Presentation on theme: "Leishmania www.sanger.ac.uk/Info/Press/2007/070617.shtml."— Presentation transcript:
Leishmania zLife cycle and transmission ecology of Leishmania zThree clinical syndromes caused by these parasites zUnderstanding host-pathogen interaction at the molecular level - LPG and its role for survival in the insect vector
The phylum Kinetoplastida zGroup of protozoans at the basis of the eukaryotic tree zMove using a single flagellum zHarbor name-giving mitochondrion with large genome which is always associated with the basal body of the single flagellum zAll kinetoplastids are parasites zTrypanosoma & Leishmania are the medically important and best studied genera in this group (but a huge variety of animals and even plants harbor kinetoplastid parasites)
Leishmania belong to the order kinetoplastida zDifferent life cycle stages in this group show characteristic morphological differences and are distinguished by the position of the basal body within the cell zHowever, the differences are not limited to morphology zDifferent stages express different set of genes from their genomes which adapt their surface, internal structure and metabolism to survival in insect or mammalian hosts trypomastigote epimastigote promastigote amastigote
Leishmania parasites exist as pro- and amastigotes zThe parasite lives in the digestive tract of sandflies as extracellular promastigote zIn the mammalian host parasites multiply as intracellular amastiogotes
procyclics and metacyclics zInfected macrophages are taken up with the blood meal and amastigotes are released by digestion, transform into procyclic promastigotes and attach to the midgut epithelium zAttached promastigotes divide rapidly (procyclics are not infective to mammals) zMetacyclic (infective) promastigotes cease replication, detach and pass forward into the pharynx from where they are regurgitated into the bite site (attached) (detached)
Leishmania infects and thrives in macrophages zWithin its mammalian host Leishmania infects macrophages and replicates as intracellular amastigoe zMacrophages are “professional” phagocytic cells and an important part of the innate immune system (they also play a role in initiating the adaptive response by antigen presentation) zThe parasite invades its host cell passively by triggering phagocytosis
Leishmania infects and thrives in macrophages Uptake of Leishmania amazonensis metacyclic promastigote by a mouse macrophage. The parasite is phagocytosed with the cell body entering first and through the formation of a long tubular pseudopod. Images were captured every 0.5 seconds over the course of 367 seconds. Courret et al
Leishmania infects and thrives in macrophages Phagocytosis of a Leishmania amazonensis metacyclic promastigote by a mouse macrophage. The parasite binds to the macrophage plasma membrane by the tip of the flagellum. It then turns around and is finally ingested via the cell body. Images were captured every 0.5 seconds over the course of 125 seconds. Courret et al
Leishmania infects and thrives in macrophages zLeishmania stimulates this process by binding elements of the complement system to its surface zBinding of complement can destroy pathogens but also tags them for phagocytosis (opsonisation: pathogen bound 3Cb is a potent ‘eat me’ signal for macrophages & neutrophils) zHowever, the parasite prevents the formation of the fully functional membrane attack complex zA molecule on the surface of the parasite seems to be responsible both for complement activation and prevention of the final attack
Leishmania infects and thrives in macrophages zOnce a pathogen is taken up by phagocytosis the phagosome usually fuses with primary lysosomes to form secondary lysosomes zWithin secondary lysosomes pathogens are killed and digested by the action of lytic enzymes and an acidic environment produced by membrane proton pumps zAdditional mechanisms of killing include the generation of oxygen radicals (‘biological bleach’)
Leishmania infects and thrives in macrophages zMacrophages are important “microbe killers”, however several pathogens have found ways to escape killing zTrypansoma cruzi -- induces phagocytosis but then escapes into the cytoplasm zToxoplasma -- active invasion, parasitophorous vacuole is never part of the endocytic pathway zMycobacterium tuberculosis -- induce phagocytosis and block lysosomal maturation zLeishmania...
