UVM Theses and Dissertations
Format:
Print
Author:
Heron, Bradley Thomas
Dept./Program:
Cell and Molecular Biology Program
Year:
2009
Degree:
M.S.
Abstract:
Amebiasis is a potentially lethal disease affecting approximately 50 million people across the world. It is caused by the protozoan parasite Entamoeba histolytica, and symptoms include colitis and amoebic dysentery. Infection begins when a person ingests food or water contaminated with amoebic cysts. When the cysts reach the small intestine the amoebas excyst into a highly motile trophozoite form that resides in the colon. The trophozoites are capable of binding to cells lining the colon through a Dgalactose1N- acetyl-D-galactosamine (Gal/GalNAc) inhibitable lectin. Following binding, the amoebas can induce host cell apoptosis and engulf the dying cells through phagocytosis. While not much is known regarding E. histolytica phagocytosis, mutant amoebas defective in phagocytosis have reduced virulence, making phagocytosis a very attractive area of study.
Previous studies have shown that inhibition of the Gal/GalNAc lectin with free Dgalactose inhibits amoebic contact-dependent cell killing and phagocytosis of live Jurkat T-lymphocytes. However, inhibition of the Gal/GalNAc lectin with free D-galactose only inhibits the phagocytosis of already apoptotic lymphocytes by approximately 40% suggesting additional receptors are involved in phagocytosis. In our attempts to identify potential ligands that stimulate E. histolytica phagocytosis we used chicken egg white ovalbumin as a negative control and discovered that amoebas not only recognize it, but efficiently phagocytose 2 pm latex microspheres coated with ovalbumin. Since there is no known reason E. histolytica should ever come in contact with ovalbumin, we believed the amoeba must recognize some portion of the protein that is similar to what they encounter in nature, such as a structure found on bacteria. The presence of one known amoebic lectin, the importance of lectin-glycoconjugate pairings in host-microbe and microbe-microbe interactions, and the fact that ovalbumin is glycosylated, led us to hypothesize that Entamoeba histolytica recognizes the glycosylation pattern found on ovalbumin.
We first compared the phagocytosis of ovalbumin coated microspheres to microspheres coated with bovine serum albumin (BSA) conjugated to Nacetylgalactosamine (GalNAc-BSA) and found that the addition of free D-galactoseinhibited the phagocytosis of GalNAc-BSA coated microspheres but minimally inhibiteduptake of ovalbumin coated microspheres. We tried a large variety of other free sugars to try to inhibit phagocytosis of ovalbumin coated microspheres, and while lactose and porcine stomach mucin showed statistically significant effects, the results were modest. To address concerns that a contaminant in the ovalbumin preparation was the basis for recognition, we used more highly purified, endotoxin-free ovalbumin and found the same results.
We next tested our hypothesis more directly by deglycosylating ovalbumin with trifluoromethanesulfonic acid (TFMS). Periodic Acid Schiff staining of TFMS-treated and untreated ovalbumin showed nearly complete deglycosylation of ovalbumin by TFMS treatment. A non-denaturing acrylamide gel showed that the ovalbumin was in fact denatured following TFMS treatment. However, further testing using untreated but heat denatured ovalbumin showed that the amoebas will still recognize and phagocytose microspheres coated with denatured ovalbumin. Finally, we compared the phagocytosis of microspheres coated with either untreated ovalbumin or TFMS-treated ovalbumin and found that TFMS treatment significantly inhibited phagocytosis of ovalbumin coated beads. These results suggest Entamoeba histolytica recognizes ovalbumin through the glycosylation structures found on the protein.
Previous studies have shown that inhibition of the Gal/GalNAc lectin with free Dgalactose inhibits amoebic contact-dependent cell killing and phagocytosis of live Jurkat T-lymphocytes. However, inhibition of the Gal/GalNAc lectin with free D-galactose only inhibits the phagocytosis of already apoptotic lymphocytes by approximately 40% suggesting additional receptors are involved in phagocytosis. In our attempts to identify potential ligands that stimulate E. histolytica phagocytosis we used chicken egg white ovalbumin as a negative control and discovered that amoebas not only recognize it, but efficiently phagocytose 2 pm latex microspheres coated with ovalbumin. Since there is no known reason E. histolytica should ever come in contact with ovalbumin, we believed the amoeba must recognize some portion of the protein that is similar to what they encounter in nature, such as a structure found on bacteria. The presence of one known amoebic lectin, the importance of lectin-glycoconjugate pairings in host-microbe and microbe-microbe interactions, and the fact that ovalbumin is glycosylated, led us to hypothesize that Entamoeba histolytica recognizes the glycosylation pattern found on ovalbumin.
We first compared the phagocytosis of ovalbumin coated microspheres to microspheres coated with bovine serum albumin (BSA) conjugated to Nacetylgalactosamine (GalNAc-BSA) and found that the addition of free D-galactoseinhibited the phagocytosis of GalNAc-BSA coated microspheres but minimally inhibiteduptake of ovalbumin coated microspheres. We tried a large variety of other free sugars to try to inhibit phagocytosis of ovalbumin coated microspheres, and while lactose and porcine stomach mucin showed statistically significant effects, the results were modest. To address concerns that a contaminant in the ovalbumin preparation was the basis for recognition, we used more highly purified, endotoxin-free ovalbumin and found the same results.
We next tested our hypothesis more directly by deglycosylating ovalbumin with trifluoromethanesulfonic acid (TFMS). Periodic Acid Schiff staining of TFMS-treated and untreated ovalbumin showed nearly complete deglycosylation of ovalbumin by TFMS treatment. A non-denaturing acrylamide gel showed that the ovalbumin was in fact denatured following TFMS treatment. However, further testing using untreated but heat denatured ovalbumin showed that the amoebas will still recognize and phagocytose microspheres coated with denatured ovalbumin. Finally, we compared the phagocytosis of microspheres coated with either untreated ovalbumin or TFMS-treated ovalbumin and found that TFMS treatment significantly inhibited phagocytosis of ovalbumin coated beads. These results suggest Entamoeba histolytica recognizes ovalbumin through the glycosylation structures found on the protein.