Figure 5. Binding of the recombinant protein D21 to gastric mucin and metacyclic trypomastigote migration.

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 methods 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).