ESSENTIALS OF GLYCOBIOLOGY LECTURE 31 MAY 6, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Cent

1. ESSENTIALS OF GLYCOBIOLOGY LECTURE 31 MAY 6, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Center College of Medicine Oklahoma Center for Medical Glycobiology “Glycans in Parasitic Infections” Dr. Cummings

2. Outline • Background on Parasite Glycobiology • Protozoans • Malaria • Trypanosomiasis • Leishmaniasis. • Others • Nematodes • Trematodes • Schistosoma sp. • Glycobiology of Other Parasites Dr. Cummings

3. Parasites can be broadly defined as any organism that lives on or in another organism without benefiting the latter. By this definition parasites include viruses, bacteria, protozoans, and helminths (worms). However, in the vernacular, the term parasite is used exclusively to describe infectious protozoans and helminths. Dr. Cummings

4. Parasites Come in All Sizes Dr. Cummings

5. World-wide distrubiton of some major parasitic diseases The figures show the total number of infections worldwide (some individuals may have more than one infection) and the number of infection-related deaths per year. • Type of Disease Estimated Human Estimated Deaths • Infections Per Year • All helminths 4.5 billion ?>500,000 • Ascaris 1 billion 20 thousand • Hookworms 900 million 50-60 thousand • Trichuris 750 million ? • Filarial worms 657 million 20-50 thousand • Schistosomes 200 million 0.5-1.0 million • Malaria 489 million 1-2 million Dr. Cummings

6. Some of the major protozoal parasites of humans Parasite Comments • 1. Amoeba Infecting Humans • Entamoeba histolytica amebic dysentery, can cause liver abscesses • 2. Intestinal and Genital Flagellates • Giardia lamblia diarrhea - one of the most common parasites in North America • Trichomonas vaginalis inflammation of reproductive organs, very common • 3. Haemoflagellates • Leshmania donovani visceral Leshmaniasis (kala-azar), hepatosplenomegaly • L. mexicana fulminating, cutaneous ulcers • L. major cutaneous ulcers • Trypanosoma brucei sp. sleeping sickness humans &cattle (African Trypanosomiasis) • T. cruzi Chagas’ Disease (South American Trypanosomiasis) • 4. Gregarines, Coccidia and Related Organisms • Plasmodium falciparum major cause of human malaria • P. vivax, P. ovale, P. malariae also cause of human malaria • Causes of Opportunistic Infections • in Immune Deficiency States • Toxoplasma gondii causes muscle and intracellular pain • Pneumocystis carinii causes interstitial cell pneumonia • Cryptosporidium parvum intracellular parasites of intestinal cells resulting in diarrhea Dr. Cummings

7. Some of the major helminthic parasites of mammals Parasite Comments • 1. Trematodes • Blood flukes • Schistosoma mansoni human schistosomiasis (affects mesenteric veins draining large intestine) • S. haematobium human schistosomiasis (affects urinary bladder plexus) • S. japonicum human schistosomiasis (affects mesentery veins in the small intestine) • Liver Flukes • Fasciola hepatica primarily infects ruminants and occasionally humans, worms live in biliary tract • Clonorchis sinensis most prevalent liver fluke in humans, can be acquired by eating from raw fish • 2. Cestodes • Taenia solium long human tapeworm acquired by eating undercooked pork • Echinococcus granulosus shorter human tapeworm acquired by eating undercooked lamb, • parasitic cysts (hydatids) occur in liver and elsewhere • Taeniaarhynchus saginatus long human tapeworm acquired by eating undercooked beef • 3. Nematodes • Ascaris lumbricoides most common intestinal roundworm in humans • Trichurus trichuiura intestinal intestinal whipworm in humans • Enterobius vermicularis tiny intestinal roundworm, causes peri-anal night itch in children • Necator americanus intestinal hookworm of humans, cause anemia • Ancylostoma duodenale intestinal hookworm of humans, cause anemia • Strongyloides stercoralis intestinal parasite - causes autoinfection • Haemonchus contortus intestinal parasite of sheep and goats • Trichinella spiralis smallest nematode parasite of humans (trichinosis) residing • in muscle fibers, parasite acquired from uncooked pork • Onchocerca volvulus filarial parasite -causes river blindness • Wuchereria bancrofti filaria live in lymph nodes causing elephantiasis • Brugia malayi filaria live in lymph nodes causing elephantiasis • Dirofilaria immitis dog heartworm Dr. Cummings

