Lack of requirement of gp82 in G strain MT invasion.

HeLa cells were incubated for 1 h with: (A) MT pretreated for 30 min with non purified anti-gp82 mAb 3F6 or mAb 5E7, or (B) purified mAb 3F6 at indicated concentrations, (C) MT in absence or in the presence of recombinant gp82 protein or GST, and processed for intracellular parasite quantification. MT invasion was significantly increased by treatment with non purified mAb 3F6 or by purified antibody at 200 μg/ml (*PD) HeLa cells were incubated for 30 min in absence or in the presence of MT. After washings, the cell extracts were analyzed by western blotting, using antibody directed to phosphorylated PKC or ERK1/2. Note that incubation with MT did not alter the phosphorylation levels of PKC or ERK1/2.</p

Activation of host cell PTK by MUC-G and G strain MT.

HeLa cells were incubated for 30 min in absence or in the presence of MT or MUC-G at 40 μg/ml. After washings, the cell extracts were analyzed by western blotting, using antibody directed to phosphorylated tyrosine proteins. Note the increased phosphorylation levels of protein bands in cells that interacted with MT or with MUC-G (black arrows). (TIF)</p

Reduced gp35/50 mucin co-localization with clathrin in sucrose-treated cells.

Untreated and sucrose-treated HeLa cells were incubated for 30 min with MT and processed for immunofluorescence microscopy, using anti-clathrin antibody and mAb 10D8. Note the clathrin-positive gp35/50 mucins (arrows) in the parasite vicinity or attached to cell membrane. Scale bar = 10 μm.</p

Inhibition of PBS⁺⁺-induced lysosome spreading by MUC-G.

HeLa cells were incubated for 30 min in RPMI containing 10% serum (R10) or in PBS++, in absence or in the presence of 40 μg/ml MUC-G or MUC-CL, and then processed for immunofluorescence analysis for detection of lysosome (green), actin (red) and nucleus (blue) by confocal microscopy. Scale bar = 20 μm. Note the PBS++-induced lysosome spreading, with accumulation of lysosomes at the cell edges (white arrows) and inhibition by MUC-G.</p

Inhibition of G strain MT invasion by pretreatment of host cells with sucrose.

(A) HeLa cells were treated for 30 min with 0.45 M sucrose in serum-free medium, washed and incubated with: (A) G strain MT in PBS++ or (B) G strain EA in R10. After 1 h, the cells were processed for intracellular parasite quantification. Values are the means ± five independent assays. HeLa cells pretreated with sucrose were significantly more resistant to invasion by MT (*PC) Untreated and sucrose-treated HeLa cells were incubated for 30 min with MT and processed for immunofluorescence microscopy, using anti-clathrin antibody and mAb 10D8. The images show clathrin (red), parasite mucins (green) and nucleus (blue). Scale bar = 30 μm.</p

Increased resistance of annexin A2-depleted cells to G strain MT invasion.

(A) HeLa cells were submitted to lentiviral transduction for annexin A2 knockdown (kd) and analyzed by western blotting. Note the depletion of annexin A2 in two independent cell lines. (B) HeLa cells depleted in annexin A2 and WT cells were incubated for 1 h with G strain MT. The amounts of intracellular parasites are shown as means ± SD of three independent assays performed in duplicate. MT invasion was significantly diminished in cells deficient in annexin A2 (*PC) Non infected annexin A2-deficient and WT cells were analyzed by immunofluorescence, to visualize actin (red), lysosome (green) and nucleus (blue). Scale bar = 30 μm. Note the altered morphology of annexin-kd1 cells, as compared to WT cells, and the distinct lysosome distribution in annexin-kd2 cells.</p

Colocalization of invading G strain MT and released gp35/50 mucins with host cell clathrin.

(A) HeLa cells were incubated for 30 min with parasites and processed for immunofluorescence microscopy, using anti-clathrin antibody and mAb 10D8. The images show clathrin (red), parasite mucins (green) and nucleus (blue). Scale bar = 30 μm. Note the colocalization of MT with clathrin. (B) A single cell with invading parasites is depicted to show the colocalization of clathrin with MT and also with shed gp35/50 mucins at the cell membrane (white arrows). Scale bar = 5 μm.</p

Schematic representation of possible mechanisms of host cell invasion by <i>T</i>. <i>cruzi</i> strains G and CL.

Host cell invasion by G strain MT involves the interaction of gp35/50 mucins with annexin A2, FAK activation and F-actin recruitment, whereas CL strain MT internalization relies on the recognition of gp82 by LAMP2, activation of PKC and ERK1/2, disruption of F-actin and spreading of lysosomes to the cell periphery.</p

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

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