Excystation of Eimeria tenella Sporozoites Impaired by Antibody Recognizing Gametocyte/Oocyst Antigens GAM22 and GAM56

Abstract

Eimeria tenella is the causative agent of coccidiosis in poultry. Infection of the chicken intestine begins with ingestion of sporulated oocysts releasing sporocysts, which in turn release invasive sporozoites. The monoclonal antibody E2E5 recognizes wall-forming body type II (WFBII) in gametocytes and the WFBII-derived inner wall of oocysts. Here we describe that this antibody also binds to the stieda body of sporocysts and significantly impairs in vitro excystation of sporozoites. Using affinity chromatography and protein sequence analysis, E2E5 is shown to recognize EtGAM56, the E. tenella ortholog of the Eimeria maxima gametocyte-specific GAM56 protein. In addition, this antibody was used to screen a genomic phage display library presenting E. tenella antigens as fusion proteins with the gene VIII product on the surfaces of phagemid particles and identified the novel 22-kDa histidine-and proline-rich protein EtGAM22. The Etgam22 mRNA is expressed predominantly at the gametocyte stage, as detected by Northern blotting. Southern blot analysis in combination with data from the E. tenella genome project revealed that Etgam22 is an intronless multicopy gene, with approximately 12 to 22 copies in head-to-tail arrangement. Conspicuously, Etgam56 is also intronless and is localized adjacent to another gam56-like gene, Etgam59. Our data suggest that amplification is common for genes encoding oocyst wall proteins.Coccidiosis in poultry is caused by protozoan parasites of the genus Eimeria. Worldwide economic losses due to these parasites have been estimated to exceed 1.2 billion U.S. dollars per annum (41). The most virulent species is Eimeria tenella, causing severe hemorrhagic enteritis by infection of the epithelium and submucosa of the ceca and, eventually, death of infected chickens (24).Eimeria infections occur by ingestion of oocysts (24). In the intestine, oocysts release four sporocysts, each containing two sporozoites. After excystation, motile infective sporozoites actively enter cells in the epithelium of the cecum. Three rounds of asexual multiplication in the epithelium and submucosa are then followed by differentiation to sexual stages of micro-and macrogametocytes (23). After fertilization of macrogametes, a complex, two-layered wall is secreted around the young oocyst by exocytosis of wall-forming body type I and type II (WFBI and WFBII) (35). While the 10-nm-thick outer oocyst wall is built up by the contents of WFBI, the 90-nm inner oocyst wall is composed mainly of glycoproteins that were stored in WFBII (31, 37). The oocyst displays a remarkable rigidity and protects the parasite from several physically and chemically adverse influences, such as commonly used disinfectants (34). A potential use of gametocyte antigens involved in formation of the oocyst wall as protective transmission-blocking vaccines has been described for Eimeria maxima (2,4,25,(38)(39)(40)46).The formation of oocyst and sporocyst walls and sporozoite excystation are rather complex processes that we are just beginning to underst...

Oocyst wall formation and composition in coccidian parasites

“…We have recently obtained similar results with both E. tenella and E. acervulina, demonstrating the conservation of these proteins and the process of wall formation (SI Belli et al, unpublished observations). Recently, Krücken et al (2008) have confirmed the presence of two E. tenella homologues of EmGam56 and, additionally, reported the discovery of a 22 kDa antigen in the macrogametocytes of E. tenella. Like the other proteins so far characterized, antibodies to this protein react with WFB2 and the inner layer of the oocyst wall.…”

“…It is notable for another reason, namely that its gene is present in extremely high copy number (in contrast to the single copies of the genes for, for example, EmGam56 and EmGam82), indicating that it may be important in oocyst wall formation via a mechanism distinct from that of the tyrosine-rich proteins. As yet, no information is available about whether this 22 kDa protein is processed into smaller polypeptides nor how it is incorporated into the oocyst wall, though its involvement in stabilizing the oocyst wall via cross-links between histidine and catechols, as seen in insect cuticles (Christensen et al 1991, Xu et al 1997, Kerwin et al 1999, is a distinct possibility (Krücken et al 2008). …”

The Glycosylation Pathway of Eimeria tenella Is Upregulated during Gametocyte Development and May Play a Role in Oocyst Wall Formation

“…7), and this may have a direct link with the stockpiling and eventual cross-linking of the oocyst wall glycoproteins Gam56 and Gam82 (6,7). It is possible that O glycosylation is required for trafficking of these proteins to the oocyst wall, a hypothesis supported by the conservation of an O glycosylation motif in both EmGam56 and EtGam56 and an additional oocyst wall protein of E. tenella, EtGam22 (19).…”

“…EmGam56 and EmGam82 are processed into smaller peptides prior to incorporation into the oocyst wall, where proposed cross-linking occurs to ensure the formation of a stable extracellular matrix (5,6). EtGam56, an E. tenella homologue of EmGam56, appears to undergo a similar process of truncation prior to oocyst wall formation (19,25), implying that this mechanism is strongly conserved within the Eimeria genus.…”

“…It is unclear why these wallforming proteins require glycosylation; however, the strong conservation of O-glycosylation motifs in both EmGam56 and EtGam56 (19) implies that it is an important process. N-Acetylgalactosamine (GalNAc) has also been implicated as the likely glycan moiety of glycoproteins present in the oocyst wall of the related apicomplexan, Cryptosporidium parvum, with a proposed role in host cell attachment (20,35).…”