8. Malaria Dr. Cummings

9. Life cycle of Plasmodium falciparum that causes Malaria 1. Sporozoites released from salivary gland of mosquito during blood meal - attach via heparan sulfate and enter liver cells of host 2. Development into the pre-erythrocytic stage 3. Cells rupture releasing merozoites 3. Merozoites attach and invade red blood cells via sialic acid recognition and enter within a vacuole 4. In the vacuole the merozoites transform intotrophozoites that digest hemoglobin to form the malarial pigment haemozoin 5. On maturation the trophozoite undergoes schizogony and forms daughter merozoites 6. After several schizogonic cycles, merozoites develop into sexually differentiated cells (male and female gametocytes). 7. Gametocytes ingested by mosquito during blood meal 8. In midgut of mosquito gametocytes become male microgametes and female macrogametes 9. Union of microgametes and macrogametes leads to zygote 10. The zygote is transformed into a ookinete that penetrates the intestinal wall and is transformed into a circular oocyst 11. Inside the oocyst the sporozoites develop from germinal cells known sporoblasts 12. The sporozoites emerge from the oocysts and migrate to salivary gland Dr. Cummings

11. Glycan-Based Vaccine to Block Toxicity of P. falciparum-derived Free GPI Glycans Dr. Cummings

12. Some major parasites and their carbohydrate-binding proteins. Parasite Stage Protein Specificity • Plasmodium merozoite EBA-175 Neu5Ac2,3Gal • falciparum • merozoite MSA-1 Neu5Ac? • sporozoite Circumsporozoite Heparan sulfate • protein • Trypanosoma trypo-mastigote Trans-sialidase Neu5Ac2,3Gal • cruzi • Penetrin Heparan sulfate • Entamoeba trophozoite Gal/GalNAc Lectin Gal/GalNAc • histolytica • 220 kD lectin chitotriose • 80 kD Hyaluronan • Giardia lamblia trophozoite taglin Man-6-phosphate • Cryptosporidium sporozoite Gal/GalNAc Gal/GalNAc • parvum lectin • Acanthamoeba 136 kDa Mannose- Man • keratitis Binding Protein Dr. Cummings

13. Leishmaniasis There are two forms of leishmaniasis. The more serious, called kala-azar or visceralleishmaniasis, affects the internal organs, causing fever, anemia, splenomegaly, and discoloration of the skin. Untreated, it can be fatal. The second, or cutaneous form, leaves deep, disfiguring sores at the site of the bite. Old World Cutaneous Leishmaniasis (OWCL) is caused by one of three main species of Leishmania protozoa: Leishmania tropica, Leishmania major and Leishmania aethiopica. These protozoan parasites invade the skin of the human leaving a characteristic scar. Visceral Leishmaniasis is primarily caused by Leishmania donovani. The parasite lives in human macrophages and is transported around the body by the lymphatic system. The parasite invades many internal structures (viscera) and can cause severe disease. Dr. Cummings

14. Leishmaniasis adult female Phlebotomus sp. (sand fly) Dr. Cummings

15. Leishmaniasis A macrophage filled with Leishmania amastigotes. Dr. Cummings

16. Leishmaniasis From Dr. Thomas Ilg Max-Planck-Institut für Biologie Dr. Cummings

17. Leishmaniasis • The disease is transmitted to humans through the bite of a sandfly infected with one of the species of Leishmania. • In the case of transmission of L.tropica and L.major to sandflies the main source of infection is desert rodents. • Sandflies are infected with L.aethiopica when they bite an infected hyrax. • L. tropica is usually transmitted to humans after the sandfly has first bitten an infected human. • L. major and L. aethiopica are mainly transmitted to humans after the sandfly has first bitten an infected animal • The basic disease of OWCL begins as a papule (mound) which develops at the site of the infected sandfly bite. • The papule enlarges and the tissue at the centre begins to die slowly. If the death of the centre is rapid this gives rise to a so-called "moist" ulcer such as of L. major. • If the centre dies slowly this gives rise to the "dry" ulcer of L. tropica. • The time between infection and the first appearance of the papule is usually a few weeks and the ulcers normally appear 2-3 months after infection. • The majority of untreated ulcers cure completely cure within 9 months and few exist longer than a year. Dr. Cummings

18. Leishmaniasis is a parasitic disease spread by the bite of the sandfly and can cause skin disease and systemic disease. The systemic form can be fatal, but treatment with antimony-containing compounds produces a high cure rate. Leishmaniasis . Cheek lesion where sand fly bite left parasites. The initial sore develops at the site of the bite and the infection spreads through the body (systemically) from that point. Dr. Cummings

19. Leishmaniasis Dr. Cummings

20. Leishmaniasis Some Lipophosphoglycans (LPGs) of Leishmania Species GPI-anchored LPG The LPG also occurs in mucin-like glycoproteins LPG side chains protein Dr. Cummings

