The relative differences of the malaria family

For decades, the origin of the deadliest of the four Plasmodium parasites that cause malaria has been an enigma.

One view is that P. falciparum has been the constant nemesis of humans and their early ancestors for millions of years.

But DNA evidence hinted that, like the viral agents of SARS and AIDS, it might be a relatively recent recruit to the ranks of human pathogens, possibly a fly-by-night scion of Plasmodium gallinaceum, acquired from the wild ancestor of modern chickens, the red jungle fowl, Gallus gallus, somewhere in Asia in the past 8000 years.

Dr Toby Sargeant, a member Professor Terry Speed’s bioinformatics research group at WEHI, has effectively eliminated P. gallinaceum as the progenitor of P. falciparum.

Sargeant’s powerful new statistical techniques for retracing evolutionary relationships between taxa hints that the host-switch may been in the opposite direction: the avian clade, including P. gallinaceum, may have been founded by a Plasmodium species that evolved in primates.

Plasmodium does have an ability to host-switch. Occasional cases of presumed P. malariae infection in Malaysia have recently been reassigned to a non-lethal Plasmodium species that infects rhesus macaques, P. knowlesii.

Malaria’s vertebrate hosts are an oddly eclectic lot: some 200 Plasmodium species form clades that specialise in parasitising rodents, birds, reptiles, and primates; two clades are primate specialists, infecting taxa as diverse as lemurs, macaques, gibbons, tamarins, and all the African and Asian great apes, including humans. Most infect more than one host species.

“If you look at the hosts’ phylogeny, there are large gaps that don’t appear to have been filled,” Sargeant says. “Ancient host-switching events seem to have been involved.

“Over the past 20 years, two questions remained unresolved. One was whether the two primate-infecting clades are closely or distantly related. Another is how the avian and reptilian malarias are related to the falciparum clade. My approach was to bring to bear as much data as possible to resolve that phylogeny.”

Sargeant began by analysing protein domains: short amino acid sequences that fold into evolutionarily conserved 3-D configurations.

“It’s not easy to say a particular gene in one species is the same as one present in another species,” he says. “You simplify the problem by looking at protein domains, for which the evolutionary origin is simpler to ascertain.

“The assumption underlying all these phylogenetic methods is that the triplet codons for amino acids are incompletely constrained, which allows changes to accumulate almost at random.

“So as related species accumulate unshared differences, we can use the relative differences to get an idea of how distantly related they are. The good thing about protein domains is that they tend to be constrained by function, so they change slowly relative to the DNA sequences that encode them.

---PB--- Evolutionary relationships

Sargeant says some slowly evolving domains remain informative over timescales of hundreds of millions of years. Others that mutate more rapidly are informative over shorter evolutionary timescales.

“So we developed methods for taking really large databases of all the instances of recognisable domains, and paired them up between sets of species,” he says.

“We use different domains for each comparative run, then from these groups of domains, you infer the tree which best describes the evolutionary relationships of all the species involved.”

As part of his PhD project, Sargeant developed search algorithms to complete a crucial census of all parasite proteins containing PEXEL elements (Plasmodium falciparum exportome elements) and hydrophobic “anchor” elements.

The combination of the two elements is the defining feature of exportome proteins. Sargeant’s techniques uncovered a number of new exportome proteins with cryptic PEXEL elements that had eluded previous searches. The patterns of duplication of these proteins suggested that the falciparum and vivax clades were more related to each other than to rodent parasites.

“Looking at shared protein domains, however, we found very strong support for falciparum and gallinaceum being more closely related to each other than to either the vivax primate clade or berghei rodent clade.

“These two observations were tied together when we looked outside Plasmodium at related Apicomplexan genera, to work out what came first, and found an indication that Plasmodium initially diversified in mammals.

“The rodent clade branched off first then both primate clades and then the avian clade branched off from the falciparum primate clade. Contrary to what was concluded from earlier phylogenetic studies, the host-switch seems to have been from primate to avian malaria.

“Another very interesting thing is that, as far as we’re aware, Plasmodium doesn’t infect any mammals apart from rodents and primates. The parasite appears to infect only African rodents, by and large, which supports the idea that malaria had its genesis in Africa.”

But if that’s the case, and the malaria parasite evolved before the rodent-primate split around 100 million years ago, Sargeant says it’s a challenge to explain why it doesn’t occur in other major mammalian groups like the carnivores and ungulates. “It could be taxonomic sampling bias, or it might be something deeper.”

---PB--- Phyologenetic condundra

The powerful new analytical techniques Sargeant has developed are applicable to all proteins, in any species. They are potentially capable of resolving some of the deepest phylogenetic conundrums in plant and animals.

For example, they could be used to peer through the fog of 50 to 80 million years of evolution, to resolve the 240-year old debate over whether fruit bats (megabats) descended from echolocating microbats, or whether fruit bats evolved independently from a gliding proto-primate, as argued by Australian neuroanatomist Professor Emeritus Jack Pettigrew.

Sargeant says his new techniques have the advantage that they do not depend on having annotated genomes from the species being compared – comparisons can be made “blind”, simply on the basis of matching shared protein domains.

“You can be somewhat opportunistic, which is important when dealing with distantly related taxa in which divergent sequences make it very difficult to identify equivalent genes for comparison,” he says.

“Methods like this are becoming more important as we get faster, cheaper sequencing. It will reach the stage where we can take a representative sample from a species, and with that, start to pick out domains.

“The most comprehensive databases currently contain about 9000 different domain patterns. Some will appear in a species, while others won’t. By studying examples of domains shared between taxa, we can infer their relationships.

“One set of domains won’t give us an accurate picture for all the species, but by overlaying data sets from taxa identified by different domains, we can build a broader and more accurate picture of relationships. Then, as we generate a set of domains for each taxon, we can place it on the tree of life.”