Sequenced parasite tells more about pathogen's dirty work

March 2, 2005 -- The combination of devastating diseases such as HIV and medical breakthroughs such as organ transplants and drugs like chemotherapy and steroid treatments has created a sea of immunocompromised people worldwide.

This, in turn, has provided a job opening for an obscure fungus discovered in the 19th century that can lead to brain infection and death in those with compromised immune systems.

Now, a team of collaborators, including two researchers from Washington University in St. Louis, has sequenced the genomes of two strains of the fungus Cryptococcus neoformans (C. neoformans), one which is virulent, the other harmless. This work provides researchers with clues on how the fungus does its dirty work and a host of genes to study for a better understanding of fungal pathogens in general.

Michael R. Brent and Tamara L. Doering examine data from the C. neoformans microarray. The husband-wife researchers had the rare chance to collaborate when they put their expertise to work on this genome sequencing project.

Estimates are that about 15 percent of people with HIV will suffer at least one life-threatening infection of C. neoformans. In Africa, that could be as much as 40 percent of HIV sufferers.

Michael R. Brent, Ph.D., Washington University professor of computer science and engineering, and Tamara L. Doering, M.D., Ph.D., associate professor of molecular microbiology in the Washington University School of Medicine , have provided key inputs into the interpretation of the genomes.

Brent and his graduate student Aaron Tenney used Brent's gene prediction software TWINSCAN to discover some 1,200 genes not found by traditional analysis methods. These predicted genes were included in a gene expression microarray that Doering and Brent designed. This new tool allows researchers to observe the expression of all Cryptococcus genes and how their expression levels change under different circumstances.

Doering, who has devoted much of her research over the past eight years to C. neoformans, has shed light on the unique polysaccharide capsule that envelops the fungus and helps it evade the body's defenses. Because the genome sequencing project identified about 30 new genes that likely are involved in building the capsule, scientists now could find ways to interfere with this process and stop infection.

The Washington University researchers are part of a long-term collaborative group on this project that includes scientists from St. Louis University, The Institute for Genomic Research (TIGR), in Rockville, Md., and the Stanford Genome Technology Center in California, among many national and international participants. Results were published. Jan. 13, 2005, in the online "express" version of the journal Science: http://www.sciencemag.org/sciencexpress/recent.shtml

The focus of Doering's laboratory is the process by which the capsule is made. Understanding that might lead to developing antifungal drugs, because the capsule is a structure unique to the virulent C. neoformansstrain.

Fungi as 'wimpy' pathogens

"As pathogens, fungi are kind of wimpy," said Doering. "Most fungi don't cause disease, and for those that do, host factors determine how severe the disease is. A typical fungal cell builds a sort of protective wall, but C. neoformans attaches a fibrous coating around that wall to interact with the host environment. This coat is called the capsule."

Researchers also identified differences between a highly virulent strain of C. neoformans and one that doesn't cause severe infection. These differences may hold the key to understanding why this particular fungus is so virulent and may help to develop effective treatments.

Brent's TWINSCAN software predicts the existence of genes by looking at two genomes in parallel and homing in on statistical patterns in the individual DNA sequences of each genome. In a Nov. 2004 issue of Genome Research, Tenney, Brent, Doering, and collaborators reported that many TWINSCAN-predicted genes that were missed by more conventional gene-finding techniques could be verified experimentally. Early results from the C. neoformans microarray support these findings on a genome-wide scale, and indicate under which conditions these genes are expressed.

"One of the new things about this genome is that it's the first genome of a single-celled organism that has complex gene structure," said Brent. "It's much more complex than Brewer's yeast, for example, more similar, in fact, to the roundworm C. elegans.

"Another remarkable aspect of this work is that it was one of the earliest genome sequencing projects where there are two genomes of subspecies for comparison. That is interesting and advantageous for gene prediction."

Brent and Doering are married. The 2004 study is their first collaboration.

"We never thought that we'd collaborate." said Brent, whose specialty in computational biology has made him valuable in the sequencing and analysis of numerous high-profile genomes.

"It's really, really fun to work together," said Doering, whose emphasis as a molecular microbiologist is on studying proteins to determine their function. "We hope our scientific paths cross again, but that's hard to predict."