Malaria’s potentially fatal weakness

The ancient war between the human immune system and the malaria parasite, Plasmodium falciparum, is fought daily, on a million individual fronts. Every individual’s private battle with the parasite is unique. Infected siblings in an African village will make different antibodies against different P. falciparum antigens.

The parasite’s ability to frustrate the immune response by switching antigenic guise has defied efforts to identify fixed antigenic targets that could be combined in a general purpose vaccine. Three decades of intense scientific effort have yielded promising candidate antigens, but a viable vaccine may be years away.

Resistant strains have left the cupboard almost bare of antimalarial drugs. DDT, for decades the frontline weapon against malaria, is gone, leaving millions of children exposed to mosquitoes that transmit the parasite. Malaria is resurgent, and killing as many as three million people a year - 90 per cent of them under the age of five.

But as so often happens in research, hope has recently sprung from an unexpected quarter: Professor Alan Cowman’s research group at the Walter and Eliza Hall Institute for Medical Research has made a discovery that raises the possibility of designing new drugs to uncloak the parasite and transform it into a personalised, live vaccine for its human host.

Cowman and co have pinpointed a potentially fatal weakness in one of the parasite’s key defensive strategies: its ability to find safe haven from prowling immune-system cells in the quiet backwaters of the bloodstream.

It does so by inducing infected red blood cells to stick to the endothelium of tiny blood vessels in major organs like the liver, intestine, placenta and, most lethally, the brain. Riding at anchor, away from the powerful currents of the bloodstream, it avoids being swept through the spleen with its hostile hordes of antibody-secreting B cells.

“It all started with our paper in Science in 2004 in which Marti et al identified a sequence required for the export of parasite proteins to the red blood cell,” Cowman says. “It allowed us to identify the P. falciparum exportome [the full complement of exported proteins]. Having identified the exportome, we wanted to work out what the important ones were doing.

Exportome proteins are identified by two unique motifs: a pentameric PEXEL (P. falciparum Export Element), and a hydrophobic element. But in many cases, time and mutation’s tides have obscured the DNA sequences specifying these elements by “static”, making them very difficult to identify by the standard approach of aligning and comparing selected DNA sequences from candidate exportome genes with model PEXEL sequences.

Toby Sargeant, a PhD student in Professor Terry Speed’s bioinformatics group at WEHI, has developed powerful new search tools that can peer through the clutter to detect PEXEL and hydrophibic elements embedded in candidate genes, considerably expanding the exportome catalogue.

“One of the interesting findings from Toby Sargeant’s PhD is that the falciparum exportome is much larger than in other Plasmodium species,” Cowman says.

The parasite re-engineers its human host’s red blood cells to express a sticky protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Knobs projecting from the surface of the infected cell are tipped with the adhesin protein. In the process, the parasite remodels the normally flexible cells into misshapen, rigid sacs that stick to the microvascular endothelmium

The transformation requires the parasite to export PfEMP1 and a supporting cast of exportome proteins through its own outer membrane, then through the erythrocyte’s membrane.

On the surface of the cell, they assemble into knob complexes, with PfEMP1 outermost. PfEMP1 anchors the infected erythrocyte to the microvasculature of the target organ.

---PB--- Yin and yang

The team has also shown that other exportome proteins are involved in remodeling erythrocytes. Instead of slipping smoothly through the microvasculature, the deformed, rigid sacs tend to “catch” in the microvasculature, presenting opportunities for PfEMP1 to bond with complementary proteins on the walls of blood vessels.

Cowman says other Plasmodium species don’t appear to require PfEMP1 and other exportome proteins for cytoadherence.

In P. falciparum, as many as 100 genes specify variants of PfEMP1 protein; each binds a complementary protein expressed on the endothelium of microscopic blood vessels in a specific organ or tissue. This complex system of yin-yang bonds specifies the organ in which the parasite will find refuge. By switching PfEMP1 variants, the parasite plays hide-and-seek with the immune system.

To mount any PfEMP variant on an erythrocyte’s surface, the parasite must deliver other exportome proteins through two membranes – its own, and the host cell’s – to be assembled into the sticky knob proteins.

The parasite proteins are transported through host erythrocytes that lack the specialised trafficking machinery of normal cells, then inserted into the erythrocyte membrane.

They form a highly organised skeleton that anchors PfEMP1 to the cell’s surface. Cowman says this transport and trafficking system for parasite proteins is unique in cell biology.

KAHRP (Knob-Associated Histidine-Rich Protein), identified by Cowman and WEHI colleague Brendan Crabb in 1997, is a key component of the knobs. Its binding with the membrane skeleton rigidifies the infected cells, causing them to lodge in tiny capillaries, obstructing them and restricting blood flow.

To determine which exportome genes and proteins were essential for PfEMP1 trafficking and function, Cowman’s team made single-gene knockout lines of the parasite – a technique he and Crabb pioneered in the late 1990s – and screened them for functional defects.

“We knocked out about 55 exportome genes, and 35 of them seemed to be essential – the parasite couldn’t do anything without them,” Cowman says.

“When we put all 55 knockout lines through a whole heap of function screens, we identified eight that are required for trafficking and function.

“These are clearly very important biologically to the parasite, which means they are potential new targets for therapeutics. It also raises the possibility of developing drugs that would create attenuated forms of living parasites, by disrupting knob assembly.”

Cowman says these non-adhering variants would be flushed into the bloodstream and carried through the spleen, where they would be exposed to antibody-secreting B-cells.

The attenuated parasites would not be able to cause disease, and would trigger the full defensive repertoire of the immune system, including antibody-secreting B cells and cytotoxic T-cells.

The attenuated parasites would effectively be transformed into a live vaccine, specific to whatever P. falciparum strain had infected that person, and eliciting an immune response unique to that individual.

“It doesn’t make sense to try to develop a vaccine targeting the exportome proteins, because most are expressed internally,” he says. “But we can make drugs that inhibit their function, preventing knob assembly.”

The beauty of this approach, says Cowman, is that the drugs wouldn’t kill the parasite – they would simply allow the immune system to have a long look at all of its surface proteins, not just a few that are can randomly switched so the parasite presents a constantly shifting target to the immune system.

The discovery could obviate the need to develop a vaccine, or vaccines, capable of protecting millions of genetically unique individuals in the world’s tropical regions against whatever unique regional or local strain of the P.falciparum they might encounter – even multi-drug resistant strains.