Leishmaniasis

Leishmaniasis

1. The problem of leishmaniasis throughout tropical and subtropical regions (Epidemiology)

   • Global distribution/incidence of leishmaniasis
     • 88 contries affected (tropics and sub-troppics)
     • 2 million new cases each year
     • 12 million people infected
     • 350 million people at risk (will increase with global warming as insect vector migrates)
     • Spectrum of disease (mild to severe)
   • Transmission
     • Parasite is transmitted by phlebotamine sand flies
       • Flies live in; moist soils, stone walls, rubbish heaps - leishmaniasis found in a variety of ecosystems
       • Only female flies spreadnthe disease
       • They are pool feeders (feed of blood/lymph from an open wound - do not penetrate the skin)
       • Parasite not in salivary gland, but in found in the anterior portion of the gut and the pharynx
     • Two main modes of transmission
       • Zoonotic (aimals to people)
         • E.g. Transmission in latin america and mediterrenean via dogs
       • Anthroponotic (human to human)
         • E.g. Indian sub continent (transmission by: transplants/transfusions, congenital and sharing of needles)
   • Causitive agent
     • 21 different species of Leismania (most notably L. donovani, major, infantum (aka chagasi in the new world) and braziliensis)
       • Morphologically indistinguishable (unlike malaria)
       • Molecularly distinct - detectable by...
         • PCR
         • Isoenzyme analysis (by many proteomic techniques)
         • Monoclonal Ab's specific to each species
     • Major parasite forms
       • Promastigote
         • Two subforms
           • Non-infectious form
             • Found in the insect gut/pharynx
             • Replicates by binary fission
           • Infectious metacyclic form
             • Invades host cells (phagocytes)
             • Non-replicating
         • 15-20um in length, flagellated (motile)
         • Structural arrangement: posterior end-nucleus-kinetoplast/flagellal pocket-anterior end
           • Flagellum: long, unattached from cell body
         • Extracellular forms
         • Elongated, with long flagellum and central nucleus
       • Amastigote
         • 2-4um in length, stumpy flagellated (non-motile), divides by binary fission (500+ parasites per host cell)
         • Intracellular form
         • Structural arrangement: posterior end-nucleus-kinetoplast/flagellal pocket-anterior end
           • Flagellum: short, barely projects beyond the flagellal pocket (non-motile)
         • Can invade macrophages
         • Transmissile from humans to insect (following blood meal)
         • Spherical with short flagellum and central nucleus

2. The lifecycle of the causitive agent (Leishmania spp - L. donovani, major, infantum, braziliensis)

   • Sandfly takes a blood meal
     • Leishmanial parasite (metacyclic promastigote in fly gut) transferred directly to the blood (unlike in chagas)
       • Infected sandflies have a dysfunctional valve at the junction of the pharynx and midgut
         • Valve cannot close meaning blood can flow backwards and forwards carrying the parasites to the wound
       • Reflux action -> expulsion of the parasite into the bite wound
         • This is aided by the secretion of a gel-like substance from the parasite which forms a physical barrier to fly feeding
           • Metacyclic promastigote phagocytised by host neutrophils
             • Trojan horse theory (for L. major)
               • Infected neutrophil undergoes apoptosis -> apoptotic bodies phagocytised by macrophages -> promastigote now in macrophage-> differentiate (amastigotes
             • Or...
               • Promastigote differentiates to amastigote in neutrophil -> replication -> ruputre -> amastigotes released and infect macrophages
             • Both ways lead to the infection of macrophages with amastigote forms
               • Amastigotes replicate in macrophages
                 • Macrophage ruptures, releasing amastigotes into bloodstream to continue cycle
                   • Sandfly takes a blood meal and ingests infected host macrophage
                     • Macrophage rupture and amastigote release in insect gut
                       • In the insect gut: amastigotes -> non-infectious promastigotes which attach to the gut wall by LPG (lipophosphoglycan) to avoid excretion
                         • Non-infectious promastigotes replicate (binary fission)
                           • Some become metacyclic promastigotes (infective) - they loose their LPG (coat change) and move to the anterior gut ready for transmission

3. Host cell invasion

   • Mechanism
     • Macrophage/neutrophil CR1 or 3 receptor binds to C3b(i)-bound GP63 on the parasite membrane
       • This stimulates phagocytosis (passive invasion of the parasite)
         • Cytochalasin D (inhibits phagocytosis by disrupting actin polymerisation) inhibit the invasion
         • Parasite is now internalised within a phagosome
           • Lysosomes fuse with the phagosome
             • Lowering the pH (~5.0)
               • Low pH triggers differntiation of metacyclic (infecitous) promastigote to amastigote
                 • Amastigote replicates in the phagosome
                   • Amastigotes are masters of modulating the phagocytes behaviour
                     • Leishmania inhibit host cell PKC which prevents NADPH oxidase complex formation
                       • Reducing the overall oxidative stress
                     • Leishmania activate SHP1 (a host cell tyrosine phosphatase)
                       • SHP1 dephosphorylates cytokine receptor-bound JAK (inactivating the JAK-STAT pathway)
                         • STAT no longer expresses genes that promote an inflammatory response
                           • Supression of the inflammatory response
             • Lysosomes acidify the phagosome, add digestive enzymes (lysosymes: proteases, nuclease and lipases) and also induce oxidative stress
               • Phagosome membrane enzyme NADPH oxidase complex produces free radicals (H2O2 and hypochlorate - which produce hydroxyl radicals un reacting with iron)
                 • Nitric oxide synthase produces nitric oxide which reacts with superoxide free radicals - producing peroxynitrite
                   • All are deadly to pathogens
   • Parasite ligand: GP63 + host C3b(i)
   • Macrophage/neutrophil target receptor: CR1 and CR3 (recognise surface bound C3b)

