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

Differential Infectivity by the Oral Route of <em>Trypanosoma cruzi</em> Lineages Derived from Y Strain

<div><h3>Background</h3><p>Diversity of <em>T. cruzi</em> strains is a central problem in Chagas disease research because of its correlation with the wide range of clinical manifestations and the biogeographical parasite distribution. The role played by parasite microdiversity in Chagas disease epidemiology is still debatable. Also awaits clarification whether such diversity is associated with the outcome of oral <em>T. cruzi</em> infection, responsible for frequent outbreaks of acute Chagas disease.</p><h3>Methods and Findings</h3><p>We addressed the impact of microdiversity in oral <em>T. cruzi</em> infection, by comparative analysis of two strains, Y30 and Y82, both derived from Y strain, a widely used experimental model. Network genealogies of four nuclear genes (SSU rDNA, actin, DHFR-TS, EF1α) revealed that Y30 is closely related to Discrete Typing Unit TcII while Y82 is more closely related to TcVI, a group containing hybrid strains. Nevertheless, excepting one A-G transition at position 1463, Y30 and Y82 SSU rDNAs were identical. Y82 strain, expressing the surface molecule gp82, infected mice orally more efficiently than Y30, which expresses a related gp30 molecule. Both molecules are involved in lysosome exocytosis-dependent host cell invasion, but exhibit differential gastric mucin-binding capacity, a property critical for parasite migration toward the gastric mucosal epithelium. Upon oral infection of mice, the number of Y30 and Y82 parasites in gastric epithelial cells differed widely.</p><h3>Conclusions</h3><p>We conclude that metacyclic forms of gp82-expressing Y82 strain, closely related to TcVI, are better adapted than Y30 strain (TcII) to traverse the stomach mucous layer and establish oral route infection. The efficiency to infect target cell is the same because gp82 and gp30 strains have similar invasion-promoting properties. Unknown is whether differences in Y30 and Y82 are natural parasite adaptations or a product of lab-induced evolution by differential selection along the 60 years elapsed since the Y strain isolation.</p></div

Differential expression of <i>T. cruzi</i> Y82 and Y30 strain surface molecules implicated in host cell invasion.

<p>Live metacyclic trypomastigotes were incubated for 1 h with mAb 3F6 (A) or polyclonal anti-J18 antibodies (B) and processed for flow cytometry analysis, or for observations at the fluorescence microscope along with paraformaldehyde-fixed saponin-permeabilized parasites. In parallel, detergent-solubilized parasite extracts were analysed by immunoblotting. C) HeLa cells were incubated for 1 h with metacyclic forms, fixed and stained with Giemsa, followed by dehydration as described in experimental procedures. The number of intracellular parasites was counted in a total of 250 cells. The values are the means ± SD of eight independent experiments performed in duplicate. Shown on the right are HeLa cells with internalized metacyclic forms (black arrows), which are surrounded by a clear space, and adherent parasites (white arrow). Scale bar = 10 µm. D) HeLa cells were incubated with metacyclic forms for 1 h, washed to remove non internalized parasites, and incubation proceeded for 24 h in DMEM containing 2% fetal calf serum, upon which the cells were fixed and stained as in (C) for parasite counting. The values are the means ± SD of four independent assays performed in duplicate. Shown on the right are HeLa cells with amastigotes (black arrows). Scale bar = 10 µm. E) HeLa cells were incubated with metacyclic forms, untreated or pretreated with mAb 3F6, and the cells were processed as in (C). The values are the means ± SD of three independent experiments performed in duplicate. The difference between the parasites treated with mAb 3F6 and the untreated control was significant (*p<0.005, **p<0.01). F) Metacyclic forms, untreated or pretreated with mAb 3F6, were incubated for 1 h with HeLa cells, non internalized parasites were washed away, and incubation proceeded for 24 h as in (D). The values are the means ± SD of three independent assays performed in duplicate. MAb 3F6 significantly inhibited parasite invasion (*p<0.005, **p<0.01).</p

Binding of the recombinant protein D21 to gastric mucin and metacyclic trypomastigote migration.

<p>A) D21 sequence was aligned to the J18 sequence previously identified as the gastric mucin binding site, with the changed residues highlighted in red. B) Microtiter plates coated with gastric mucin were incubated with the indicated proteins, at 20 µg/ml, and the assay proceeded as described in the <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001804#s2" target="_blank">methods</a> section. GST was used as control. Values, given relative to the binding of J18, which were arbitrarily fixed to 100, are the means ± SD of three independent assays performed in duplicate. C) Polycarbonate transwell filters coated or not with gastric mucin were placed onto parasite-containing wells. After 60 min, samples from the filter chamber were collected and the number of parasites counted. Values are the means ± SD of three independent assays. The difference between Y30 migration through non coated and gastric mucin-coated filter was significant (*p<0.05). (D) Transwell filters coated with gastric mucin were placed onto parasite-containing wells, and at different time points, the number of parasites collected in the upper chamber was counted. Values are the means ± SD of four independent experiments. E-G) Transwell filters coated with gastric mucin alone or mixed with the recombinant protein J18 or T07 (E), with synthetic peptide P7 or P7* (F), with the recombinant protein J18 or D21 (G), were placed onto Y82 parasite-containing wells. After 60 min, samples from the filter chamber were collected and the number of parasites counted. Values are the means ± SD of three independent experiments. Significant difference was found between the control and the gastric mucin mixed with J18 or P7 (*p<0.0005).</p

Host cell invasion by Y30 and Y82 strain metacyclic forms through similar mechanisms.

<p>A) and B) HeLa cells were incubated with the indicated recombinant protein at 40 µg/ml. After 15 min, metacyclic trypomastigotes were added and incubation proceeded for 1 h before fixation and staining with Giemsa, as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001804#pntd-0001804-g001" target="_blank">Fig. 1C</a>. The number of internalized parasites was counted in a total of 250 cells. Values are the means ± SD of three independent assays performed in duplicate. The difference between cells treated with the recombinant protein and the untreated control was significant (*p<0.05). C) HeLa cells were preincubated for 30 min with the indicated drug, at 50 nM. After removal of the drug, the parasites were added and the incubation proceeded for 1 h, before processing for parasite counting as above. The values are as in A and B. Parasite invasion in drug-treated cells was significantly reduced (*p<0.05, **p<0.01).</p

Network genealogy of two <i>T. cruzi</i> nuclear gene sequences.

<p>Analysis of SSU rDNA (<b>A</b>) and actin (<b>B</b>) was performed to verify the genetic diversity of Y30 and Y82 strains. Red boxes indicate Y82 haplotypes, blue boxes Y30 haplotypes, purple boxes G (TcI) haplotypes, green boxes CL Brener (TcVI) haplotypes and the pink arrow indicates the Y strain (TcII) cluster. In haplotype names, the suffix R indicates the copy detected by direct sequencing of PCR products from genomic DNA while the other indicates the sequence obtained from cloned PCR products. The shaded area indicates the hybrid zone between the polar TcI and TcII. Numbers above links indicate bootstrap frequencies from 1000 pseudoreplicates. Networks were inferred using SplisTree 4 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001804#pntd.0001804-Huson1" target="_blank">[24]</a> as described in Material and Methods.</p