21. Trypanosomiasis Life Cycle of Trypanosoma brucei gambiense Dr. Cummings

22. Trypanosomiasis Blood smear of infected patient (flagellated trypanosomes) tsetse fly of the genus Glossina (carrier of Trypanosomiasis) Dr. Cummings

23. Trypanosomiasis Trypanosomes are Covered with a Dense Array of a GPI-Anchored Protein Termed the Variant Surface Glycoprotein or VSG (each species of Trypanosomes differs in the modifications of the GPI core) Dr. Cummings

24. Trypanosomiasis The major surface molecules of T. brucei bloodstream and procyclic forms. The cartoons represent 20 nm Å~ 20 nm portions of plasma membrane. The blue components of the VSGs represent the two GPI anchors that attach the VSG dimers to the membrane. The mature GPI anchors of the procyclins have very complex side chains and some procyclins contain small N-linked oligosaccharides beyond the polyanionic rod domain, as shown here. From Ferguson (2000) Proc. Natl. Acad. Sci. U.S.A 97, 10673-10675 Dr. Cummings

25. Trypanosomiasis From Ferguson (2000) Proc. Natl. Acad. Sci. U.S.A 97, 10673-10675 The GPI biosynthetic pathways of T. brucei and mammalian cells. This is a consensus view based on the work of many groups, reviewed in refs. 11 and 23. (A) The shaded area represents the pathway in T. brucei procyclic cells and the nonshaded area represents the additional fatty acid remodeling steps and attachment to VSG unique to T. brucei bloodstream forms. (A and B) The location of the MT-III enzyme, encoded by the TbGPI10 and PIG-B genes in the parasite (A) and mammalian (B) cells, respectively, is indicated in red. Dr. Cummings

26. Life Cycle of Schistosomes Dissemination via fecal matter Transformation to schistosomula and shedding of tail (minutes) Adult worms Egg-laying Skin Residence (hours to days) Eggs Sexual Maturity-Pairing of Skin penetration of definitive host Vertebrate stage Penetration of Veins Migration Against Blood Flow to Inferior Mesenteric Veins Replicative stage in invertebrates Travel to Right Ventricle Miracidium Cercariae released from snail Hepatic Portal Vein Skin Penetration by Cercaria Travel to Pulmonary Capillaries (Lung stage (48-72 h) Residence in Liver Sinusoids (2 wks) Sporocyst (replication in snail) Penetration and infection of Biomphalaria snail (intermediate host) Travel to left Ventricle Enter Systemic Arterial Circulation Dr. Cummings

27. Patient with Hepatosplenic Disease Adult Schistosomes 6-14 mm long

28. Life Cycle of Schistosoma mansoni

29. Schistosomiasis Some Major Schistosome Glycans Known to be Highly Antigenic in Infected People and Animals Dr. Cummings

30. Schistosomiasis Expression of Glycan Antigens on the Surface of 3-h old Schistosomula of Schistosoma mansoni Dr. Cummings

31. Schistosomiasis Complement-mediated Killing of Schistosomula Using Monoclonal Antibody to LDN Propidium Iodide - A DNA-staining dye (Red Fluorescence) From Nyame et al (2003) Exp Parasitol. 104:1-13. Dr. Cummings

32. Schistosomiasis Flow Cytometric Analysis of Complement-mediated Killing of Schistosomula Using Monoclonal Antibody to LDN Propidium Iodide staining From Nyame et al (2003) Exp Parasitol. 104:1-13. Dr. Cummings

33. A protein conjugate vaccine candidate containing the LDN antigen for vaccination against schistosomiasis can be generated by chemo-enzymatic steps that remodel a core glycopeptide derived from a commercially available glycoprotein. Generation of Glycan Conjugates for Testing as Vaccine Candidates From Nyame et al (2004) Arch. Biochem. Biophys. In Press Dr. Cummings

34. A protein conjugate vaccine candidate containing the LDN antigen for vaccination against schistosomiasis can be generated by chemo-enzymatic steps that remodel a core glycopeptide derived from a commercially available glycoprotein. Generation of Glycan Conjugates for Testing as Vaccine Candidates From Nyame et al (2004) Arch. Biochem. Biophys. In Press Dr. Cummings

35. Induction of IgG responses to LDNF antigen in mice immunized with an LDNF-BSA conjugate Dr. Cummings

36. Examples of Parasite Glycans From Nyame et al (2004) Arch. Biochem. Biophys. In Press Dr. Cummings

37. Examples of Parasite Glycans From Nyame et al (2004) Arch. Biochem. Biophys. In Press Dr. Cummings

38. Examples of Parasite Glycans From Nyame et al (2004) Arch. Biochem. Biophys. In Press Dr. Cummings