Plasmodium parasites, responsible for malaria in humans, have a complex lifecycle that is dependent on mosquito and human hosts. In human blood, the merozoite stage of the parasite invades red blood cells (erythrocytes), growing and multiplying before rupturing the cell and escaping to infect other erythrocytes. It is this profound effect on erythrocytes that is responsible for the symptoms of malaria – fevers, chills and anaemia. Untreated, the disease can be fatal and drug resistance is an increasing problem. With up to half a billion people infected each year and nearly a million deaths, mostly in sub-Saharan Africa, there is an urgent need for new treatments.
Researchers at Harvard School of Public Health (HSPH) were attempting to identify the mechanism by which Plasmodium falciparum merozoites enter erthyrocytes, but instead found a parasite protein that is essential for escape from the cells. When the protein, P. falciparum calcium-dependent protein kinase (PfCDPK5), was suppressed the parasites were trapped in the host cell and unable to infect new cells. In further experiments the team showed that these merozoites were still able to invade erythrocytes if released from their host cell by other means, indicating separate mechanisms for invasion and egress from erythrocytes.
The findings reveal an essential step in the biology of P. falciparum and suggest a new, parasite-specific, drug target for fighting one of the world’s most common and dangerous infections. Whilst many scientists are looking for inhibitors of parasite egress and invasion of red blood cells, no anti-malarial drugs yet target these stages of the parasite lifecycle.
The lifecycle of all Plasmodium species is complex and involves a round of replication in host erythrocytes. The clinical manifestations of malaria are linked to this stage in the lifecycle and are associated with rupture of the infected erythrocytes. During this growth phase, the parasite enters the erythrocyte and then releases several hundred effector proteins into the cytoplasm. These key virulence proteins provide a suitable environment for multiplication and allow the parasite to evade the host immune system. Proteins destined for export contain a conserved pentameric motif known as PEXEL and, when this is cleaved in the endoplasmic reticulum, the protein can be transported into the host cell. Two independent studies by scientists in the US and Australia have now shown that the protease responsible for cleaving the PEXEL motif is the aspartyl protease, plasmepsin V. Cleavage reveals an export signal at the amino terminus of the cargo protein which is then transported into the host cell cytoplasm, likely through a channel in the parasite’s outer membrane. Since export of the effector proteins is essential for the erythrocytic stage of the plasmodium life-cycle, drugs that block plasmepsin V should provide an effective treatment for malaria.
Histamine is a biogenic amine which acts as a mediator of immune responses as well as acting as a neurotransmitter in the CNS. Histamine plays a role in a variety of physiological processes including allergic reactions, gastric acid secretion, bronchoconstriction and neurotransmission. Four histamine receptors, termed H1, H2, H3, and H4, have been identified. The H1 receptor mediates most of the pro-inflammatory effects of histamine whereas the anti-inflammatory and immunosuppressive activities of histamine are mainly dependent on stimulation of the H2 receptor. The H4 receptor is expressed predominantly on haematopoietic cells and agonists of this receptor induce chemotaxis of mast cells and eosinophils as well as production of IL-16 by T-cells. H4 stimulation has also recently been shown to cause inhibition of airway resistance and inflammation in a murine model of allergic asthma. Unlike the other histamine receptors, H3 receptors are expressed mainly on neurons of the peripheral and central nervous system where they control the synthesis and release of histamine and also influence the release of other neurotransmitters including dopamine, γ-aminobutyric acid, noradrenaline, acetylcholine, serotonin and tachykinins.
In human infection with Plasmodium falciparum, as well as in murine models of malaria, increased levels of histamine have been shown to be associated with severity of infection. Histamine signalling through H1 and H2 receptors increases the susceptibility of mice to infection with lethal strains of Plasmodium berghei and mice genetically deficient in the histidine decarboxylase gene – and thus lacking histamine – are highly resistant to severe malaria whether infected by mosquito bites or via injection of infected erythrocytes. A study recently published in the journal PLoS ONE has now investigated the role of the H3 receptor in the inflammatory response in the brain during P. berghei infection in mice. Compared with wild type mice, mice deficient in the H3 receptor showed an accelerated onset of cerebral malaria, increased brain pathology and more pronounced loss of blood brain barrier integrity associated with earlier death. H3 receptors tightly regulate release of histamine and other neurotransmitters and neuronal histamine activity was found to be significantly higher in naive knockout mice than in wild type mice.
In further studies, the H3 receptor agonist, (R)-alpha-methylhistamine, was found to be effective in reducing progression to cerebral malaria. In wild type mice, both the H1 receptor antagonist, levocetirizine, and the H2 receptor antagonist, cimetidine, were found to be effective in reducing clinical symptoms and mortality caused by cerebral malaria. This protection was attributed, in part at least, to down-regulation of inflammatory response-associated genes such as IFN-γ and TNF-α. The beneficial role of the H3 receptor in controlling histamine levels and limiting disease was demonstrated by the higher effectiveness of cimetidine and, to a lesser extent, levocetirizine in wild type compared with H3 receptor knockout mice. The authors propose that H1 or H2 receptor antagonists, either alone, or together with an H3 receptor agonist if one becomes available, might be used alongside anti-malaria medicines as preventative therapies against the development of cerebral malaria, especially in areas where malaria transmission is seasonal.
