Fig 1.
Entamoeba histolytica evokes mucin secretion via cysteine protease 5.
LS174T colonic cells metabolically labeled with 3H-glucosamine were assayed for mucin secretion in response to live WTEh, EhCP5- and WTEh treated with the cysteine protease inhibitor E64. The phorbol-ester PMA (1μM) was used as a positive control to elicit mucin hypersecretion. A. Mucin secretion was assessed at various time points with 5 x 104 Eh/well. B. After 2 h, supernatants containing secreted mucins were precipitated 10% TCA and 1% PTA and loaded onto a Sepharose 4B column. 1mL fractions were collected to assess the molecular weight profile of secreted 3H-mucin following Eh infection. The column was calibrated with Blue dextran [BD; 2,000,000Da that elutes in the void volume (Vo fractions 16–19) consistent with high molecular weight mucins] and Thyroglobulin (Ty; 669,000Da) and BSA (66,000Da). C. Area under the curve was calculated for the Vo of the Sepharose 4B column for each condition and plotted as percent from baseline secretion. D. Secreted components (SC) from Eh and recombinant CP5 were assayed for their ability to induce mucin secretion in a dose-dependent manner. E. Live Amoebapore-deficient Eh and EhCP5- complemented with exogenous rCP5 were assayed for their ability to induce mucin secretion. F. Specific inhibitors for EhCP1 (WRR483; 20μM) and EhCP4 (WRR605; 20μM) incubated with Eh and mucin secretion quantified. ***p <0.001.
Fig 2.
αvβ3 is the critical CP5 receptor on goblet cells responsible for mucin secretion.
Mucins from LS174T goblet cells were metabolically labeled with 3H-glucosamine and assayed for mucins secretion in the presence of A, GRGDSP (50μM) and B, αvβ3 function-blocking antibody (LM609 5μg/mL) for 2 h with live Eh. These were compared to the appropriate controls GRADSP and mouse IgG respectively. C. Eh induced mucin secretion in the presence of a specific αvβ3 inhibitor P11 (10 μM) with either GRADSP (50μM) or GRGDSP (50μM). D. αvβ3 binding to various adhesive proteins as assessed by ELISA. Vitronectin and laminin are the positive and negative controls respectively. For P11 inhibition, the inhibitor was included when αvβ3 was added and used at a concentration of 1μM. ***p <0.001, **p <0.01, *p <0.05.
Fig 3.
CP5-αvβ3 engagement leads to SRC and FAK activation.
A. LS174T cells were pre-treated with SRC family kinase inhibitors (PP1 and PP2; 5μM) along with the appropriate control (PP3; 5μm) and assayed for the ability to secrete mucins following metabolic labeling with 3H-glucosamine. B. The role of FAK in facilitating mucin secretion was assessed by pre-treating LS174T cells with the FAK inhibitor FAK14 (25μM). C. LS174T cells were infected with 2x105 Eh/well with WTEh in the presence and absence of FAK14 for 20 minutes, then immunostained for phospho-FAK and SRC. The dotted white line indicates the position of Eh. The arrows indicate a site of Eh contact that induces phosphorylation of SRC and FAK. D, E. Quantification of confocal images was performed by measuring the concentration of phosphorylated FAK and SRC within the host cell in the immediate contact site by Eh. Scale bar 10 μm. ***p <0.001, **p <0.01, *p <0.05.
Fig 4.
E. histolytica activates PI3K via CP5 to evoke mucin secretion.
A. Mucins from LS174T goblet cells metabolically labeled with 3H-glucosamine were treated with the PI3K inhibitor wortmannin (10μM) and assayed for secretion of 3H-labeled mucins after 2 h in response to Eh. B. PIP3 accumulation was quantified by counting radiolabeled PIP3 extracted from confluent LS174T monolayers in response to secreted components (50μg/mL) from WTEh, EhCP5-, WTEh + E64 and the positive control epidermal growth factor (50ng/mL). Control cells were uninfected basal resting cells. C. Confocal microscopy on fixed monolayers infected with WTEh and EhCP5- were imaged with a Phospho-PI3K antibody to detect activation. The dotted white line indicates the position of Eh. Scale bar 10 μm. D. Flow cytometry analysis of LS174T cells labeled with PE-Phospho-PI3K antibody after stimulation with 50μg/mL WTEh secreted components. As above with Phospho-PI3K, PIP3 was visualized by E, confocal microscopy and F, Flow cytometry. The PIP3 antibody was routinely observed to strongly cross-react with Eh and contact with host cells did not affect this off target staining. Arrows indicate the Eh-goblet cell contact site that contains P-PI3K or PIP3 respectively. G, H. Quantification of confocal images was performed by measuring the concentration of phosphorylated PI3K and PIP3 within the host cell in the immediate contact site by Eh. ***p <0.001, **p <0.01.
