Abstract
Cryptosporidium parvum, an Apicomplexan parasite of the mammalian gut epithelium, causes a diarrheal illness in a wide range of hosts and is transmitted by contamination of food or water with oocyst-laden feces from an infected animal. We have identified a glycosylinositol phospholipid from the sporozoite stage of the parasite that is frequently recognized by serum antibodies from human cryptosporidiosis patients. The humoral immune response is dominated by IgG1 subclass antibodies but can also include IgA and IgM antibodies. The glycosylinositol phospholipids were purified by butanol extraction of a Triton X-114-soluble fraction followed by octyl-Sepharose column chromatography and preparative high performance TLC and were shown to include at least 5 species. By using mass spectrometry and radiolabeled neutral glycan analysis, we found that the structure of the dominant glycosylinositol phospholipid antigen contained a C18:0 lyso-acylglycerol, a C16:0-acylated inositol, and an unsubstituted mannose3-glucosamine glycan core. Other diacyl species were also identified, most notably a series of glycosylinositol phospholipids having an acyl-linked C20:0 to C28:0 lipid on the inositol ring. Less abundant species having three acyl-linked fatty acids and species with an additional 1-3 hexoses linked to the mannose core were also observed. We are currently working to determine the role that these glycolipids may play in the development of disease and in the clearance of infection.
Highlights
Cryptosporidium parvum is an Apicomplexan protozoan parasite that has been recognized as a major cause of diarrheal illness in humans and in livestock around the world [1,2,3]
We report that many cryptosporidiosis patients have a serum antibody response to sporozoite-derived glycosylinositol phospholipids (GIPLs), 1 The abbreviations used are: GIPL, glycosylinositol phospholipid; GPI, glycosylphosphatidylinositol; OS, octyl-Sepharose; HPTLC, high performance thin layer chromatography; PVDF, polyvinylidene difluoride; PBS, phosphate-buffered saline; PI, phosphatidylinositol; GC-MS, gas chromatography-mass spectrometry; JBAM, jack bean ␣-mannosidase; ASAM, Aspergillus satoi ␣-mannosidase; Electrospray-Mass Spectrometry (ES-MS), electrospraymass spectrometry; Collision-induced dissociation (CID), collision-induced dissociation; AHM, 2,5-anhydromannitol; mAb, monoclonal antibody
While examining the IgG antibody reactivity to the subset of those antigens that were extracted into Triton X-114 detergent, we noted that an additional antigen having a molecular mass of Ͻ6-kDa was recognized by serum IgG antibodies from ϳ50% of the patients (Fig. 1 and data not shown)
Summary
Cryptosporidium parvum is an Apicomplexan protozoan parasite that has been recognized as a major cause of diarrheal illness in humans and in livestock around the world [1,2,3]. In a study of C. parvum infections in human volunteers, antibody responses to these antigens were associated with protection from diarrheal symptoms [20]. Both of these antigens are associated with the sporozoite surface, and subsets of the antigens can be partially purified from sonicated oocysts by phase partitioning into Triton X-114 detergent [19]. GIPLs and related structures that anchor some surface proteins into the membrane, glycosylphosphatidylinositol (GPI) anchors, are present in large quantities in the surface membranes of many protozoan parasites and have been recognized recently as important effectors of the host immune response during infection We report that many cryptosporidiosis patients have a serum antibody response to sporozoite-derived GIPLs, This paper is available on line at http://www.jbc.org. Glycosylinositol Phospholipid Antigens from C. parvum and we report the purification and structural analysis of the sporozoite GIPL antigens
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