Animal parasites such as malaria have complex life cycles and, so far, most attempts to control infection have centred on preventing the parasite from entering host cells. Writing in the journal Science, a team led by Dr Doron Greenbaum at the University of Pennsylvania has now focussed on an alternative treatment approach – locking the parasites inside the host cell. The team found that Plasmodium falciparum, the species responsible for the majority of human infections, and also the one that causes the most virulent form of malaria, uses a host protease to escape from cells. The protozoa replicate within a vacuole in infected cells and must escape to begin a new lytic cycle. The team used a variety of techniques to show that P falciparum makes use of host cell calpain proteases to facilitate escape.
The team were also interested to find out whether the distantly related parasite, Toxoplasma gondii adopts a similar strategy. Disease caused by T gondii infection is usually mild and self-limiting, but can be fatal to the unborn child if contracted during pregnancy. They found that in the absence of calpain, the parasites could not escape the infected cell, just as they had observed for malaria parasites.
Greenbaum plans to continue to explore the practicality of calpain as a target for anti-parasitic drugs. P falciparum has become increasingly resistant to anti-malarial drugs and targeting a host protein may afford less scope for the development of resistance. Calpains are a family of calcium-dependent cysteine proteases whose physiological roles are poorly understood.
Although carbohydrates were previously thought of as little more than an energy source, it is now recognised that they play a key role in many biochemical processes including intercellular recognition, immune function, fertilisation and certain types of cancer. From a synthetic viewpoint, the structural complexity that makes carbohydrates important in so many biological processes poses significant challenges. Unlike oligonucleotides and peptides, carbohydrates can form branched as well as linear structures, and there are a staggering number of ways in which monosaccharides can be combined. Increasingly powerful and versatile methods have been developed to allow the synthesis of pure oligosaccharides in the laboratory, but the process requires regioselective protection of hydroxyl groups as well as stereoselective assembly of glycosidic linkages and is technically difficult and very time consuming. Speaking at the annual meeting of the American Chemical Society in Salt Lake City, Dr Peter H Seeberger has described the development of a fully automated carbohydrate synthesiser that should make complex carbohydrates more accessible. A simple cleavage, deprotection and purification protocol provides rapid access to naturally occurring and synthetic oligosaccharides and is suitable for use by non-experts.
One application that Seeberger’s group have been addressing with the new technology is the development of a vaccine against malaria. Fatalities caused by the malaria parasite, Plasmodium falciparum, are thought to result, at least in part, from a reaction to the malaria toxin, glycosylphosphatidylinositol (GPI). Anti-GPI vaccination was found to protect against fatality in mice and clinical trials of an anti-toxin vaccine to protect against the inflammation and anaemia associated with malarial infection are scheduled for 2010 in Mozambique and Tanzania. Seeberger also believes that carbohydrate-based vaccines could be used against other serious infectious diseases, including antibiotic-resistant infections and HIV.
The malaria parasite, Plasmodium falciparum, has limited capacity for de novo amino acid synthesis and relies on degradation of host haemoglobin for a supply of these essential building blocks. Haemoglobin is first degraded into di- and tri-peptides by the action of a number of cysteine-, aspartyl-, and metallo-proteases. These small peptide fragments are then further hydrolysed to release free amino acids by the action of the metallo-exopeptidases, PfA-M1 (an alanyl aminopeptidase) and PfA-M17 (a leucine aminopeptidase).
A team from the University of Monash has recently described the X-ray crystallographic structure of truncated recombinant PfA-M1 at a resolution of 2.1 Å. Comparison of structures of PfA-M1 bound to the known inhibitors, bestatin (Ki 500nM) and hPheP[CH2]Phe (Ki 80nM), with the native structure showed that the enzyme did not undergo any global conformational rearrangements on binding either inhibitor. It is proposed that substrate access is achieved by means of the C-terminal domain vortex, and that control of substrate hydrolysis can be achieved, and depends on, the size of this channel. hPheP[CH2]Phe, which provides effective protection in a murine model of malaria, also inhibits PfA-M17 and the authors suggest that inhibiting both PfA-M1 and PfA-M17 may be less likely to allow the development of drug-resistant malaria. The fact that the site of action of PfA-M1 is outside the digestive vacuole, together with the comparative ease of identifying drug-like inhibitors of metallo-proteases, makes PfA-M1 an attractive target for new anti-malarial therapies.
The study is published in the February 5th Early Edition of PNAS.
Malaria is a global health problem and causes 2 – 3 million deaths each year. Mosquito bites allow malaria parasites to enter the bloodstream. Within 30 minutes, the parasites are transported to the liver where they enter cells and start to reproduce rapidly. Following release from hepatocytes, the parasites re-enter the bloodstream and infect red blood cells, triggering the pathology that is associated with malaria.
The receptor on human liver cells that allows the malaria parasites to enter hepatocytes has been identified as the scavenger receptor (SR-B1). This receptor normally transfers cholesteryl esters and other lipids from high density lipoprotein (HDL) in the bloodstream into liver cells. A new study shows that, in cell culture experiments as well as experiments in mice, blocking the SR-B1 receptor dramatically reduced the ability of the malaria parasite to infect liver cells. The researchers used RNA interference (RNAi), monoclonal antibodies, and small molecule inhibitors to demonstrate the importance of the SR-B1 receptor for entry of malaria parasites into hepatocytes. The study demonstrates that blocking the SR-B1 receptor may offer a new approach to the prophylaxis of malaria.
Targeting host mechanisms promises better protection against the emergence of resistant strains of the malaria parasite but, in this case, should be balanced against the atherosclerotic potential of long term blockade of the SR-B1 receptor.