Fig 5.
PKCδ is activated downstream of PI3K and is necessary for EhCP5 induced mucin secretion.
A. Mucins from LS174T goblet cells metabolically labeled with 3H-glucosamine were treated with the broad spectrum PKC inhibitor bisindolylmaleimide I (10μM), infected with Eh for 2 h and assayed for 3H-labeled mucin secretion. A panel of PKC inhibitors including Rottlerin (20μM), Bisindolylmaleimide IX (10μM) and Gö6983 (10μM) was used to discern the critical isoform in response to WTEh secreted components (50μg/mL). B. WTEh secreted components (50μg/mL) and recombinant EhCP5 (5μg) were added to LS174T cells and activation of PI3K and PKC determined over a 15-minute period. C. Confocal microscopy on fixed monolayers infected with WTEh and EhCP5- were imaged with a Phospho-PKCδ antibody to detect activation. The dotted white line indicates the position of Eh. Arrows indicate regions of interest where PKCδ is phosphorylated at the Eh-goblet cell contact site. Scale bar 10 μM. D. Quantification of confocal images was performed by measuring the concentration of phosphorylated PKCδ within the host cell in the immediate contact site by Eh. E. LS174T cells were pretreated with either the PI3K inhibitor wortmannin or PKC inhibitor bisindolylmaleimide I and stimulated with WTEh secreted components over a 15 minute period. *** p <0.001, **p <0.01.
Fig 6.
EhCP5 signaling results in activation of MARCKS.
A. LS174T cells were infected with live WTEh or EhCP5- for 15 minutes and subsequently immunostained for phospho-MARCKS and actin. Arrows indicate intracellular phospho-MARCKS in cells that are contacted by live WTEh. The dotted white line indicates the position of Eh. Arrows indicate key regions of the cell that contains activated intracellular MARCKS that is phosphorylated. Scale bar 10 μm. B. Quantification of confocal images was performed by measuring the concentration of threshold phosphorylated PKCδ staining within the host cell in the immediate contact site by Eh. C. Mucin granules were extracted and purified from LS174T cells previously stimulated with 50μg/mL-secreted components from WTEh along with subcellular fractionation of both the plasma membrane and cytoplasm. The following markers were used: Mucin for granule contents, Vamp8 for granule membrane, Integrin αv for the plasma membrane and GADPH for the cytoplasm. D. The gating strategy and calibration controls for flow cytometry of mucin granules using markers of known sizes 300nm (Blue), 1μm (Red) and presence of mucin within the granule as visualized with the lectin WGA (Black). E. Mucin granules were extracted from monolayers stimulated with 50μg/mL-secreted components from WTEh, EhCP5-, WTEh+E64 or PMA (1μM) for 20 minutes and analyzed by flow cytometry using the gating strategy shown in 5D. Mean fluorescence intensity was measured from PE-Phospho-MARCKS whereby PMA served as the positive control. F. siRNA was used to disrupt MARCKS and PKCδ protein expression at concentrations of 20, 5, 1nM and compared to 20nM non-targeting control pool (CP). GAPDH serves as a loading control. G. 3H-glucoasmine metabolically labeled LS174T cells were transfected with siRNA against MARCKS, PKCδ or control pool and assayed for mucin secretion in response to live Eh. Control cells were treated with the transfection reagent in the absence of siRNA. *** p <0.001, *p <0.05.
Fig 7.
EhCP5-αvβ3 signaling cascade is activated in an ex vivo model of Eh infection.
Colonic explants were infected ex vivo with live WTEh, EhCP5- or WTEh+E64 for 15 minutes and immunostained for A, phospho-PI3K or B, PKCδ. Arrows indicate sites in the colonic explants that show activation of each respective kinase. UEA1 was used to detect secreted and goblet cell resident mucin and DIC were used to detect live Eh that contacted the epithelial cell surface. Arrows indicate critical sites of contact in which PI3K and PKCδ were activated. Scale bar 20 μm.