Malaria pathogenesis

Parasite 'turns women into sex kittens' By Jane Bunce, December 26, :27pm, Article from: AAP A COMMON parasite can increase a women's attractiveness to the opposite sex but also make men more stupid, an Australian researcher says. About 40 per cent of the world's population is infected with Toxoplasma gondii, including about eight million Australians. … Until recently it was thought to be an insignificant disease in healthy people, Sydney University of Technology infectious disease researcher Nicky Boulter said, but new research has revealed its mind- altering properties. "Interestingly, the effect of infection is different between men and women,'' Dr Boulter writes in the latest issue of Australasian Science magazine. "Infected men have lower IQs, achieve a lower level of education and have shorter attention spans. They are also more likely to break rules and take risks, be more independent, more anti-social, suspicious, jealous and morose, and are deemed less attractive to women. "On the other hand, infected women tend to be more outgoing, friendly, more promiscuous, and are considered more attractive to men compared with non-infected controls. "In short, it can make men behave like alley cats and women behave like sex kittens''. … (

Apicomplexan invasion zActive, parasite driven process zDepends on parasite actin/myosin motility (conveyor belt model) zInvolves secretion of micronemes (attachment, motility), rhoptries (PV & MJ formation) and dense granules (makes PV into a suitable home) zSets up a parasitophorous vacuole which initially is derived from the host cell cell-membrane zA moving junction is formed which screens out host membrane proteins from the PV, the PV is fusion incompetent and the parasite protected

Two great movie clips summarizing the malaria life cycle: zDevelopment in the human: zhttp:// human.htmlhttp:// human.html zDevelopment in the mosquito: zhttp:// z(make sure to watch these before the next exam)

Malaria II zMalaria the disease zPathogenesis of severe falciparum malaria zDrugs used to treat malaria and the development of drug resistance

Malaria the disease zHuman malaria is primarily a blood disease, however it causes pathology in a variety of organs & tissues zAll disease is due to the parasites development within the red blood cell (merozoite, trophozoite, schizont). zOther stages are important for transmission but they do not contribute to pathogenesis

Malaria the disease z9-14 day incubation period zFever, chills, headache, back and joint pain zGastrointestinal symptoms (nausea, vomiting, etc.)

Malaria the disease

zMalaria tertiana: 48h between fevers (P. vivax and ovale) zMalaria quartana: 72h between fevers (P. malariae) zMalaria tropica: irregular high fever (P. falciparum)

Malaria the disease zSymptoms intensify zIrregular high fever zAnxiety, delirium and other mental problems zSweating, increased pulse rate, severe exhaustion zWorsening GI symptoms zEnlarged spleen and liver

Malaria the disease Irritability, loss of reflexes, neurological symptoms similar to menigitis, coma 20% fatality Progressive severe drop of hematocrit, poor oxygen Supply for organs and tissues Dwindling urine, high urea Level in serum, hyperventilation Coma, poor prognosis Cerebral malariaSevere anemiaRenal failure 3 Severe manifestations

Malaria the disease WHO-TDR

Pathogenesis of malaria zIn highly endemic areas: high mortality among children due to severe anemia, children who survive beyond the first years show decreasing parasitemia and disease (this immunity is not sterile and depends on constant exposure) zIn areas with less infection pressure: malaria is an epidemic disease with varying intensity. Adults and children are equally susceptible and death in adults is mostly due to cerebral malaria

Pathogenesis of malaria Parasitemia Cerebral malaria Anemia Age

Pathogenesis of cerebral malaria zCerebral malaria is characterized by multiple brain hemorrhages (vessel rupture and bleeding)  Excessive serum and tissue levels of TNF  and INF  (two important immune hormones driving inflammation) are associated with severe malaria zSome researchers believe this inflammation is the main cause for pathology (remember the immunology introduction: an overshooting immune response against a chronic pathogen that can not be cleared can cause severe disease)

Sequestration & cytoadherence zThe second model suggests sequestration to be the main culprit zIn P. falciparum infections only early stages (rings) are found in the peripheral blood zTrophozoites and schizonts are sequestered to the post- capilary venules by attachment to the endothelium Ring stages

Pathogenesis of falciparum malaria zParasite infected RBC become ‘sticky’ and adhere to endothelial cells zThis phenomenon takes about hours to develop after parasite invasion zUnder high flow (here modeled using a microfluidic device) this first results in rolling and then in attachment z(movie courtesy of Dr. Pradip Rathod University of Washington)

