Fig 1.
Phosphatase activity is needed for C. parvum infectivity.
A) phospho-Tyrosine blot analysis using whole cell lysates from C. parvum infected FHs 74 Int cells at 2h and 4 h post infection. Tyrosine-phosphorylated proteins decrease at higher molecular weights upon infection. B) Treatment of FHs 74 Int cells with the phosphatase inhibitor, sodium orthovanadate significantly decreases C. parvum infectivity. The number of infections per field of view (at 200X magnification) are represented on the y-axis. Values are means ±SD for three independent experiments. Statistical significance criteria set at: *p<0.05, **p<0.02.
Fig 2.
Upon C. parvum infection, SHP-2 activity and expression increase; and SHP-2 co-localizes with the C. parvum sporozoite.
A) Active binding partners specific to the SH2 domain of SHP-2 increases after 2 h of C. parvum infection. Values represent mean ±SD for two independent experiments. B) C. parvum infection upregulates SHP-2 expression at 2h and 4 h post-infection, and levels decrease to pre-infection levels by 6 h. C) Densitometry of Western blots from three independent experiments reflect normalized fluorescence intensity units of SHP-2 expression using GAPDH as a loading control. Data represented are means ±SD for three independent experiments. Statistical significance criteria set at: *p<0.05. D-F) Single confocal microscopic images demonstrating that SHP-2 and the C. parvum sporozoite co-localize. G) 3D compiled z-stack image of C. parvum infected FHs 74 Int cells, showing that SHP-2 localizes apically and around the C. parvum sporozoite. Cy5/Red, C. parvum; Cy3/green, SHP-2. Scale bar, 10 μm.
Fig 3.
Inhibition of SHP-2 activity decreases C. parvum infectivity.
A) Dose-dependent decrease of Infections per field of view (y-axis) after treatment of FHs 74 Int cells with increasing concentrations of the SHP-2-specific inhibitor, SHP-2 PTPase Inhibitor. Values are means ±SD for three independent experiments. Statistical significance criteria set at: *p<0.05. B) SHP-2 inhibitor treatment does not alter SHP-2/sporozoite co-localization in the few infections sites that still form after SHP-2 PTPase Inhibitor treatment. Cy5/Red, C. parvum; Cy3/green, SHP-2. Scale bar, 10 μm.
Fig 4.
Paxillin, a substrate of SHP-2, does not co-localize with C. parvum infection site, but is moderately dephosphorylated upon infection.
A) Paxillin does not co-localize with the C. parvum sporozoite. Cy5/Red, C. parvum; Cy3/green, paxillin. Scale bar, 10 μm. B) Although expression of total paxillin does not change after infection, paxillin is moderately dephosphorylated for up to 6 hours post-infection. C) Densitometry of Western blots from three independent experiments, with normalized fluorescence intensity units of phospho-paxillin expression, using total paxillin as baseline, and GAPDH as a loading control. Data represented are means ±SD for three independent experiments. Statistical significance criteria set at: *p<0.05.
Fig 5.
Inhibition of Src kinases and paxillin leads to dose-dependent decreased infection by C. parvum.
A) Number of infections per field of view decrease after exposure of host cells to increased levels of the inhibitor, Saracatinib (AZD0530). Values are means ±SD for three independent experiments. Statistical significance criteria set at: *p<0.05, **p<0.02. B) Merged confocal images of FHs 74 Int cells labeled for paxillin and C. parvum, show decreased number of infections with increased concentrations of Saracatinib. Red, C. parvum; green, paxillin. Scale bar, 10 μm.
Fig 6.
Inhibition of SHP-2 activity promotes paxillin phosphorylation.
A) Paxillin phosphorylation levels increase in the presence of 300 and 700 μM SHP-2 PTPase Inhibitor. B) Densitometry of Western blots from three independent experiments, showing normalized fluorescence intensity units of phospho-paxillin expression, using total paxillin as baseline, and GAPDH as a loading control. Data represented are means ±SD for three independent experiments. Statistical significance criteria set at: *p<0.05.
Fig 7.
Diagramatic representation summarizing SHP-2-mediated regulation of C. parvum infectivity.
The C. parvum sporozoite binds to the apical surface of intestinal cells. Upon C. parvum infection, SHP-2 co-localizes with the C. parvum sporozoite, forming a cap over the sporozoite with finger-like projects extending down the sides of the parasite. This interaction increases the rate of C. parvum infectivity through SH2-mediated SHP-2 activity. Whether the interaction of SHP-2 with the sporozoite also participates in forming the parasitophorous vacuole remains to be established. In parallel, SHP-2 moderately dephosphorylates cytoplasmic paxillin, and possibly other proteins, to facilitate C. parvum infection.