Leishmania infects and thrives in macrophages z… Leishmania just doesn’t seem to care zAmastigotes thrive in what looks like a fully matured lysosome with acidic pH and abundant lysosomal hydrolases zAmastigotes rapidly divide and will infect new macrophages after rupture of host cell zThe dense surface coat covering Leishmania seems to protect the parasite from the action of the lytic enzymes zHowever, with help from T cells macrophages can be stimulated to kill the parasite
A TH1 response is required for parasite control and healing zStimmulation with different cytokines leads to the development of two types of T-cells specialized for different immune responses zTh1 and Th2 strongly downregulate each other zThis polarization has important consequences for the downstream response and can spell life or death zNon healing Leishmania infections are characterized by a strong TH2 response (remember this was the response useful to get rid of worms by antibody and hypersensitivity) zHealing infections are characterized by TH1 zThe parasites seems to manipulate this balance in his favor, we don’t understand yet how that is done
Leishmania distribution (all species)
American soldiers contracted Leishmaniasis in Iraq & Afghanistan A REGION INFLAMED: Hundreds of U.S. Troops Infected by Parasite Borne by Sand Flies, Army Says By DONALD G. MCNEIL JR. Published: December 6, 2003 Hundreds of American troops in Iraq have been infected with a parasite spread by biting sand flies, and the long-term consequences are still unknown, Army doctors said Friday … Skin Disease Strikes Iraqi Children Thursday, February 14, 2008 By MARIA CHENG, AP Medical Writer LONDON — At least 275 children in southern Iraq have been infected with a disfiguring skin disease, an outbreak some health officials are blaming on the war's devastating effect on the public health system.
Leishmania is transmitted by sand flies (Phlebotomidae) zSand flies are minute diptera (they are more closely related to mosquitoes than “real” flies and only females bite) zOnly mosquito nets with fine meshwork hold them off zIn the wild sand flies often breed in rodent burrows zOld world Phlebotomus, new world Lutzomya zCan also transmit Bartonella bacilliformis and Papatsi virus
Ecology of old world Leismaniasis zClose contact of humans and their domestic animals can provide optimal conditions for sand flies and Leishmania transmission (stables provide good breeding ground for larvae) zIn urban environments infection is mostly human to human zIn rural areas Leishmaniasis can be a zoonosis zInfection in dogs is quite frequent in the Mediterranean
Ecology of new world leishmaniasis zIn the new world most people get infected while working or hunting in the forest zHere wild animals including rodents, monkeys and sloths provide a reservoir for the parasite zA transmission pattern within a population of wild animals that result in occasional infection of humans is called sylvatic
A variety of species and species complexes causes disease in humans Visceral Leishmaniasis Leishmania donovani Leishmania infantum Leishmania chagasi Cutaneous Leishmaniasis Leishmania tropica Leishmania major Leishmania aethiopica Mucocutaneous Leishmaniasis Leishmania brazieliensis Leishmania mexicana Leishmania amazoniensis
Three syndromes associated with Leishmania infection in humans
Visceral Leishmaniasis or Kala Azar zSystemic infection of reticulo-entdothelial cells (mostly macrophages) throughout multiple internal organs and the blood
Kala Azar - Visceral Leishmaniasis zCaused by the L. donovani complex zGeneral infection of macrophages in the entire RES zWeeks to months incubation period zThe lead symptom is abdominal swelling due to hepato- and splenomegaly zHigh fever. Fever often oscillates with a peak every second day zProgressive drastic weight loss (kachexia) zDarkening of the skin zMortality of untreated disease %
Post Kala-Azar Dermal Leishmaniasis zSequel of visceral leishmaniasis which may manifest years after successful treatment and resolution of Kala Azar. zDermal lesions may contain parasites in great numbers
Cutaneous Leishmaniasis zInfection remains restricted to the initial site of infection (the bite site)
Cutaneous Leishmaniasis is usually self-limiting zOld world oriental sore is caused by parasites of the L. tropica complex. (similar disease in the new world is caused by L. mexicana) zA chronic but self-limiting dry ulceration at the site of the bite zUlceration starts months after infection zParasites are not found outside the lesion zA granuloma is formed which finally leads to healing leaving a depressed scar zNearly absolute resistance to reinfection zInoculation to vaccinate has long been practiced in the middle east
Espundia or mucocutaenous leishmaniasis zCaused by L. braziliensis z~20% of infected patients develop ulcers of the oral and nasal mucosa
