The researchers said their findings provide important additional insight into the origin of the Vancouver Island outbreak, which began in 1999. Moreover, the evidence that sex played an important role in the pathogen's expansion may provide a useful model for the evolution of infectious diseases and parasites more generally, they said.
The team reported its findings October 9, 2005, in an advanced online publication of Nature. The work was supported by the National Institute of Allergy and Infectious Disease.
After extensive genetic analysis of fungal samples, the researchers suggest that mating between two less harmful fungal strains of the same sex or "mating type" produced the more virulent form. That strain has now taken hold and appears to be spreading -- perhaps driven by unique conditions in the Vancouver area, they said.
"While the number of people infected so far does not approach that of many other infectious diseases, this fungus is invading the central nervous systems of people who have no other apparent risk factors except having taken a walk in the park on Vancouver Island," said Joseph Heitman, M.D., Ph.D. "A year after infection, some of these people still have not fully recovered.
"The fungus appears to have become entrenched in the Vancouver Island area," he added. "It is unlikely to disappear, and all indications are that it is spreading. Our findings suggest that sex played a role in the expanded geographic range for this pathogen."
Since it was first documented in 1999, C. gattii has infected at least 100 people on Vancouver Island and the Canadian mainland and led to four deaths. The fungus, which lives in trees and soil, has also infected a variety of domestic and marine animals, including dogs, cats, llamas and porpoises.
C. gattii is closely related to the more widespread infectious yeast, Cryptococcus neoformans. The potentially life-threatening C. neoformans invades the central nervous system to cause disease. It most commonly affects immune-compromised patients such as organ transplant recipients and cancer patients -- whose immune systems are crippled by immunosuppressive drugs or chemotherapy -- and people with HIV/AIDS. In contrast, C. gattii infects individuals with apparently normal immunity. Symptoms include persistent headaches, coughing and night sweats. In rare cases, C. gattii causes cryptococcal disease, pneumonia, meningitis or death.
While C. neoformans is found worldwide in association with pigeon droppings, the rarer C. gattii is normally restricted to tropical and subtropical areas, often in association with Eucalyptus trees.
"It is suspected that the infectious propagules of Cryptococcus are airborne spores," Heitman said. "Such spores are produced during sexual reproduction, though mating of the fungus has never been observed in nature."
In plants and animals, sexual identity is governed by sex chromosomes, Heitman explained. In fungi, however, sexual identity is determined by so-called "mating type loci," genes arranged contiguously, but which typically do not span an entire chromosome. Cryptococcus exists in two mating types, "a" and "alpha," determined by a single genetic region, or locus.
Earlier studies by the Duke team found that most Vancouver Island outbreak isolates are sexually fertile, but all are of one "sex," a trend that would seem to preclude the normal sexual cycle. A recent laboratory study led by Heitman's group suggested a possible explanation: the related yeast C. neoformans can undergo same-sex mating between two alpha partners.
Among clinical and environmental isolates of the fungus from British Columbia, the researchers identified two forms: an extremely virulent major strain, which accounted for 95 percent of all samples, and a less virulent and less common strain, which made up the other five percent.
By comparing select gene sequences that spanned the genomes of the Vancouver Island fungi to samples collected from around the world, the team traced the rarer type to identical isolates in Australia. The major form matched a sample taken from an infected person in Seattle 30 years ago and another collected from a Eucalyptus tree in San Francisco in 1992.
What's more, the Canadian strains shared approximately half of their genetic makeup, suggesting that the two might be related. Further analysis confirmed this initial finding, suggesting that the two C. gattii strains in Vancouver Island are either siblings or that one is the parent and the other the progeny.
"Given that the minor outbreak form also exists in multiple locations in Australia, while the major outbreak form has only been found in the Pacific Northwest, we favor the hypothesis that the minor type represents one of two parental strains that gave rise to the major outbreak isolate," said study author James Fraser, Ph.D., also of Duke. "The second parent strain remains to be discovered."
Additional examination of the mating type locus provided evidence that the major outbreak isolate may have resulted from same-sex mating, Heitman added. In a traditional sexual cycle, all alpha progeny inherit identical alpha genes from their alpha parent. However, the mating type locus of the two strains from Vancouver Island – both of the alpha mating type -- differed at numerous sites, they found.
"Sex within the same mating-type may confer an evolutionary advantage when the opposite mating type is unavailable," Heitman said. "Other human pathogens or parasites may harbor cryptic same-sex cycles that contribute to produce progeny with altered virulence, geographic or host range or other advantageous characteristics."
Collaborators on the study include Steven Giles, Emily Wenink, Scarlett Geunes-Boyer, Jo Rae Wright, Stephanie Diezmann, Andria Allen, Jason Stajich, Fred Dietrich and John Perfect, all of Duke.
Journal
Nature