By Levi Clancy for Student Reader on
Intestinal Protozoan Parasites Giardia lamblia & Entamoeba histolytica
|G. lamblia||E. histolytica|
|Excystation Site||Triggered by gastric acid.||Lower small intestines.|
|Colonization Site||Upper small intestine.||Large bowel lumen. Sometimes invades mucosa, liver, lung and brain.|
|Nutrients||Absorbs from host.||Phagocytosis of RBCs & tissue bacteria.|
|Ingestion||Infection is acquired by ingestion of mature cysts|
|Excystation||Cysts pass through the stomach, where gastric acid (low pH) induce them to excystate and emerge in the lower stomach or upper small intestine as a trophozoite.|
|Colonization||The trophozoites colonize the small intestine and are the disease-causing stage.|
|Adherence||The trophozoite adheres to the epithelia via its ventral adhesive disc, and can cause malabsorption from the intestines and diarrhea.|
|Encystation||Trophozoites that migrate to the distal small intestine are triggered by high pH and bile salts to undergo encystation. The resulting cyst is non-dividing and has a rigid cell-wall that allows it to survive outside the host.|
|Defecation||Cysts are passed in the feces of the host and mature and survive externally until they are ingested by another host.|
Giardia lamblia Life Cycle
Infection of the host results when cysts are ingested. Trophozoites (growing, motile parasite stage) emerge from the cyst via excystation in the proximal small intestines. Some trophozoites colonize the small intestine, while others travel to distal intestine where they encyst so that they may be defecated. The life cycle is completed when a new host ingests these defecated cysts.
Giardia lamblia Transmission
Person-to-person contact and ingesting contaminated water are the most frequent methods of contracting this fecally transmitted infection. Since Giardia lamblia infects many mammals, mountain and forest streams are often contaminated with cysts defecated by local wildlife. Filtration, boiling or chemical treatment of stream water is critical for preventing Giardia lamblia infection.
Giardia TrophozoiteThe Giardia lamblia trophozoite has two nuclei, each with equal DNA content and activate transcription. The trophozoite also has eight flagella in four pairs. Additionally, the Giardia lamblia trophozoite has an axostyle and ventral adhesive disc. It reproduces by binary ﬁssion and can undergo encystation to become a cyst.
In vivo, occurs in stomach/upper small intestine motile, feeding trophozoite emerges in the small intestines. In vitro, induced by: low pH, as is found in the stomach; or protease treatment at pH 8, mimicking the upper small intestines.
In vivo, occurs in lumen of small intestines. Trophozoites undergoes developmental change, synthesizes and exports cyst wall components to its outer surface. In vitro, it is induced by: high pH and high bile salt, mimicking lower small intestines.
The Giardia lamblia cyst has a protective coat that allows the parasite to exist outside the host; this is necessary for transmission. Unlike the trophozoite, it has four nuclei, no visible flagella and stains for cyst-specific proteins. The Giardia lamblia cyst is resistant to cold, chlorine and disinfectants; but is sensitive to heat, freezing and desiccation. The cyst wall is composed of leucine-rich cyst wall protein (CW1-4) and polymers of galactosamine and N-acetylgalactosamine.
Encystation involves synthesis of a cell wall requiring specific secretion of cyst wall components. Encystation-speciﬁc secretory vesicles (ESV) are induced at the beginning of encystation. ESVs deliver cyst wall components (CW1 and CW2) to the trophozoite surface. Inhibition of encystation blocks transmission of giardiasis.
VSP & Antigenic Variation
Giardia lamblia's plasma membrane is covered by Variant Specific Protein (VSP). There are 50-150 different VSP genes encoded in the Giardia lamblia genome. Regulated expression of these genes results in antigenic variation. Antigenic variation is the ability of a single parasite to change the proteins its presents to the host's immune system. There is a 30-250 kDA, 25 amino acid membrane spanning region that is conserved; it is cysteine-rich and is laden with CXXC motifs.