Espundia or mucocutaenous leishmaniasis zCaused by L. braziliensis z~20% of infected patients develop ulcers of the oral and nasal mucosa zProgression of the ulceration is slow but steady, ultimately destroying all soft parts of the nose, the lips, and the soft palate zDeath can occur through secondary bacterial infection
Diagnosis & Treatment zGold standard of diagnosisis demonstration of parasites in the blood or lymph (kala azar) or in scrapings of the ulcer (cutaneous versions) zTreatment for all forms of Leishmaniasis is not satisfactory at the moment zVisceral Leishmaniasis (pentavalent antimonials) cutaneous (amphotheracin B, a liposome formulation is better tolerated but much more expensive) zThe available drugs have significant side effects and resistance has emerged (pentavalent antimonials) zHIV coinfection further complicates treatment zBetter and more affordable drugs are urgently needed
A not-for-profit drug company? zNew hope for better treatment includes paramomycin an old off- patent antibiotic which has shown promising activity in studies conducted by the Institute of One World Health zParamomycin interferes with the growth of a number of bacteria and protozoa (the mode of action is not fully understood) zIntravenous injection has shown significant cure in kala azar patients in India (this was the first large scale clinical trial for this disease) zOne World Health develops this drug for kala azar together with an indian drug maker and the indian health authorities z (http://www.oneworldhealth.org)
procyclics and metacyclics
Parasite interacts with the sand fly midgut zPromastigotes attach and detach to the midgut epithelium zAttachment helps them to remain in the insect gut when the blood meal is passed zHowever, later they need to detach to move to the pharynx and proboscis for infection zHow is this accomplished at the molecular level?
Lipophosphoglycans play important roles in Leishmania pathogenesis zLipophosphglycan or LPG is the dominant molecule on the surface of Leishmania zLPG is not a protein but a glycolipid zThe molecule is anchored in the membrane by a lipid to which a long chain of highly hydrophilic sugar-phosphate repeats are attached zThe structure of LPG changes over the life cycle to adapt to various functions zLPG is a pathogenesis factor in the mammalian host and important for the life cycle within the sandfly
Structural modification of LPG during the sand fly cycle zLPG is structurally modified during the developmental process of metacylogenesis (parasites decide to stop replicating and begin to pre- adjust to the mammalian host) zLPG in metacylics has 2-3 times the number of repeat units zSidechains with terminal galactose (squares) are downregulated and the remaining galactose residues are capped with another terminal sugar (arabinopyranose) zAre these chemical changes responsible for the pathogens attachment phenotype?
Only LPG from procyclics attaches to the midgut zPhosphoglycans were isolated from procyclics (those that attach) and metacyclics (those that detach and infect) and were labeled with a dye (resulting in fluorescence seen as ‘white’ in the lower panels) zOpened sandfly midguts were incubated with PG from procyclics (A/B) and metacyclics (C/D) and detected with an antibody zOnly procyclic phosphoglycan binds to the midgut
LPG binds to the microvilli of the sandfly midgut epithelium OK, LPG appears to be the molecule on the parasite responsible for the attachment of parasites to the sandfly midgut, but what kind of host molecule does it bind to?
LPG binds to a species specific galectin in the sandfly midgut zA gene for an abundantly expressed galactose binding protein or lectin (galectin) was identified in a sandfly sequencing project zA specific antibody against the galectin reacts with the midgut of the sandfly species from which it was isolated (but not from other species) zHigh resolution microscopy shows that the protein(red in lower panel) is found on the luminal side of the midgut epithelium Cell 119:329-41
LPG binds to a species specific galectin in the sandfly midgut zGalectin (labeled with a fluorescent dye) binds specifically to procyclic Leishmania major parasites zHowever little binding is detected when incubated with metacyclic parasites (the stage that detaches and moves to the the proboscis to infect the mammalian host, V1met) zThere is little binding to a mutant parasite (Spock) which lacks LPG z(B lower) anti-galectin also blocks binding of procyclic parasites to the midgut epithelium zNew studies show that differences in galectins and LPG in different sandfly and parasite species might govern host specificity Cell 119:329-41
Modification of LPG modulate interaction of the parasite and midgut galectin
Summary zLeishmania species cause three clinical syndromes depending on the spread of the infection in the body zLeishmania ‘provoke’ phagocytosis by macrophages and develop intracellular in an fully acidified lysosome zLPG-galectin interaction and modification of LPG regulate attachment and detachment of parasites in the sandfly host