Leishmaniasis

1. Leishmaniasis Silvia Reni B. Uliana

2. Leishmania FamilyTrypanosomatidae GenusLeishmania Sub-genera Leishmania Viannia More than 30 species already described cutaneous leishmaniasis visceral leishmaniasis

3. cutaneous leishmaniasis visceral leishmaniasis Geographical Distribution 14 million cases each year, distributedin 88 countries (350 million peopleat risk). http://www.who.int/emc-documents/surveillance/docs/whocdscsrisr2001.html

4. Visceral leishmaniasis / AIDS

5. Amastigote Reservoir Mammal bite bite Insect Promastigote Life cycle (MAN) Assexual

6. insect Promastigotes Extracellularforms living in the intestine of the mosquito In the mosquito’s gut they go through a differentiation process called metacyclogenesis.

7. Amastigote Intracellularforms “Ideal” host cell:macrophage Amastigotes are found inside macrophages at various sites: skin, lymph nodes,spleen, liver, bone marrow.

8. Os amastigotas são liberados no intestino do vetor e transformam-se em promastigotas procíclicos. Estes se reproduzem inicialmente por divisão binária e posteriormente se diferenciam em promastigotas metacíclicos. O hospedeiro vertebrado se infecta pela picada do vetor. Os promastigotas invadem macrófagos circulantes ou teciduais e se reproduzem como amastigotas. O vetor ingere macrófagos infectados com o sangue ingerido Mecanismo de infecção O macrófago parasitado eventualmente se rompe, liberando amastigotas que passam a infectar outros macrófagos, circulantes ou teciduais.

9. Clinical syndromes Cutaneous leishmaniasis – L. braziliensis, L. amazonensis, L. guyanensis L. major Diffuse leishmaniasis – L. amazonensis Mucocutaneous leishmaniasis – L. braziliensis Visceral leishmaniasis – L. chagasi, L. donovani New World species Old World species

10. Cutaneousleishmaniasis L. amazonensis L. mexicana L. guyanensis L. braziliensis Reservoir: wild rodents, marsupials (opossum), sloth, anteater

11. Diffuse leishmaniasis L. amazonensis Reservoir: wild rodents

12. Mucocutaneous leishmaniasis L. braziliensis Reservoir: ? Wild rodents

13. Visceral leishmaniasis L. chagasi L. donovani L. infantum Reservoir:fox, dog, humans

14. Some tricks performed by Leishmania S.C. Ilgoutz, M.J. McConville / International Journal for Parasitology 31 (2001) 899±908 906

15. Some tricks performed by Leishmania -LPG form McConville MJ in Molecular Biology of Parasitic Protozoa, ed. DF Smith, M Parsons multiplication inside the gut – adherence - procyclic being “available” to take the next ride - metacyclic

16. Immune system Complement C3bi Lysis complex O’Garra and Arai (2000) Trends Cell Biol.

17. the innate defense mechanisms… LPG and complement lysis complex

18. “getting home…” - the macrophage! Interaction with C3bi receptor via complement andgp63 inhibition of the respiratory burst burst

19. “shall we make it cosy?…” – the parasitophorous vacuole Fusion with lisossomes: gp63 is a protease (other cysteino-proteases) LPG and other GL induce the production ofTGF- and IL-10 AND… inhibit the production of NO “the macrophage army is ready…” DISEASE

20. Diagnosis • Biopsy – low sensitivity which species ? • Culture – slow isoenzymes, monoclonal antibodies PCR ELISA – cross-reaction, sensitivity

21. TOXICITY TIME RESISTANCE Treatment • pentavalent antimonials • anphotericin B

22. PATIENT - treatment VACCINE VECTOR – partially effective in urban areas CONTROL RESERVOIR - wild

23. What is the difference ? Why do they sometimes behave “badly” ? Clinical syndromes Cutaneous leishmaniasis – L. braziliensis, L. amazonensis, L. guyanensis L. major Diffuse leishmaniasis – L. amazonensis Mucocutaneous leishmaniasis – L. braziliensis Visceral leishmaniasis – L. chagasi, L. donovani New World species Old World species

24. Crithidia fasciculata L. donovani L. tarentolae C. fasciculata T. cruzi T. brucei 99 60 L. tarentolae 100 96 98 L. (L.) amazonensis 95 L. (L.) donovani L. (L.) chagasi 0.1 E. gracilis Briones et al 1992, MBP Orlando et al 2002 MIOC

25. AIDS Transplantation Persistence ? “reactivation” (atypical clinical picture) Immune-deficiency associated Primary infection (with or without symptoms) Where ? How ?

26. VIRULENCE Ilgoutz and McConville (2001) Int J Parasitol How do they do all this ?

27. L S 2.1 kb rRNA Characterisation of the meta 1 gene in L. major • Single copy gene • RNA expression upregulated in metacyclic promastigotes 10 20 30 40 L.maj MEMKNLLGKH KIVSVNGKPA PAGVTVEFKA SENSGSVYMH L.don ....H..... .V.L...R.. .......... G......H.. L.ama ....S.I..R RVL....R.. .......... .....TIQ.. 50 60 70 80 L.maj AKVANIMNGP LKLANRKLSG ALVSTMMLGS DDLMNIENAL L.don .R.......Q .R.E...... .......... .......... L.ama .N...F...Q ...G...... T......... .......... 90 100 110 L.maj SQGFMEGMTY TVKDGGKLTL QSKTHIIMLV PA* L.don .......... .......... .......K.. ..* L.ama I....D.... ..H....... K.N..T.K.. ..* • Gene conserved in both Old and New World Leishmania species

28. Deduced meta 1 aminoacid sequence • No significant aminoacid or nucleotide identity with any known protein. • No glycosilation sites. • Hydrophilicity plot with no distinguishing features. • Predicted molecular mass 11.95 kDa.

29. What is the function of the meta 1 protein in Leishmania ? A- Lines with double replacement of the meta 1 gene were not viable ESSENTIAL GENE

30. L.maj LaHE L S A L S A AI M kDa _ 18.4 _ 14.3 _ 6.2 B- Overexpressing the meta 1 protein • High level expression of the L. major meta 1 protein was obtained in L. amazonensis promastigotes, by stable maintenance of an episome. • The levels of expression were high in both log phase and stationary phase promastigotes.

31. C- Testing the recombinant lines for a phenotype • No change in growth curves • No differences in in vitro infection of macrophages

32. WT ARHyg Hyg3’ HE WT HE Meta 1 overexpressor in vivo BALB/c 107 promastigotes A 20 Lesion Index 15 10 5 4 6 8 10 12 14 16 Weeks B BALC/c 106 amastigotes 25 Lesion Index 20 15 10 5 4 6 8 10 12 14 16 Weeks C C57Bl/6 106promastigotes 6 Lesion index 5 WT HE 4 3 2 1 6 12 24 Weeks

34. L. major Chromosome 16