ESSENTIALS OF GLYCOBIOLOGY

LECTURE 31

MAY 6, 2004

Richard D. Cummings, Ph.D.University of Oklahoma Health Sciences Center

College of MedicineOklahoma Center for Medical Glycobiology

“Glycans in Parasitic Infections”

Dr. Cummings

Background on Parasite GlycobiologyProtozoansMalariaTrypanosomiasisLeishmaniasis.OthersNematodesTrematodesSchistosoma sp.Glycobiology of Other Parasites

Outline

Dr. Cummings

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

Dr. Cummings

Parasites Come in All Sizes

Type of Disease Estimated Human Estimated Deaths InfectionsPer 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

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

1. Amoeba Infecting Humans Entamoeba histolytica amebic dysentery, can cause liver abscesses

2. Intestinal and Genital FlagellatesGiardia lamblia diarrhea - one of the most common parasites in North AmericaTrichomonas vaginalis inflammation of reproductive organs, very common

3. HaemoflagellatesLeshmania donovani visceral Leshmaniasis (kala-azar), hepatosplenomegalyL. mexicana fulminating, cutaneous ulcersL. major cutaneous ulcersTrypanosoma brucei sp. sleeping sickness humans &cattle (African Trypanosomiasis)T. cruzi Chagas’ Disease (South American Trypanosomiasis)

4. Gregarines, Coccidia and Related OrganismsPlasmodium falciparum major cause of human malariaP. vivax, P. ovale, P. malariae also cause of human malaria

Causes of Opportunistic Infections in Immune Deficiency StatesToxoplasma gondii causes muscle and intracellular painPneumocystis carinii causes interstitial cell pneumonia Cryptosporidium parvum intracellular parasites of intestinal cells resulting in diarrhea

Parasite Comments

Some of the major protozoal parasites of humans

Dr. Cummings

1. TrematodesBlood flukesSchistosoma 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 FlukesFasciola hepatica primarily infects ruminants and occasionally humans, worms live in biliary tractClonorchis sinensis most prevalent liver fluke in humans, can be acquired by eating from raw fish

2. CestodesTaenia solium long human tapeworm acquired by eating undercooked porkEchinococcus granulosus shorter human tapeworm acquired by eating undercooked lamb,

parasitic cysts (hydatids) occur in liver and elsewhereTaeniaarhynchus saginatus long human tapeworm acquired by eating undercooked beef

3. NematodesAscaris lumbricoides most common intestinal roundworm in humansTrichurus trichuiura intestinal intestinal whipworm in humansEnterobius vermicularis tiny intestinal roundworm, causes peri-anal night itch in childrenNecator americanus intestinal hookworm of humans, cause anemiaAncylostoma duodenale intestinal hookworm of humans, cause anemiaStrongyloides stercoralis intestinal parasite - causes autoinfectionHaemonchus contortus intestinal parasite of sheep and goatsTrichinella spiralis smallest nematode parasite of humans (trichinosis) residing

in muscle fibers, parasite acquired from uncooked porkOnchocerca volvulus filarial parasite -causes river blindnessWuchereria bancrofti filaria live in lymph nodes causing elephantiasisBrugia malayi filaria live in lymph nodes causing elephantiasisDirofilaria immitis dog heartworm

Parasite Comments

Some of the major helminthic parasites of mammals

Dr. Cummings

Malaria

Dr. Cummings

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 stage3. Cells rupture releasing merozoites3. Merozoites attach and invade red blood cells via sialic acid recognition and enter within a vacuole4. In the vacuole the merozoites transform into trophozoites that digest hemoglobin to form the malarial pigment haemozoin5. On maturation the trophozoite undergoes schizogony and forms daughter merozoites6. After several schizogonic cycles, merozoites develop into sexually differentiated cells (male and female gametocytes).7. Gametocytes ingested by mosquito during blood meal8. In midgut of mosquito gametocytes become male microgametes and female macrogametes9. Union of microgametes and macrogametes leads to zygote10. The zygote is transformed into a ookinete that penetrates the intestinal wall and is transformed into a circular oocyst11. Inside the oocyst the sporozoites develop from germinal cells known sporoblasts12. The sporozoites emerge from the oocysts and migrate to salivary gland

