Malaria in the Mosquito Senior Director Quality Systems and Entomology Research Sanaria Inc. Rockville MD. 1 Malaria: one of the world s foremost killers Every year 1 million children die of malaria 250 million people become ill with malaria At least $12B annually in economic activity (GDP) is lost due to malaria in Africa alone At least 30,000 international travelers and military personnel fall ill to malaria annually Billingsley - malaria in the mosquito 2 Malaria P. falciparum responsible for more deaths in children in the world than any other single infectious agent 3000-5000 children will die today of malaria, 1-3 million in the next year 3 The screen versions of these slides have full details of copyright and acknowledgements 1
Malaria endemic countries Malaria transmission occurs throughout Malaria transmission occurs in some places Malaria transmission is not known to occur Malaria in areas where conditions allow parasite multiplication in the vector Restricted to tropical and subtropical areas, altitudes below 1,500 m Distribution might be affected by climatic changes, especially global warming, and population movements Plasmodium falciparum and P. malariae are encountered in all shaded areas Plasmodium vivax and P. ovale occupy complementary niches, P. ovale in Sub-Saharan Africa, P. vivax in the other areas 4 5 On some peculiar pigmented cells found in two mosquitos fed on malarial blood Surgeon-Major Ronald Ross, British Medical Journal, December 1897 For the last two years I have been endeavouring to cultivate the parasite of malaria in the mosquito. The method adopted has been to feed mosquitos, bred in bottles from the larva, on patients having crescents in the blood, and then to examine their tissues for parasites similar to the haemamoeba in man. The study is a difficult one, as there is no a priori indication of what the derived parasite will be like precisely, nor in what particular species of insect http://resources.bmj. the experiment will be successful, while the investigation requires com/bmj/readers/bac k-ssues-and-archive a thorough knowledge of the minute anatomy of the mosquito. Hitherto the species employed have been mostly brindled and grey varieties of the insect; but though I have been able to find no fewer than six new parasite of the mosquito, namely a nematode, a fungus, a gregarine, a sarcosporidium (?), a coccidium (?), and certain swarm spores in the stomach, besides one or two doubtfully parasitic forms, I have not yet succeeded in tracing any parasite to the ingestion of malarial blood, nor in observing special protozoa in the evacuations due to such digestion. 6 The screen versions of these slides have full details of copyright and acknowledgements 2
The Sporogonic cycle Human Liver Stages Gametocytes, male microgametocytes female Liver cell Infected Mosquito Stages (macrogametocytes) liver cell Ingested by Anopheles mosquito Ruptured oocyst Mosquito takes during a blood meal a blood meal Exo-erythrocytic Cycle (injects sporozoites) In the mosquito midgut Oocyst Release of sporozoites Microgametocytes Ruptured schizont undergo exflagellation (emergence from RBC) Schizont Microgametes penetrate Sporogonic cycle the macrogametes zygotes Human Blood Stages Zygotes become motile Immature trophozoite Ookinete and elongated ookinetes (ring stage) Mosquito takes a blood meal Ookinetes which invade Microgametocyte (injects gametocytes) the midgut wall of the mosquito where they develop Erythrocytic into Cycle oocysts Mature Microgamete entering Oocysts grow, rupture, trophozoite macrogamete and release sporozoites Exflagellated Sporozoites Ruptured make their way to salivary glands macrogamete schizont Inoculation of the sporozoites Schizont into a new Gametocytes = Infective Stage P. vivax human host perpetuates the malaria Gametocytes life cycle = Diagnostic Stage P. ovale P. malariae From - http://www.dpd.cdc.gov/ 7 Malaria in mosquito The mosquito midgut Exflagellation Ookinete recognition and penetration of the mosquito midgut Mosquito immune responses The midgut and oocyst formation Sporozoites in the salivary glands The dynamics of malaria infections in the mosquito 8 Malaria in the mosquito 9 The screen versions of these slides have full details of copyright and acknowledgements 3
The mosquito midgut 10 11 Mosquito midgut morphology 12 The screen versions of these slides have full details of copyright and acknowledgements 4
Midgut morphology - digestive cells Single cell layered epithelium Apical plasma membrane folded into microvilli Lots of synthetic and secretory machinery (RER, Golgi, vesicles) Midgut lumen Peritrophic Matrix Nuclei each with notable nucleolus Baso-lateral plasma membrane folded into basal labyrinth -water removal Cells rest on continuous basal lamina Cells separated from lumen by peritrophic matrix Basal lamina Haemocoel 13 Anopheles midgut ultrastructure Lumen Microvilli Apical vesicles Nucleus Epithelial cell Basal lamina Muscle layer 14 Microvilli and peritrophic matrix Anopheles stephensi Peritrophic matrix (mostly carbohydrate chitin/glcnac) Microvilli 15 The screen versions of these slides have full details of copyright and acknowledgements 5
