Apical organelle discharge by Cryptosporidium parvum is temperature, cytoskeleton, and intracellular calcium dependent and required for host cell invasion

Abstract

The apical organelles in apicomplexan parasites are characteristic secretory vesicles containing complex mixtures of molecules. While apical organelle discharge has been demonstrated to be involved in the cellular invasion of some apicomplexan parasites, including Toxoplasma gondii and Plasmodium spp., the mechanisms of apical organelle discharge by Cryptosporidium parvum sporozoites and its role in host cell invasion are unclear. Here we show that the discharge of C. parvum apical organelles occurs in a temperature-dependent fashion. The inhibition of parasite actin and tubulin polymerization by cytochalasin D and colchicines, respectively, inhibited parasite apical organelle discharge. Chelation of the parasite's intracellular calcium also inhibited apical organelle discharge, and this process was partially reversed by raising the intracellular calcium concentration by use of the ionophore A23187. The inhibition of parasite cytoskeleton polymerization by cytochalasin D and colchicine and the depletion of intracellular calcium also decreased the gliding motility of C. parvum sporozoites. Importantly, the inhibition of apical organelle discharge by C. parvum sporozoites blocked parasite invasion of, but not attachment to, host cells (i.e., cultured human cholangiocytes). Moreover, the translocation of a parasite protein, CP2, to the host cell membrane at the region of the host cell-parasite interface was detected; an antibody to CP2 decreased the C. parvum invasion of cholangiocytes. These data demonstrate that the discharge of C. parvum sporozoite apical organelle contents occurs and that it is temperature, intracellular calcium, and cytoskeleton dependent and required for host cell invasion, confirming that apical organelles play a central role in C. parvum entry into host cells.

FIG. 1.

Labeling of C. parvum sporozoites that were maintained at 4 or 37°C in the absence of host cells. Freshly excysted sporozoites were incubated in culture medium at 4 or 37°C in the absence of host cells for 2 h and then fixed for immunofluorescence staining. Sporozoites collected immediately after excystation were used as a control. (A to C) Fluorescent images of sporozoites stained with antibodies against C. parvum. Y271, a polyclonal antibody which recognizes the whole protein profile of C. parvum sporozoites, showed strong staining in the apical region of control sporozoites (A1, arrowheads) or those that were maintained at 4°C for 2 h (A2, arrowheads), but not in sporozoites that were maintained at 37°C for 2 h (A3, arrows). Both 4E9 (a monoclonal antibody which recognizes microneme-associated gp900 and gp40 proteins) and CP2 (an antibody against membranous structures) also showed strong staining in the apical region of control sporozoites (B1 and C1, arrowheads) or those that were maintained at 4°C for 2 h (B2 and C2, arrowheads), but not in sporozoites that were maintained at 37°C for 2 h (B3 and C3, arrows). (D) Quantitative analysis of fluorescence intensities in the apical region of sporozoites after treatment with antibodies. (E) Viability of C. parvum sporozoites when maintained at 4 or 37°C. Ctrl, control; *, P < 0.05, compared with control or sporozoites maintained at 4°C; error bars, standard errors of the means. Bars = 1 μm.

FIG. 2.

Discharge of parasite-associated molecules into the supernatant when C. parvum sporozoites were maintained at 4 or 37°C in the absence of host cells. Freshly excysted sporozoites were incubated in culture medium at 4 or 37°C in the absence of host cells for 2 h, and supernatants were collected for Western blotting. Whole-cell lysates of freshly excysted sporozoites were used as a control. Supernatants from C. parvum sporozoites that were maintained in culture medium at 37°C for 2 h showed multiple bands with Y271 (A) and three bands (40 kDa, about 200 kDa, and >400 kDa) with 4E9 (B). Supernatants from C. parvum sporozoites that were maintained at 4°C for 2 h did not show any detectable bands with both antibodies. Ctrl, control.

FIG. 3.

Morphological changes of C. parvum sporozoites after incubation at 4 or 37°C in the absence of host cells, as revealed by transmission electron microscopy. (A and B) Sporozoites before and during excystation showed typical characteristics of a rhoptry (r), dense granules (dg), and micronemes (mn) around the apical region. (C) Sporozoites continued to preserve apical organelles around the apical region of sporozoites immediately after excystation. (D) When the sporozoites were maintained in the culture medium at 37°C for 2 h in the absence of host cells, much fewer dense granules and micronemes were found in the apical region. The single rhoptry in the apical region was detected in only a few of the sporozoites. (E) Sporozoites incubated at 4°C for 2 h preserved the apical organelles in the apical region. (F to H) Quantitative analyses showed a significant decrease in dense granules, micronemes, and rhoptries in sporozoites that were maintained at 37°C for 2 h compared with sporozoites examined immediately after excystation (control) or maintained at 4°C for 2 h. Ctrl, control; *, P < 0.05, compared with control or sporozoites that were maintained at 4°C; error bars, standard errors of the means. Bars = 0.1 μm.

