Immunodominant proteins in Giardia lamblia

Abstract

The protozoan parasite Giardia lamblia is a major cause of diarrhoea in humans and other mammals. Infection starts by ingestion of infectious cysts that excyst in the upper part of the small intestine and attach to intestinal epithelial cells. In the intestine the trophozoites multiply by binary fission and trigger a form of diarrhoea called giardiasis. Humoral immunity has been shown to be important for clearance of the infection, but only a few antigens have been identified and characterized.

In this thesis we have identified several immunoreactive proteins by performing the first large-scale proteomics study performed in G. lamblia. Sixteen immunoreactive proteins were identified using serum samples from 93 patients with acute giardiasis, infected during a waterborne outbreak in Sweden. Several of the identified proteins (i. e. alpha-1-, alpha-2-, alpha-7.1-giardin, OCT and ADI) were produced in recombinant form, mixed and used successfully as a diagnostic tool to screen sera from infected patients. The anti-Giardia IgG titers in serum from patients with acute giardiasis could be correlated to recombinant alpha-giardin immunoreactivity. The immunoreactive region of alpha-1 giardin was mapped to amino acids 160-200. In similarity to other annexins, alpha-1 giardin was shown to be plasma membrane associated. In addition, immunoelectron and immunofluorescence microscopy revealed that some alpha-1 giardin is displayed on the surface of excyzoites (recently excysted cells). Recombinant alpha-1 giardin displayed a Ca2+-dependent binding to glycosaminoglycans and bound specifically to thin sections of human small intestine.

By a genome survey and phylogenetic analyses 14 additional (21 in total) genes coding for proteins related to alpha-giardins were identified. Several alpha-giardin genes localized to the same chromosomal fragment, indicating a molecular evolution through gene duplication and subsequent functional divergence. RT-PCR analysis, during the G. lamblia life cycle, revealed large differences in the mRNA expression levels among the alpha-giardins. The alpha-giardin proteins localized to different cytoskeletal components such as flagella, adhesive disc and plasma membrane, thus pointing toward a function in cell motility, attachment and membrane stability. The cytoskeleton is crucial for parasite survival and virulence. In this respect, the ?-giardins are excellent targets for the development of a G. lamblia-specific drug.

Additionally, the influcence of differentiation on antigenic variation of the immunodominant variant specific surface proteins (VSPs) was studied. The results showed that there are two independent switch events during in vitro differentiation of isolates from the two major human Giardia genotypes, one during encystation and one during excystation. During encystation, the dominantly expressed VSP transcript within the trophozoite population is down regulated, whereupon a new encystation/cyst specific VSP is expressed late in enycstation. In addition, results obtained by analysing single cell parasites during vegetative growth and excystation support the idea that one major VSP is expressed per trophozoite at any given time. Also, the switching during excystation appears to take place after the excysted parasite has undergone its first cell division step. Notably, the switch events during differentiation occur at about the same time as nuclear division and DNA replication. In this respect, we hypothesize that differentiation driven antigenic variation is influenced by nuclear division and/or DNA replication events.