Leishmania zLeishmania: a large group of kinetoplastid parasites causing a variety of syndromes zPhosphoglycans: important molecules for parasite development and pathogenesis zClassic model for polarized T-cell response (Th1/Th2, will be covered later in the immunology part of this course)

Leishmania belong to the order kinetoplastida zGroup of flagellates at the basis of the eukaryotic tree zHarbor name-giving mitochondrion with large genome which is always associated with the basal body of the single flagellum zTrypanosoma & Leishmania are the medically important and best studied genera in this group trypomastigote epimastigote promastigote amastigote

Leishmania parasites exist as pro- and amastigotes zThe parasite lives in the digestive tract of sand flies as promastigote zIn the mammalian host parasites multiply as intracellular amastiogotes

Leishmania infects and thrives in macrophages zHow do they get in and how to the avoid being killed?

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 (opsonization: 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 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 is transmitted by sand flies (Phlebotomidae) zSand flies are minute diptera (only females bite) zThey do not fly well and stay close to the ground zIn the wild sand flies often breed in rodent burrows zOld world Phlebotomus, new world Lutzomya zCan also transmit Bartonella bacilliformis and Papatsi virus

A variety of species and species complexes causes disease in humans

Kala Azar - Visceral Leishmaniasis zCaused by the L. donovani complex zGeneral infection of macrophages in the entire RES zWeeks to months incubation period zAbdominal swelling (hepato- and splenomegaly zOften but not always fever occurs in two daily peaks zProgressive weight loss zDarkening of the skin zMortality of untreated disease %

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 start months after infection zParasites are not found outside the lesion zNearly absolute resistance to reinfection (however, there is long term persistence and persistence is required for resistance)

Espundia -- 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, the soft and the soft palate zDeath occurs usually through secondary bacterial infection

Leishmania produce a unique glycoconjugate zLeishmania parasites can be labeled at surprizing efficiency with radioactive sugars and inositol zLabel is incorporated into a large glycoconjugate which is acid labile and has a lipid anchor zThis lipophosphoglycan (LPG) completely covers the surface of the the promastigote

Lipophosphoglycans share structural features with GPIs

Lipophosphoglycan zMajor surface molecule of Leishmania zFour domains: z1-O-alkyl-2-lyso-phosphatidylinositol anchor zGlycan core zDisaccharide phosphate repeat units zOligosaccharide cap

LPG shows structural variation among Leishmania species

Biosynthesis of LPG

What does LPG “not” do? zLPG protects parasites in the sand fly midgut zLPG attaches parasites to the sand fly midgut epithelium zLPG protects against complement attack zLPG enhances uptake into macrophages zLPG interferes with macrophage signaling preventing oxidative burst zLPG protect from toxic macrophage products

Is LPG a pathogenesis factor? Stan Falkow’s virulence postulates zPathogenesis is reasonably associated with the expression of a certain virulence factor zInactivation of the gene should result in loss of virulence zRestoration of the gene should fully restore virulence

Identification of LPG genes by genetic complementation

Mutants in LPG biosynthesis as tools to study LPG enzymology & function

To proof the third postulate has been a challenge zLpg- mutants show significant loss in virulence both in in vitro macrophage infections as well as in in vivo experiments zBut: Leishmania tends to loose virulence in culture anyway, and expression of the WT gene did not always fully restore virulence zChemical mutants might be ‘over-mutated’ and carry multiple mutations (some might not be related to LPG but affect virulence) zPresence of several LPG related molecules further complicate the issue

A number of Leishmania glycoconjugates share structural features with LPG

Knock outs by gene targeting as a cleaner way to obtain mutants

Lpg1 k.o. in L. major has an effect on virulence

Lpg1 k.o. in L. major has an effect on virulence

Lpg1 k.o. in L. major has an effect on virulence

In Leishmania mexicana LPG seems dispensable for infection of mice zlpg1 knock outs infect macrophages in vitro and mice in vivo as well as wt suggesting LPG is not needed zlpg2 knock outs do the same suggesting that PG repeats in general are not needed zOverall, it appears that the main role for LPG might lie in the interaction with the sandfly and not the mammalian host

procyclics and metacyclics zPromastigotes attach to and metacyclics detach from the midgut epithelium

procyclics and metacyclics zInfected macrophages are taken up with the blood meal and amastigotes released by digestion transform into procyclic promastigotes which attach to the midgut epithelium zAttached promastigotes divide rapidly zMetacylcic promastigote detach and pass forward into the pharynx from where they are regurgitated into the bite site

Structural modification of LPG during the sand fly cycle zLPG is structurally modified during metacylogenesis zLPG in metacylics has 2-3 times the number of repeat units zSide chains with terminal galactose are down- regulated in favor of chains with terminal arabino- pyranose

Only phosphoglycans from procyclics attach to the midgut zOpened midguts were incubated with PG from procyclics (A/B) and metacyclics (C/D) and detected with an antibody Pimenta et al., Science. 256:

Phosphoglycan binds to the microvilli of the epithelium Pimenta et al., Science. 256:

Phosphoglycan repeats inhibit attachment of procyclics zOpened midguts were incubated with radiolabeled procyclic promastigotes z(A) 2-5 procyclic PG, 6+7 metacyclic PG (lipid removed)  (B) 1, control, 2, pPG, 3 no  Gal, 4 with  Gal, 5, second  Gal, 6 ara  (C) commercial saccharides: 2 free Gal, 3-5  and  digalactosides, 6 similar Man disaccharide Pimenta et al., Science. 256:

LPG is not essential for early survival but for retention z(A + B) number of promastigotes present in midgut upon disection (in B 5x higher inoculum is used) Sacks et al., PNAS 97:

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 protein encoded by this gene 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 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