Pathogenesis of falciparum malaria zCytoadherence seems to be the main culprit for pathogenesis zInfected RBCs will adhere to the endothelium as well as to each other and cause clogging and hemorhaging zNote that high cytokine levels induce expression of endothelial adhesins -- inflammation makes the endothelia ‘stickier’ zAdherence and inflammation reinforce each other in an unholy circle causing pathology

Knobs and cytoadherence zCytoadhrence correlates with the presence of “knobs” (left column) on the surface of the infected RBC zThe right column shows a RBC infected with a knob- less strain which does not cause cerebral malaria zKnobs are made up of parasite derived proteins knobsknob-less

Knobs and cytoadherence zPfEMP1 (P. falciparum erythrocyte membrane protein) is found in knobs and is responsible for cytoadherence and rosetting zPfEMP1 is a large membrane protein anchored in the RBC membrane with the bulk extending into the blood stream zVarious domains of PfEMP1 have been shown to bind to ligands on the endothelia of the vasculature and the placenta zPfEMP1 is an important pathogenesis factor

Knobs and cytoadherence

zThe parasite exports PfEMP1 and other proteins (this picture is showing Knob associated protein) into the RBC and its surface to form knobs zF: in early rings protein is in the parasite and the parasitophorous vacuole, G,H: in trophozoites it is found first within the RBC cytoplasm and then at the RBC membrane (I).

Knobs and cytoadherence zHow precisely the parasite transports proteins through the RBC is still under study zHowever it is clear that the parasite has not only to provide the cargo but also the transport machinery as the RBC has reduced its capability for membrane transport and secretion zAll these proteins are initially secreted by the parasite into the parasitophorous vacuole zA recently discovered gatekeeper in the vacuole membrane appears to shuttle proteins across (PTEX Plasmodium translocon of exported proteins) zMaurer’s clefts (parasite induced membranous structures in the RBC) appear to be an important bridgehead acting in the sorting and trafficking of exported proteins Reiff & Striepen, Nature 459:918

Immunity to malaria zThere is no sterile immunity to malaria zPatients produce strong antibody responses to PfEMP1 which is exposed to the immune system on the surface of the infected RBC. zWhy is the immunity to malaria relatively weak? zPfEMP1 is encoded by a large multigene family (VAR genes) and parasites switch to new variants (antigenic variation again) zThe parasite genome encodes 60 VAR genes, only one is expressed at a time (allelic exclusion)

Immunity to malaria zSuccessful vaccination in humans has been achieved with large doses of irradiated sporozoites, however that is likely not practical zMany approaches have been and are explored to stimulate immunity against sporozoites (infection blocking), merozoites (disease blocking) or gametocytes (transmission blocking) zNone has yielded a satisfactory and safe human vaccine yet zThe most promising new strategies use genetic manipulation to engineer attenuated parasites strains (parasites that enter cells and induce immunity yet fail to develop fully and cause disease) zFor now, control depends heavily on drug therapy

Chinchona the source of quinine zPeruvian Indians appear to have been the first to know about the medicinal effects of quinine, they chewed Chinchona bark while working in the mines as forced laborers for the Spanish zJesuits brought the bark back to Europe to treat febrile diseases zIn the early 1600s the bark was used to treat the fever of the Countess of Chinchon and became well known as Jesuit’s powder or Peruvian bark zInitial preparations were often quite variable in the amount of active ingredient resulting in varying effects

Chichona the source of quinine zHigh demand had brought the Chinchona tree almost to extinction in the wild zCharles Ledger a trader in Peru send out Manuel Incra Macrami to collect seeds from a stand of special trees they had found earlier zAfter three years Manuel came back with 15 kg of seeds which they sold for 100 guilders to the Dutch consul as the British were not interested zC. ledgeriana formed the basis of a very profitable Dutch quinine monopoly which lasted until World War II

Chloroquine the wonder drug zChloroquine, a synthetic quinine analog developed by German and American chemists during WWII, was a very potent drug that was cheap to make, stable, and had no serious side effects zChloroquine was a major component of the 60/70s malaria eradication campaign zNone of the drugs developed since come close to chloroquine