FIG. 4.

Effects of colchicine, CD, and BAPTA-AM on discharge of apical organelle contents by C. parvum sporozoites into the supernatants. Freshly excysted sporozoites were incubated in culture medium at 37°C in the presence or absence of CD, colchicine, or BAPTA-AM for 2 h, the supernatants were collected for Western blotting, and the parasites were fixed for immunofluorescence staining. (A to D) Fluorescent images of sporozoites staining with antibody Y271 in the absence of drugs (A) or in the presence of CD (B), colchicine (C), or BAPTA-AM (D). (E) Quantitative analysis of fluorescence intensities in the apical region of sporozoites after treatment with drugs and with antibodies. (F) Viability of C. parvum sporozoites. (G) Western blot detection of parasite-associated proteins in the supernatants after treatment with the reagents. (H) Protein levels in the supernatants after treatment with drugs. *, P < 0.05, compared with sporozoites maintained at 4°C; #, P < 0.05, compared with sporozoites maintained at 37°C; Col, colchicine; B-AM, BAPTA-AM; error bars, standard errors of the means. Bars = 1 μm.

FIG. 5.

Effects of temperature, colchicine, CD, and BAPTA-AM on gliding motility of C. parvum sporozoites. Freshly excysted sporozoites were incubated in culture medium in the presence or absence of CD, colchicine, or BAPTA-AM for 30 min and then maintained on poly-l-lysine-coated glass slides for 5 min at 37°C. The sporozoites were then fixed, and their gliding trails were detected by immunofluorescence staining with the Y271 antiserum. (A to E) Fluorescent images of sporozoites showing gliding trails when maintained at 18°C (A) or incubated at 37°C (B) or in the presence of CD (C), colchicine (D), or BAPTA-AM (E) at 18°C. (F) Quantitative analysis of gliding motility of C. parvum sporozoites. Ctrl, control (sporozoites incubated at 18°C); *, P < 0.05, compared with control sporozoites maintained at 4°C; Col, colchicine; B-AM, BAPTA-AM; error bars, standard errors of the means. Bars = 5 μm.

FIG. 6.

Manipulation of C. parvum apical organelle discharge affects parasite host cell attachment and invasion. Freshly excysted C. parvum sporozoites were either incubated in culture medium in the absence of cholangiocytes at various temperatures for 0 to 24 h or incubated in culture medium at 18°C for 30 min in the presence or absence of CD, colchicine, or BAPTA-AM before exposure to host cells. After treatment, the sporozoites were then exposed to host cells to measure infectivity. (A) No significant difference in attachment rates was found for sporozoites incubated at different temperatures. (B) A significant decrease in attachment and invasion rates was detected in sporozoites after incubation at 37°C. (C) No decrease in attachment rates was detected when sporozoites treated with drugs were exposed to fixed cholangiocytes compared with those for untreated controls. (D) Inhibition of C. parvum sporozoite actin and tubulin polymerization by colchicine and CD or depletion of intracellular calcium by BAPTA-AM resulted in a significant decrease in the attachment and invasion rate of C. parvum with cultured cholangiocytes. The cotreatment of sporozoites with a Ca²⁺ ionophore (A23187) in the presence of extracellular Ca²⁺ restored the infectivity of C. parvum sporozoites. *, P < 0.05, compared with sporozoites maintained at 4°C; #, P < 0.05, compared with sporozoites treated with BAPTA-AM; Col, colchicine; B-AM, BAPTA-AM; error bars, standard errors of the means.

FIG. 7.

Immunogold labeling of CP2 protein in infected cholangiocytes at the host cell-parasite interface. Panels B and D show a higher magnification of the boxed regions in panels A and C, respectively. The anterior portion of the parasite reveals an accumulation of gold particles in the anterior vacuole region (arrowheads in panel B) as well as at the host-parasite interface (dense band) (arrows in panel B), suggesting the release of parasite factors from the apical region. (C and D) No labeling was found in the controls when the primary antibody was omitted. (E and F) Attachment-invasion assay in the presence of a CP2 antibody. Infection in the presence of an anti-CP2 antibody did not inhibit attachment, yet the invasion of cholangiocytes was significantly inhibited. *, P < 0.05, compared with control serum; error bars, standard errors of the means. Bars = 0.5 μm (A and C) and 0.1 μm (B and D).