ASM: Plasmodium's newest cousin

Nearly 12 years ago evolutionary biologist Geoff McFadden of the University of Melbourne stunned malaria researchers by announcing he had found a degenerate chloroplast in Plasmodium falciparum, the deadly agent of human malaria. The deadliest of five Plasmodium species that infect humans, P. falciparum kills more than a million children under the age of two every year.

McFadden's team later confirmed that the apicoplast has retained some of the biosynthetic machinery of the original chloroplast by showing that the broad-spectrum herbicide glyphosate (Roundup) kills P. falicparum.

The herbicide kills plants by disrupting the chloroplast-based shikimate pathway, which synthesises the amino acids tryptophan, phenylalanine and tyrosine.

The 175-odd species of Plasmodium have an oddly eclectic menagerie of vertebrate hosts that includes humans, non-human primates, bats, porcupines, squirrels, birds, and reptiles.

All are obligate blood parasites that never see the light of day, yet McFadden's discovery of an unpigmented chloroplast showed Plasmodium had evolved from a light-harvesting ancestor. What could it have been?

The answer has now been found at the bottom of Australia's most famous harbour.

As a PhD student with Associate Professor Dee Carter's research group of the School of Molecular and Microbial Biosciences at the University of Sydney, Robert Moore was attempting to isolate a dinoflagellate alga, Symbiodinium, from a scleractinian coral, Plesiastrea versipora, that grows in Sydney Harbour at a depth of around three metres.

All coral polyps host symbiotic zooxanthellae, specialised photosynthetic dinoflagellates that live in their tissues, harvesting light and synthesising carbon compounds for their hosts, in return for a steady supply of carbon dioxide. Symbiodinium is the dominant genus among the zooxanthellae.

"At the time Bob was very interested in trying to develop a system to culture Symbiodinium," Carter says.

"We were analysing the population genetics of Symbiodinium to look at sexual recombination in the genus, to determine if we were dealing with a single species with diverse genetics, or a population of different species with diverse genetics.

"Bob was picking out single cells, to see if he could find anything different, and found an unusual dinoflagellate. He started sequencing its DNA, and it looked very interesting, so he continued the work during his first postdoctoral appointment in the US."

The divergent DNA sequence indicated they had discovered an unrecognized genus of zooxanthellae. In a recent paper in Nature, they formally named it Chromera velia.

---PB--- Light on Chromera

Electron microscopy of the Chromera photosynthetic organ, or plastid, revealed it is enclosed by four membranes - the same arrangement that Geoff McFadden found in the Plasmodium apicoplast.

The chromera plastid is pigmented solely by chlorophyll a. Carter suggests the absence of chlorophyll b, found in all higher plants and most marine algae, indicates Chromera is vulnerable to light stress, and would be killed by bright sunlight near the surface - consistent with it living in a protected environment where it is exposed only to diffuse sunlight.

The Chromera DNA sequence also has a very unusual feature. Nearly all higher plants and marine algae use the codon UGG to specify a tryptophan residue. Chromera uses UGA - a feature unique to Apicomplexa, including Plasmodium.

The combination of these two characters suggests Chromera is a photosynthetic cousin of apicomplexan parasites, which have dispensed with chlorophyll altogether, but retain a degenerate plastid, the apicoplast.

Chromera and Plasmodium are both members of a recently recognised super-group of protists, the Alveolata, which incorporates foraminerifa, ciliates, dinoflagellates and Apicomplexa.

With the discovery of Chromera, the mists around the origins of Plasmodium have begun to lift.

On this evidence, Carter and her colleagues place Chromera somewhere between Apicomplexa and dinoflagellates. More than a decade ago, Geoff McFadden guessed that Plasmodium had descended from a red alga, probably a dinoflagellate.

Not all dinoflagellates are comfortably settled in symbiotic relationships. Free-living species flourish in the shallows of the world's oceans and seas, and are notorious as the toxic agents of so-called "red tides" that episodically poison fish and oysters.

People who eat have dined on predatory fish from tropical reefs in the wake of natural disturbances like cyclones have sometimes developed a bizarre food-borne illness called ciguatera, that reverses the sensations of hot and cold.

Predatory fish like barracuda, Spanish mackerel and grouper sit at the apices of marine food webs that concentrate ciguatoxin from the toxic dinoflagellate Gambierdiscus.

The uniquely toxic dinoflagellate Pfiesteria thrives in polluted seawater, and has caused massive fish kills in polluted rivers and estuaries in the north-eastern US.

Pfiesteria emits toxic molecules into water and air that have caused skin rashes and chronic mental confusion and memory loss in fishermen, and in scientists who unwisely cultured the so-called "Cell from Hell" in aquaria in poorly ventilated laboratories.

---PB--- Evolutionary arc

The evolutionary arc from toxic dinoflagellate to marine symbiont to deadly blood parasite is not improbable, given natural nature's inventiveness and opportunism.

Female Anopheles mosquitoes deliver the human-infecting species of Plasmodium as they seek a high-protein blood meal - various other blood-feeding insects including Culex midges provide the same service for Plasmodium species that infect other mammals, birds and reptiles.

Plasmodium takes temporary lodgings in the salivary glands of its intermediate host, and is careful not to kill its vector, so it can be delivered into its vertebrate host's bloodstream. Mosquito and midge larvae live in fresh or brackish water, like some dinoflagellates. Join the dots ...

Chromera is a photosynthetic dinoflagellate, that has adapted to life as intercellular symbiont in colonies of single-celled coral polyps, but as Robert Moore showed, is easily grown in culture, independently of its host.

Coral polyps pass their friendly dinoflagellates on to their daughters at meiosis, but zooxanthellae are facultative symbionts that can also exist as free-living cells in seawater.

At water temperatures above 32 degrees, stressed polyps expel their zooxanthellae back into the water - or the zooxanthellae quit the partnership. The result is coral bleaching. When the crisis passes, polyps reinstate the partnership by recruiting free-living zooxanthellae.

Plasmodium is a non-photosynthetic, intracellular parasite of vertebrate red blood cells that drifts in a fluid with the ancient salt signature of seawater.

The Apicomplexa include familiar waterborne pathogens like Cryptosporidium - which has dispensed altogether with its apicoplast - and Toxoplasma gondii, the urine-borne agent of cat-scratch disease, which can cause severe developmental abnormalities in babies if the mother is infected during pregnancy.

Carter et al observe in the introduction to their paper that the discovery of Chromera provides a powerful model with which to study the evolution of parasitism in Apicomplexa.

"This discovery shows that you can find some very interesting things in the Australian environment - unexpected things," Carter says. "It shows the value of curiosity-driven research.

"If we had told anybody we were going to look for the closest relative of the world's deadliest human parasite in the sea, we might have been laughed out of the room."

Carter believes the Chromera paper is the first taxonomic paper published in Nature for a long time. "Taxonomy has been so undervalued that it has been devastated [as a branch of science]," she says.

"We had some grant money from the Australian Biological Resources Survey, which is the only Australian agency that funds taxonomy at the moment, because the previous Federal Government just wasn't interested. The amount of money ABRS has to distribute is minuscule."

Plasmodium is tricky to grow, and cannot be replicated in vitro because of its multi-stage life cycle. Robert Moore has now demonstrated that its endosymbiont cousin, Chromera, with which it shares basic biosynthetic pathways, is easily grown and replicated in the laboratory.

The authors predict C. velia will be of practical use for high-throughput screening of prospective anti-apicoplast drugs - potentially a big bang for the few bucks invested by ABRS in the project.