Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima

Abstract

The oocyst wall of apicomplexan parasites protects them from the harsh external environment, preserving their survival prior to transmission to the next host. If oocyst wall formation could be disrupted, then logically, the cycle of disease transmission could be stopped, and strategies to control infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography, together with the detection of a UV autofluorescence in intact oocysts, implicates dityrosine- and possibly DOPA-protein cross-links in oocyst wall hardening. In addition, the identification of peroxidase activity in the wall-forming bodies of macrogametes supports the hypothesis that dityrosine- and DOPA-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode cuticles, yeast cell walls, mussel byssal threads, and sea urchin fertilization membranes.

FIG. 1.

Developmental processing of the gametocyte 56-kDa glycoprotein, gam56, to a ∼33-kDa oocyst wall protein in E. maxima. (A) Immunoblot of extracts from gametocytes (gam), unsporulated oocysts (u-oocyst), and sporulated oocysts (sp-oocyst) probed with mouse anti-r56 antibody or control normal mouse serum (C2). Gels were loaded with 5 × 10³ parasite equivalents for all stages. (B) Immunoblot of purified oocyst wall fragments probed with chicken anti-APGA antibody, control chicken serum (C1), mouse anti-r56 antibody, or control normal mouse serum (C2).

FIG. 2.

N-terminal sequences of oocyst wall proteins in E. maxima. (A) Coomassie blue-stained SDS-polyacrylamide gel of purified oocyst wall fragments. (B) Schematic representation of where the N-terminal amino acid sequences of oocyst wall proteins wp33, wp8, wp10, and wp12 map to the gametocyte glycoproteins gam56 and gam82. The numbers above the sequences of the wall proteins refer to amino acid numbering pertaining to the precursor proteins, gam56 (GenBank accession number AY129951) and gam82 (GenBank accession number AY179510). The N-terminal sequence for oocyst wall protein wp29 is given below the immunoblot in panel A because it shares no homology with gam56 or gam82.

FIG. 3.

UV autofluorescence detection of E. maxima oocysts. (A) Purified sporulated oocysts visualized under 330- to 385-nm UV light. (B) Immunofluorescence detection of recognition of intact sporulated oocysts by anti-56-kDa MAb (1E11-11). (C) Bright-field image of sporulated oocysts. (D) Merged image of purified gametocytes visualized under 330- to 385-nm UV light and under bright-field microscopy. (E) Purified oocyst and sporocyst wall preparation visualized under 330- to 385-nm UV light. Magnifications, ×200 (A to D) and ×1,000 (E).

FIG. 3.

UV autofluorescence detection of E. maxima oocysts. (A) Purified sporulated oocysts visualized under 330- to 385-nm UV light. (B) Immunofluorescence detection of recognition of intact sporulated oocysts by anti-56-kDa MAb (1E11-11). (C) Bright-field image of sporulated oocysts. (D) Merged image of purified gametocytes visualized under 330- to 385-nm UV light and under bright-field microscopy. (E) Purified oocyst and sporocyst wall preparation visualized under 330- to 385-nm UV light. Magnifications, ×200 (A to D) and ×1,000 (E).

FIG. 4.

Detection of dityrosine and DOPA in hydrolysates of E. maxima oocysts by HPLC with UV and fluorescence detection. Oocysts were prepared and subsequently hydrolyzed to free amino acids as described in Materials and Methods. The hydrolysates were subsequently subjected to HPLC analysis with a gradient HPLC system as outlined in Materials and Methods, with the column eluent examined by serial fluorescence (traces A and B) and UV-visible (traces C and D) detectors, with the latter used to identify the parent amino acid tyrosine and the former used to identify the oxidation products dityrosine and DOPA. Peaks in the oocyst hydrolysates were assigned to tyrosine, dityrosine, and DOPA on the basis of their retention times and characteristic UV-visible or fluorescence spectra (traces B and D). Quantification was achieved by integration of peak areas and comparison with standard curves (traces A and C) generated with authentic standards. Peaks from authentic samples of tyrosine (p-tyrosine), dityrosine, and DOPA are as indicated (arrows). In trace D, the arrow indicates the peak corresponding to tyrosine, and the other peaks correspond to other products of tyrosine oxidation.

FIG. 5.

Peroxidase activity in the wall-forming bodies of macrogametes of E. maxima. (A and B) Intestinal tissue sections taken at 164 h postinfection with E. maxima and stained with diaminobenzidene in the presence of hydrogen peroxide. Magnifications, ×200 (A) and ×400 (B). (C and D) Intestinal tissue sections taken at 164 h postinfection and stained with diaminobenzidene with and without pretreatment with 3% hydrogen peroxide. Magnification, ×400. WFB, wall-forming body.