16 Water removal from midgut Mosquito Naturietic Peptide targets malpighian tubules Water Na + Na + Water Bloodmeal in Water Na + Na + MNP stimulates Na + channels on BLM 17 Microvilli and peritrophic matrix Anopheles stephensi Peritrophic matrix (mostly carbohydrate chitin/glcnac) Microvilli 18 The screen versions of these slides have full details of copyright and acknowledgements 6
Peritrophic matrix composition Chitin (poly-b-(1,4)-n-acetyl-d-glucosamine) Major carbohydrate structure (but not so evident in Anopheles) Proteoglycans Core linear proteins with multiple polysaccharide side chains Highly charged, bulk structures Proteins Little known May have chitinase or chitin synthase activities Ae-Aper50 - Chitin-binding protein from Aedes aegypti Ag-Aper14 - Chitin-binding protein from An. gambiae 19 Localisation of Ae-Aper50 & Ag-Aper14 Before feeding After feeding 20 Anopheles gambiae aminopeptidase 1 (AgAPN1) expression and recognition by α-agapn1 PAbs Dinglasan R R et al., PNAS 2007; 104: 13461-13466 2007 by National Academy of Sciences 21 The screen versions of these slides have full details of copyright and acknowledgements 7
Digestive enzymes Primary hydrolases N Endopeptidases Cleave proteins into peptides Alkaline: Trypsin, Chymotrypsin Secondary hydrolases Cleave single or paired (rare) amino acids from end of peptides Carboxypeptidase: can be acid or alkaline types Aminopeptidases Protein in bloodmeal Primary hydrolases C 22 Spatial separation of enzymes A B Abdomen cross section Trypsin and chymotrypsin released from cells into edge of midgut Digest into bloodmeal Aminopeptidase and carboxypeptidase released but AP also remains on microvilli Endo Peritrophic Space Peritrophic Matrix High zone of enzymes at periphery of gut Ecto Peritrophic Space Large molecules do not pass PM, Midgut Epithelium therefore gradient for absorption Haemocoel PM may act as semi-permeable barrier Salivary gland 23 Life and death in the mosquito Vaughan 2006, Trends Parasitol 23: 63-70 24 The screen versions of these slides have full details of copyright and acknowledgements 8
Anopheles midgut basal lamina Collagen IV Perlecan Laminin Nidogen 25 Exflagellation 26 Exflagellation (2) Emergence of microgametes from microgametocyte Three rounds of replication of its nuclear DNA in ten minutes Eight-fold replication Eight thread-like male gametes formed Takes place in blood meal of mosquito Occurs within 20 minutes of blood feeding Ubiquitous - all parasites in practically all mosquitoes 27 The screen versions of these slides have full details of copyright and acknowledgements 9
Factors triggering exflagellation Occurs spontaneously after exposure to air temperature (2-3 o C) pco 2 ph (8-8.3) Xanthurenic acid 28 Ookinete 29 Plasmodium berghei ookinetes 30 The screen versions of these slides have full details of copyright and acknowledgements 10
In vitro invasion Ookinetes move on cell surface Ookinetes invade cells indistinguishable from others by microscopy http://www.pnas.org/cgi/content/full/97/21/11516/dc1?maxtoshow=&hits=10&hits=10&resultformat= &fulltext=plasmodium+movie&searchid=1079952085525_1793&stored_search=&firstindex=0 31 In vitro invasion (2) Ookinetes burrow deep into the cell Midgut cell becomes damaged and bursts open 32 Chitinase in ookinete Chitinase localises to the ookinete surface and micronemes 33 The screen versions of these slides have full details of copyright and acknowledgements 11
Ookinete invading PM High trypsin zone Peritrophic matrix Prochitinase released at cell surface Chitinase active here Ookinete Midgut Cell 34 α-agapn1 antibodies block plasmodium transmission to the mosquito Feed antibodies with parasites to the mosquito Antibodies recognize AgAPN1 Wait 6-9 days and count oocysts to see how many ookinetes made it through the cells If the concentration of antibodies is high enough, get >80% inhibition of infection Dinglasan R R et al., PNAS 2007; 104: 13461-13466 35 Ookinetes in the midgut Ookinete interactions with the mosquito midgut Ookinetes have very intimate interaction with the midgut wall A set of surface molecules (Pfs25, Pfs 48/45) present on the ookinete surface and are important in invasion These proteins, as well as AgAPN1, are targets of Transmission Blocking Vaccines - vaccines that block transmission of malaria through the mosquito but do not protect the individual from infection or disease Role of molecules in parasite biology and vaccine candidates identified by gene knock out 36 The screen versions of these slides have full details of copyright and acknowledgements 12
CTRP KO - loss of infectivity to mosquito Circumsporozoite and TRAP-related protein Thought to be involved in gut invasion Looked for oocysts No oocysts in CTRP knock-outs B EMBO J. 1999 Nov 15; 18 (22): 6221-7 KO WT 37 In vitro invasion Ookinetes burrow deep into the cell Midgut cell becomes damaged and bursts open 38 The time bomb theory Midgut cells responding with an immune peptide do not co-localise with invaded ookinetes But they do express V-ATPase and NOS: typical of damaged cells showing a programmed cell death response Cells associated with invading ookinetes bulge out of the epithelium - damaged/killed 39 The screen versions of these slides have full details of copyright and acknowledgements 13