Life cycle of Plasmodium falciparum that causes Malaria

Dr. Cummings

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

Dr. Cummings

Plasmodium merozoite EBA-175 Neu5Ac2,3Galfalciparum

merozoite MSA-1 Neu5Ac?

sporozoite Circumsporozoite Heparan sulfate protein

Trypanosoma trypo-mastigote Trans-sialidase Neu5Ac2,3Galcruzi

Penetrin Heparan sulfate

Entamoeba trophozoite Gal/GalNAc Lectin Gal/GalNAchistolytica

220 kD lectin chitotriose

80 kD Hyaluronan

Giardia lamblia trophozoite taglin Man-6-phosphate

Cryptosporidium sporozoite Gal/GalNAc Gal/GalNAcparvum lectin

Acanthamoeba 136 kDa Mannose- Mankeratitis Binding Protein

Parasite Stage Protein Specificity

Some major parasites and their carbohydrate-binding proteins.

Dr. Cummings

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

adult female Phlebotomus sp. (sand fly)

Leishmaniasis

Dr. Cummings

A macrophage filled with Leishmania amastigotes.

Leishmaniasis

Dr. Cummings

From Dr. Thomas IlgMax-Planck-Institut für Biologie

Leishmaniasis

Dr. Cummings

• 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

.

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

Leishmaniasis

Dr. Cummings

Some Lipophosphoglycans (LPGs) of Leishmania Species

Leishmaniasis

The LPG also occurs in mucin-like glycoproteins

GPI-anchored LPG

protein

LPG side chains

Dr. Cummings

Life Cycle of Trypanosoma brucei gambiense

Trypanosomiasis

Dr. Cummings

tsetse fly of the genus Glossina (carrier of Trypanosomiasis)

Blood smear of infected patient(flagellated trypanosomes)

Trypanosomiasis

Dr. Cummings

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)

2Man1, 2Man1

PO3-CH2-CH2- - -NH CO Protein

Man1,4GlcNH21

R2

R3R4

6

6 4 3 2

R1

NH3+

In .T brucei ; VSG R1, R2 , and R24 = , OH R3 = Gal0-5 and the lipid is a diacyl-glycerolIn .T cruzi 1 7; G R1 = , 2, Man R R3 and R4 = OH and the lipid is undefined In . L major ; PSP R1, R2, R3 and R4 = /OH and the lipid is alkylacyl-glycerol

-

6 -myoInositol-1 PO3

Diacylglycerol Plasma Membrane

-

Inside

Outside

Trypanosomiasis

Dr. Cummings

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

Trypanosomiasis

Dr. Cummings

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, 10673-10675

Trypanosomiasis

Dr. Cummings

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 ininvertebrates

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 PulmonaryCapillaries (Lung stage

(48-72 h)

Travel to leftVentricle

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

Adult SchistosomesPatient with

Hepatosplenic Disease

6-14 mm long

Life Cycle of Schistosoma mansoni

Some Major Schistosome Glycans Known to be Highly Antigenic in Infected People and Animals

Schistosomiasis

Dr. Cummings

Expression of Glycan Antigens on the Surface of 3-h old Schistosomula of Schistosoma mansoni

Schistosomiasis

Dr. Cummings

Complement-mediated Killing of Schistosomula Using Monoclonal Antibody to LDN

Propidium Iodide - A DNA-staining dye (Red Fluorescence)

Schistosomiasis

Dr. CummingsFrom Nyame et al (2003) Exp Parasitol. 104:1-13.

Flow Cytometric Analysis of Complement-mediated Killing of Schistosomula Using Monoclonal Antibody to LDN

Propidium Iodide staining

Schistosomiasis

Dr. CummingsFrom Nyame et al (2003) Exp Parasitol. 104:1-13.

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. CummingsFrom Nyame et al (2004) Arch. Biochem. Biophys. In Press

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

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

Induction of IgG responses to LDNF antigen in mice immunized with an LDNF-BSA conjugate

Dr. Cummings

Examples of Parasite Glycans

Dr. Cummings

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

Examples of Parasite Glycans

Dr. Cummings

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

Examples of Parasite Glycans

Dr. Cummings

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