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

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Presentation on theme: "ESSENTIALS OF GLYCOBIOLOGY LECTURE 31 MAY 6, 2004 Richard D. Cummings, Ph.D. University of Oklahoma Health Sciences Center College of Medicine Oklahoma."— Presentation transcript:

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

5 Type of DiseaseEstimated Human Estimated Deaths InfectionsPer Year All helminths4.5 billion?>500,000 Ascaris 1 billion20 thousand Hookworms900 million50-60 thousand Trichuris750 million? Filarial worms 657 million20-50 thousand Schistosomes 200 million million Malaria 489 million1-2 million 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. Dr. Cummings World-wide distrubiton of some major parasitic diseases

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

7 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 sinensismost prevalent liver fluke in humans, can be acquired by eating from raw fish 2. Cestodes Taenia soliumlong human tapeworm acquired by eating undercooked pork Echinococcus granulosusshorter human tapeworm acquired by eating undercooked lamb, parasitic cysts (hydatids) occur in liver and elsewhere Taeniaarhynchus saginatuslong 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 vermicularistiny 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 volvulusfilarial parasite -causes river blindness Wuchereria bancroftifilaria live in lymph nodes causing elephantiasis Brugia malayifilaria live in lymph nodes causing elephantiasis Dirofilaria immitisdog heartworm ParasiteComments Some of the major helminthic parasites of mammals Dr. Cummings

8 Malaria Dr. Cummings

9 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 into trophozoites 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 Life cycle of Plasmodium falciparum that causes Malaria Dr. Cummings

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

12 Plasmodium merozoiteEBA-175Neu5Ac  2,3Gal falciparum merozoiteMSA-1Neu5Ac? sporozoiteCircumsporozoiteHeparan sulfate protein Trypanosomatrypo-mastigoteTrans-sialidaseNeu5Ac  2,3Gal cruzi PenetrinHeparan sulfate Entamoeba trophozoiteGal/GalNAc LectinGal/GalNAc histolytica 220 kD lectinchitotriose 80 kDHyaluronan Giardia lambliatrophozoitetaglinMan-6-phosphate Cryptosporidium sporozoiteGal/GalNAcGal/GalNAc parvum lectin Acanthamoeba 136 kDa Mannose-Man keratitis Binding Protein ParasiteStageProteinSpecificity Some major parasites and their carbohydrate-binding proteins. Dr. Cummings

13 There are two forms of leishmaniasis. The more serious, called kala- azar or visceral leishmaniasis, 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. Leishmaniasis Dr. Cummings

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

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

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

17 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. Leishmaniasis 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. 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. Leishmaniasis Dr. Cummings

19 Leishmaniasis Dr. Cummings

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

21 Life Cycle of Trypanosoma brucei gambiense Trypanosomiasis Dr. Cummings

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

23 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) Trypanosomiasis Dr. Cummings

24 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, Trypanosomiasis Dr. Cummings

25 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. From Ferguson (2000) Proc. Natl. Acad. Sci. U.S.A 97, Trypanosomiasis Dr. Cummings

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

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

28 Life Cycle of Schistosoma mansoni

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

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

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

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

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 Dr. Cummings From Nyame et al (2004) Arch. Biochem. Biophys. In Press

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 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 Dr. Cummings From Nyame et al (2004) Arch. Biochem. Biophys. In Press

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

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


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