Fig 1.
Profile of purified gp35/50 mucins from T. cruzi strains G and CL.
Purified mucins were analyzed by silver staining or Schiff staining of SDS-PAGE gel, or by western blot using the indicated gp35/50 mucin-specific mAbs.
Fig 2.
Inhibition of T. cruzi G strain MT internalization and host cell lysosome spreading by purified G-MUC.
(A) HeLa cells were incubated for 1 h with MT of G or CL strain, in absence or in the presence of mucins purified from G strain (G-MUC) or CL strain (CL-MUC), and the number of intracellular parasites was quantified. Values are the means ± four independent assays. Note the significant decrease in G strain MT invasion in the presence of G-MUC (*P<0.001). (B) HeLa cells, grown in ELISA microtiter plates, were fixed and incubated for 1 h with MUC-G or MUC-CL at the indicated concentrations. Binding of mucin was revealed by mAb 2B10. The assay was performed in triplicates. (C) HeLa cells were incubated for 30 min with G strain or CL strain MT and processed for immunofluorescence analysis for detection of actin (red), nucleus (blue) and mucin (green). Confocal microscopy visualization, under 63x objective, showed thick F-actin filaments in cells incubated with G strain and overall disruption of F-actin in cells incubated with CL strain. Arrows indicate the site of MT entry with disrupted cortical actin. Scale bar = 10 μm. (D) HeLa cells were incubated for 30 min in absence or in the presence of MUC-G and processed for detection of actin (red), nucleus (blue) and lysosome (green). Thick actin bundles were observed in cells incubated with MUC-G. Scale bar = 20 μm.
Fig 3.
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.
Fig 4.
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 (*P<0.01, **P<0.005). (C) 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.
Fig 5.
Binding of G strain mucins to annexin A2 and activation of host cell FAK.
(A) Protein A/G magnetic beads, crosslinked to mAb 10D8 directed to gp35/50 mucins, were incubated for 1 h with MT lysate and afterwards with HeLa cell extract for 1 h. The eluate corresponding to immunoprecipitate (IP) was analyzed by western blot (WB), along with the flowthrough samples corresponding to HeLa cell extract or MT. The blot was revealed with anti-annexin A2 antibody or with mAb 10D8. Note that both annexin A2 and gp35/50 mucins were detected in IP from beads crosslinked to mAb 10D8 (red arrows). (B) HeLa cells were incubated for 30 min in absence or in the presence of MT or purified gp35/50 mucins at 40 μg/ml. After washings, the cell extracts were analyzed by western blotting, using antibody directed to phosphorylated FAK. Note the increased phosphorylation levels of FAK in cells that interacted with MT or with gp35/50 mucins. (C) HeLa cells, untreated or pretreated with FAK inhibitor at indicated concentrations, were incubated for 1 h with MT and the number of internalized parasites was counted. Values are means ± SD of three independent assays performed in duplicate. MT invasion was significantly reduced in cells pretreated with 40 μM (*P<0.005).
Fig 6.
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.
Fig 7.
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 (*P<0.0001) or EA (*P<0.005). (C) 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.
Fig 8.
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.
Fig 9.
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 (*P<0.05), and decreased in the presence of r-gp82 (*P<0.005). (D) 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.
Fig 10.
Schematic representation of possible mechanisms of host cell invasion by T. cruzi 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.