The time bomb theory of ookinete invasion Ookinetes use a protease - Subtilisin 2 to invade cells The cell responds to invasion by upregulating inos and releasing NO NO in turn up-regulates the production of peroxidases; Both are produced to kill the ookinete but The cell becomes damaged, dies and buds off into the lumen The epithelium repairs using an actin ring Oocysts form below cells in a healthy, repaired epithelium 40 The time bomb theory of ookinete invasion (2) Time See: Han, Y.S., et al., 2000, EMBO J. 19: 6030-6040 Kumar S., et al., 2003, Proc. Natl. Acad. Sci. USA 100: 14139-14144 Han, Y.S. and Barillas-Mury, C. 2002, Insect Biochem. Molec. Biol. 32: 1311-1316 41 Oocyst 42 The screen versions of these slides have full details of copyright and acknowledgements 14
Plasmodium berghei: oocysts 43 Ookinete to oocyst transfromation Meis et al., 1987, Parasitol Res. 73: 500-506 44 Collagen IV expressed during oocyst growth Collagen expression oportion of S7 expression C as a pro 6h 24h 48h 72h 5d 8d 12d 15d 21d Time post feeding Collagen IV essential component of basal lamina Helps epithelium maintain polarity Expressed as oocyst grows away from the epithelial wall Expressed in older (ca. 25 days) mosquitoes 45 The screen versions of these slides have full details of copyright and acknowledgements 15
Transmission electron micrograph of immunogold labelling of laminin in a P. berghei oocyst 14 days post-infection; A) Oocyst resting upon the basal lamina B) Higher magnification image of A. Laminin is found close to the membranes of, or within, developing sporozoites (arrows); Laminin is also present on basal lamina around the midgut and individual muscles blocks Me = midgut epithelium, BL = basal lamina, Sp = sporozoites, Cap = capsule, Mu = muscle Nacer et al., Parasites & Vectors 2008, 1: 33 doi:10.1186/1756-3305-1-33 46 Interaction between plasmodium and mosquito midgut basal lamina Nacer et al., Parasites & Vectors 2008, 1: 33 doi:10.1186/1756-3305-1-33 47 Insect immunity 48 The screen versions of these slides have full details of copyright and acknowledgements 16
Mosquito targets the parasite Parasite interactions with the mosquito immune system Malaria is susceptible to immune attack from the mosquito Immune proteins are poorly specific, usually causing damage by punching holes in the parasite membrane, e.g. SHIVA-3, or agglutinating the parasites to prevent their movement Thus- parasite in general is susceptible to inhibition of invasion and to killing 49 Insect immunity Encapsulating strain heavily studied Set of 46 genes in Pen1 (Plasmodium encapsulting) region of A. gambiae genome Other families of genes described based on gene structure - e.g., complement-like thioester found from cell line family in genome TEP1 secreted around the ookinete but is only produced by hemocytes 50 Sporozoite 51 The screen versions of these slides have full details of copyright and acknowledgements 17
Sporozoites in salivary glands Around 20% of sporozoites make it to salivary glands At the salivary glands, sporozoites 1. Attach to the basal lamina 2. Invade the space between the basal lamina and the basal epithelial cell plasma membrane, a process associated with the loss of the sporozoite's s thick coat 3. Penetrate into the salviary gland cell and reside within a vacuole 4. Escape through the apical cell membrane 5. Enter the secretory cavity 6. Release themselves from mosquito cell membrane by an unknown mechanism 7. Assemble into bundles within the secretory cavity 8. Enter the secretory duct to be deposited into the host with saliva Current opinion in microbiology 2009, 12: 394-400 BL: Basal Lamina DW: Duct Wall N: Nucleus; n: nucleolus PM: Plasma Membrane SC: Secretory Cavity 52 A sporozoite based malaria vaccine No transmission to mosquito No late liver stage infection No blood stage infection 53 Pf IRR SPZ-Pf challenges (5-14 infected mosquitoes) # Immunizing bites # Protected/ # Challenged # Protected/ # Challenges > 1000 immunizing bites 33/35 1 st challenge 13/14 (93%) 13/14 (93%) Re-challenge <10 wk 6/6 (100%) 15/15 (100%) Re-challenge 23-42 wk 5/6 (83%) 5/6 (83%) <1000 immunizing bites 5/15 54 The screen versions of these slides have full details of copyright and acknowledgements 18
A sporozoite vaccine Limited studies 35 challenges in 14 people Protective immunity as good as protective immunity of any vaccine for any indication Currently being developed, manufactured and testing in clinical trials (Sanaria) Hoffman et al., JID, 2002; Luke and Hoffman, J Exp Bio, 2003 55 Dynamics of malaria in the mosquito 56 Life and death in the mosquito (a) (b) Parasite number (log scale) Time since gametocyte ingestion Trends in parasitology Baton & Ranford-Cartwright 2005, Trends Parasitol 22: 574-580 57 The screen versions of these slides have full details of copyright and acknowledgements 19
Appetite is reduced during sporogonic cycle Anderson et al., 1999, Proc Roy Soc B 266: 1729-33 58 Malaria in the mosquito 59 60 The screen versions of these slides have full details of copyright and acknowledgements 20