Chloroquine the wonder drug zDuring its development within the RBC the malaria parasite ingests the cytoplasma of its host zNote that in this schematic (and in real micrographs) the red color of the blood cell gets considerably lighter -- at the same time malaria pigment accumulates zThe parasite digests large ammounts of hemoglobin to cover part of its amino acid needs

Chloroquine the wonder drug zRBC cytoplasm is taken up by endocytosis zThe endocytotic vesicles fuse with the food vacuole (a homolog of the secondary lysosome) were hemoglobin digestion occurs zDigestion frees large ammounts of heme zHeme is toxic to the parasite and is neutralized by polymerization into the malaria pigment or hemozoin zChloroquine accumulates in the food vacuole (it’s a weak base and like all lysosomes the FV is an acidic compartment) zChloroquine is thought to interfere with the polymerization and detoxification of heme

Resistance to chloroquine

Mechanisms of drug resistance

zChanges in target enzyme (e.g. decreased affinity to drug) zOverexpression of target (amplification) zDecreased activation of drug zChanges in accessibility (less import, or more export of drug)

Resistance to chloroquine zGenetic studies have shown that resistance is linked to the transporter protein PfCRT zStudies using parasite cultures suggests that a series of point mutations in PfCRT are responsible for resistance zThis putative transporter localizes to the membrane of the food vacuole zLarge field studies have found strong association of these mutations with chloroquine resistance zIt is now thought that the “natural” role of PfCRT is to export small peptides from the food vacuole PfCRT, resistance mutations highlighted

Antifolates as malaria drugs zThe synthesis of certain building blocks of DNA requires reduced folate (more specifically the syntheisis of dTMP) zNo reduced folate -- no DNA zThe malaria drug Fansidar uses a drug combination to hit the same target pathway twice zCombinations that are more effective than the sum of their individual activities are called synergistic dUMP Tetrahydrofolate Nucleotide synthesis dTMP

Antifolates as malaria drugs GTP Dihydrofolate Tetrahydrofolate Folate synthesis Folate ‘recharging’ Parasite

Antifolates as malaria drugs Dihydrofolate Tetrahydrofolate Nucleotide synthesis GTP Dihydrofolate Tetrahydrofolate Folate synthesis Folate ‘recharging’ ParasiteHuman

Antifolates as malaria drugs Dihydrofolate Tetrahydrofolate Nucleotide synthesis GTP Dihydrofolate Tetrahydrofolate Folate synthesis Folate ‘recharging’ ParasiteHuman Sulfonamide

Antifolates as malaria drugs Dihydrofolate Tetrahydrofolate Nucleotide synthesis Human GTP Dihydrofolate Tetrahydrofolate Folate synthesis Folate ‘recharging’ Parasite Sulfonamide Pyrimethamine

Antifolates as malaria drugs zFirst strike: Folate synthesis. We can’t make folate and take it up with food as a vitamin. The parasite makes it and is therefore susceptible to sulfonamides which block synthesis zSecond strike: After each use dihydrofolate has to be reduced again (think of it as recharging). The enzyme which does this (dihydrofolate reductase) is different in human and parasite zThe drug pyrimethamine inhibits parasite DHFR but not human DHFR zFansidar combines pyrimethamine with sulfadoxine zA very similar drug combination is used to treat toxoplasmosis GTP Dihydrofolate Tetrahydrofolate Folate synthesis Folate ‘recharging’ Parasite Sulfonamide Pyrimethamine

Antifolate resistance developed very fast

Combinations of Artemisinin and other antimalarials are promising zExtracts of Artemisia annua (sweet wormwood) have long been used in traditional Chinese medicine to treat fever zChinese investigators extracted the active ingredients and showed that they and there chemical modifications are powerful antimalarials zHowever monotherapy results in high level of recrudescence zCombining Artemisinin with other drugs have been very successful especially for severe malaria zArtemisinin acts very fast which helps to reduce mortality and get patients out of their coma quickly

Summary zSevere forms of malaria include: severe anemia in kids, and cerebral and renal malaria in adults zSevere pathogenesis is related to adherence of infected RBC to entothelia zAdherence is mediate by knobs in the RBC surface made up by parasite proteins (PFEMP1) zPFEMP1 undergoes antigenic variation zChloroquine resistance has been a public health catastrophe zChloroquine accumulates in the food vacuole and prevents heme polymerization, resistance is linked to mutations in a transport protein in the food vacuole membrane