Presence and Location of Proteins
|Tagging||Use recombinant DNA techniques to introduce a tag in the gene of interest. The tag is usually <15 amino acids and inserted into a non-critical domain of the protein. The plasmid with the gene encoding this protein is introduced into the cell and immunoﬂuorescence assays (IFA) using an antibody that speciﬁcally recognizes the “tag” are conducted on ﬁxed cells to detect and localize the protein.|
|Fluorescence||Use recombinant DNA to fuse a large naturally ﬂuorescent protein (ie, GFP) to the protein of interest. The plasmid with the gene encoding this fusion protein is introduced into the cell and living cells can be viewed, in real time, to watch movement of the GFP-fusion protein.|
|Conventional IFA||Isolate the protein of interest or its gene and make a recombinant protein. Use the protein to make an antibody that recognizes it and use microscopy on ﬁxed cells to detect the protein.|
|Immunoblots||Cell lysate is fractionated via SDS-PAGE, then electrophoreticaly transferred from gel onto a membrane matrix. The membrane is reacted with an antibody of interest. This verifies protein presence but does not reveal localization.|
Applying These Techniques: Antigenic Variation
Antigenic variation was studied via conventional IFA against the VSP TSA417.
|Clone||A TSA417 plasmid was transfected into E. coli.|
|Purify||Affinity chromatography was used to purify the protein.|
|Antibody||An animal was used to make a TSA417 antibody.|
|Giardia||G. lamblia were chemically fixed onto slides.|
|Reaction||Fixed cells were treated with anti-TSA417 antibody.|
|Label||Fourescent secondary antibody labelled bound cells.|
Over 85% of Giardia lamblia are positive for TSA417, indicating the presence of this surface antigen.
Assay For Presence of a Specific RNA
Northern blots and RT-PCR can assay for presence of steady-state RNAs. To examine whether gene expression is being controlled at the transcription level, this procedure is used:
|Purify||RNA is purified from all stages of in vitro switching.|
|Separate||RNAs are size-fractionated via gel electrophoresis.|
|Blot||RNAs are transferred to a blot and specific RNAs are detected with labelled probes.|
Antigen (VSP) Switching Occurs
|Induction||TSA417+ cells were induced to encyst/excyst.|
|TSA417?||The newly differentiated cells were stained for surface TSA417.|
|Previously TSA417+ cells become TSA417- upon encystation or excystation. Antigen switching occurred during differentation.|
How Is VSP Switching Controlled?
Selective intracellular transport to the cell surface?
Does IFA results address this?
Do the Immunoblot results address this?
Protein Stability or Production?
Rapid, speciﬁc degradation of speciﬁc VSP proteins?
Selective translation of speciﬁc VSP mRNA?
RNA Stability or Production?
Rapid, speciﬁc degradation of speciﬁc VSP mRNA?
Selective transcription of speciﬁc VSP mRNA?
A nature paper published in 2008 that suggests RNAi (post-transcriptional regulation) is used to assure only a single VSP protein is made at any given time 1.
Conclusions on Antigenic Switching, Part I
During encystation/excystation expression of the “old” VSP is turned-off. The newly emerged trophozoite, following encystation/excystation, expresses a “new” VSP on the surface. Antigenic switching occurs during differentiation - upon completion of the life cycle when a parasite is transmitted from one host to another. Differentiation-associated switching of surface antigens may explain the common occurrence of repeated giardiasis.
Conclusions on Antigenic Switching, Part II
Previously infected hosts can be re-infected by their own cysts because new variants are not recognized by existing antibodies and can thus recolonize the intestines. Antigenic Variation allows the parasite to thrive within a community allowing it to reinfect hosts previously infected by other variants. Enhances the probability of parasites being transmitted to reservoir animals that are partially immune to other VSP variants.
1Prucca et al. (2008) Antigenic variation in Giardia lamblia is regulated by RNA interference. Nature 456: 750-754.