4. Disease pathlogy

   • Cutaneous leishmaniasis
     • Most common manifestation (5-10 million cases)
       • Called; orient/bay sore, uta, baghdad/dehli/kandahar/lahore boil
     • Starts as raised, painless red lesion at site of bite
       • Lesion can ulcerate
         • Wet ulcer = L. major (typically in rural areas)
         • Dry ulcer = L. tropica (typically seen in citites)
         • No pus (unless bacterial infection follows)
           • Ulcer self-heals, leaving a scar (esp. L. major)
             • Oce healed - individual is immne for life
             • Can become diffuse
   • Visceral leishmaniasis
     • Also known as kala azar (literally black sickness); or dumdum fever
       • Typically caused by: L. donovani (india/pakistan) or infantum (mediterreanean; L. chagasi in latin america)
     • Most severe form
       • 200-500,000 cases per year
         • 50,000 deaths per year
           • Mortality = 10-25%
     • Generalised infection of the reticuloendothelial system (phagocyte cells)
     • Also involves; liver, spleen, bone marrow and lymph nodes
       • Fever
       • Hepatosplenomegaly with lymphadenopathy
       • Anaemia, leukopaenia (WBC deficiency) and thrombocytopaenia (platelet deficiency)
       • Progresses to; malaise, tiredness, lassitude (a state of mental tiredness) and weakness
         • Patient exhibits wasting (even despite a good apetite)
           • Common presentation = distended abdomen (hepatosplenomegaly) with wasted arm musculature
             • Secondary infections are common
   • Diffuse leishmaniasis
     • Two types;
       • Diffuse cutaneous leishmaniasis (DCL)
         • Limited to cutaneous surfaces - but metastasises over the entire body
           • Non-ulcerated, scaley,lesions (nodular)
             • Caused by a lack of immune response (anergy)
               • Amastigotes abundant in lesions
         • E.g. L. aethiopica and mexicana
       • Mucocutaneous leishmaniasis (MCL)
         • Primarily a cutaneous form that metastasises to the mucocutaneous junctions (e.g. Mouth/nose/soft palate or anus/genitalia)
           • Can occur weeks or years after initial infection
             • Tissue is grossly destroyed causing disfigurement
               • Secondary bacterial infections are common
         • Mostly L. braziliensis
   • Disease severity factors
     • Depends on the immune status of the individual
       • T-helper lymphocytes (TH1 and TH2) response determines pathology
         • TH1 response
           • Cellular immune response stimulated (e.g. Cytotoxic T-lymphocytes)
             • No/low Ab production
             • Mild infection (cutaneous)
               • Full recovery
         • TH2 response
           • Stimulates a humoral (Ab) immune response
             • High Ab production
               • Succumb to infection (visceral form)
           • Particularly nasty species (e.g. L. donovani) actually trigger a switch from TH1 to TH2 response

5. Immune evasion of Leishmania spp.

   • There are several mechanisms used in the avoidance of the innate immune repsonse
     • The complement cascade (discussed in Chagas mind map)
       • Following differentiation in the fly gut from non-infective promastigote to metacyclic (infectious) promastigote the parasite coat changes
         • Non-infectious promastigote: short LPG chains with low levels of surface protein GP63
         • Infectious metacyclic promastigote: has much longer LPG chains (conceal the membrane proteins) and a higher level of GP63 surface protein
           • Long LPG
             • Blocks the MAC forming within the parasite membrane
             • Lipophosphoglycan (LPG) - around 3-5million copies per cell
             • Structure
               • Oligosaccharide capping complex
                 • Phosphoglycan domain
                   • Recurring motif of [galactose-mannose-phosphate]n
                     • Features side chains (Galactosyl sidechains)
                       • These galactosyl side chains bind galectin in the insect gut (during differentiation into the infectious promastigote these side chains are capped)
                         • Arabinosyl caps (prevents binding of infectious form in the insect gut)
           • GP63
             • Binds to activated C3b and inactivates it
               • Preventing C3 convertase formation
                 • However, used the bound C3b(i) to attract macrophages for invasion
                   • Macrophages phagocytose anything that is bound to C3b (detected by CR1 and CR3 receptors)
                     • This is called opsonisation
                     • CR = comement receptor
             • Zinc metalloproteinase