PULLMAN – Researchers at WSU have shown that the ability of some pathogens to “superinfect” animals that were already infected is the driving force behind greater genetic diversity in those pathogens. Their results shed light on the evolution of some of the most debilitating pathogens of humans and animals, including those that cause malaria, sleeping sickness and syphilis.

Their report appeared in the Feb. 12 issue of the Proceedings of the National Academy of Sciences.

The research team, led by National Academy of Sciences member Guy Palmer, worked with a blood parasite that afflicts cattle. Anaplasma marginale is the most prevalent tick-borne pathogen of cattle worldwide, infecting more than two-thirds of the cattle in some regions. Anaplasma’s ability to spread in such areas has puzzled researchers, because most potential hosts it encounters will already be infected and thus able to mount an immune response against the newcomer.

“If you’re in an area where everybody’s already infected, it’s kind of a dead end,” said Palmer of the problem faced by Anaplasma in what might be called a saturated market. “You’ve got to find a way to get in [to those hosts]. One way to get in is to make sure that you’re different enough that you look like a different pathogen.”

The research team found that for Anaplasma, the key to being able to superinfect was having a different form of a gene that codes for a major surface protein. Previous work in Palmer’s lab showed that the protein, called Msp2, is the part of the pathogen that most of the host’s defenses are directed against.

Each Anaplasma organism has five unique genes for Msp2, which it cuts and splices together in various combinations to create new versions of the protein. Just as the host’s immune system gears up to recognize and attack one form of Msp2, the genes shuffle again to produce a new form that the immune system won’t recognize. That process continues throughout the life of the host, enabling the pathogen to remain in the host indefinitely.

The WSU researchers had also previously observed that one cow may carry infection by more than one strain of Anaplasma. In such superinfections, not all combinations of strains were found to co-exist within one cow. The reason for that wasn’t clear.

In the current study, the research team set out to discover what conditions make superinfection possible. They first examined the amount of variation in the genes for Msp2 in five strains of Anaplasma. They found that the sequences coding for Msp2 varied as much between strains as within a given strain. The variation within a given strain was expected; it’s what allows the pathogen to change its appearance and evade attack by a host’s immune system. The degree of variation between strains was somewhat surprising, said Palmer. It would not help a strain succeed within an individual host, but Palmer and his co-workers thought it could be related to the pathogen’s need to infect new hosts that are already infected with Anaplasma.

To find out if that was the case, the researchers tested pairs of Anaplasma strains to see whether one strain could infect a host that was already carrying a long-term infection by another strain. They found that if the two strains had the same repertoire of Msp2 genes, no superinfection occurred. The second strain just couldn’t gain a foothold in the host, probably because the host’s immune system recognized it and promptly disposed of it. However, if the second strain had a different set of Msp2 genes than the strain that caused the original infection, it did superinfect. Palmer said the different set of Msp2 genes allowed the second strain to look different enough from the first strain that it was not immediately attacked as a known invader.

Since some pairs of superinfecting strains had no overlap in their Msp2 genes while others differed by only a single gene, the question became, “How different do they have to be?” Palmer said. The answer, it turned out, was that a single different Msp2 gene was enough to allow superinfection. Upon closer inspection, the researchers found that the second strain to enter a cow left all of its Msp2 genes silent except the one that the first strain did not have. The researchers concluded that the presence of that one different Msp2 gene allowed the second strain to escape immune attack long enough to establish an infection.

Palmer said the ability to superinfect provides the “driving force to be different”—conferring a strong selective advantage on strains that evolve at least one form of the Msp2 gene that is different from the forms found in other strains. The advantage would be especially strong in areas where Anaplasma is widespread, he said.

While focusing on Anaplasma, Palmer said the study has implications for understanding other pathogens as well.

“I think the principle is broadly applicable to a large number of organisms,” Palmer said. The organisms that cause malaria, sleeping sickness, relapsing fever, gonorrhea, and syphilis probably do much the same thing, he said. Like Anaplasma, all those organisms persist for a long time in one host by modifying their surface proteins so they can stay a step ahead of the host’s immune response.

Palmer said influenza virus is perhaps the best-known example of a pathogen that can re-infect hosts who have already been infected. The need to exploit already-infected hosts had been thought to be important in more complex pathogens as well, said Palmer, but the current study was one of the first to examine that possibility in detail. Tracking the development of new genetic strains of virus has been possible because viral genomes are very small and relatively easy to sequence and study. More complex pathogens like Anaplasma, a bacterium whose genome is about a thousand times larger than that of a flu virus, have been much harder to analyze.

The paper: “Superinfection as a driver of genomic diversification in antigenically variant pathogens,” by James E. Futse, Kelly A. Brayton, Michael J. Dark, Donald P. Knowles Jr., and Guy H. Palmer. Proceedings of the National Academy of Sciences, vol. 105:2123-2127. http://www.pnas.org/cgi/doi/10.1073/pnas.0710333105