Peptide p7 inhibits host cell invasion by metacyclic trypomastigotes in the presence of gastric mucin.

<p>A) Parasites were incubated with HeLa cells in absence or in the presence of gastric mucin, plus the indicated peptides at 200 µg/ml. After 1 h incubation, the cells were fixed and stained with Giemsa for quantification of internalized parasites. Values are the means ± SD of 4 independent experiments. The inhibitory effect of peptide was significant (*) for p7, P = 0.005, and for p10, P<0.05. B). Metacyclic trypomastigotes were incubated with HeLa cells in the presence of gastric mucin, plus peptide p7 at the indicated concentrations and the reaction proceeded as above. Values are the means ± SD of 3 independent experiments. The difference in invasion rate in absence and in the presence of p7 was significant (*) at all concentrations, P<0.05. C) HeLa cells were incubated with metacyclic forms in the presence of gastric mucin, plus peptide p7 or p7* which has the same composition of p7 but a scrambled sequence. Values are the means ± SD of 4 independent assays performed in duplicate or triplicates.</p

Specific binding of the recombinant protein J18 to gastric mucin through its central domain.

<p>A) The recombinant protein J18, containing the full length <i>T. cruzi</i> gp82 peptide sequence, was added to microtiter plates coated with gastric or submaxillary mucin at the indicated concentrations. Values are the means of triplicates of one representative assay out of three. Variation between triplicates was <5%. (B) C03, a recombinant protein of <i>T. cruzi</i> gp82 family with 59.1% identity with J18, was used for gastric mucin-binding assay. C) Gastric mucin preparations at pH 2.5 and pH 7.2 were used to coat microtiter plates and then binding of J18 was performed. In (B) and (C), the values are the means of triplicates (variation <5%). D) Schematic representation of recombinant proteins based on gp82 molecule. Shown are the GST-fused constructs containing the full-length gp82 sequence (J18) or lacking either the amino-terminal portion (J18b) or the central domain spanning residues 257–321 (J18*). E) Binding of J18, J18b and J18* to gastric mucin was compared. Values are the means of triplicates (variation <10%).</p

Inhibitory effect of peptide p7 on oral <i>T. cruzi</i> infection.

<p>A) Balb/c mice were divided in control (n = 5) and experimental (n = 5) groups, peptide p7* were given orally to control mice and peptide p7 to experimental animals 15 min before infection with metacyclic trypomastigotes by the oral route and the number of circulating parasites counted. Variations in the parasitemia levels between mice are indicated. B) Histological sections of the mouse stomach, collected 4 days after infection, were stained by hematoxylin and eosin and the number of amastigote nests (white arrow) was counted in 7 equivalent tissue sections. The representative results are shown, with bars corresponding to the variation in the number of parasites nests between sections.</p

qRT-PCR of infestin.

<p>Adult insects infected with <i>T. cruzi</i> and uninfected <i>T. infestans</i> 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 <i>t</i> test was performed for statistical analysis.</p

Functional classification of transcripts that originated from downregulated contigs.

<p>Functional classification of transcripts that originated from downregulated contigs.</p

Functional classification of transcripts that originated from upregulated contigs.

<p>Functional classification of transcripts that originated from upregulated contigs.</p

A pie chart showing the species distribution of BLASTx hits of the <i>T. infestans</i> clusters for several organisms.

<p>A pie chart showing the species distribution of BLASTx hits of the <i>T. infestans</i> clusters for several organisms.</p

Analysis summary of <i>T. infestans</i> ESTs.

<p>Analysis summary of <i>T. infestans</i> ESTs.</p