Playbook for a pathogen

DURHAM, N.C.—Cryptococcus neoformans, an encapsulated mushroom-like fungus responsible for a million cases of pneumonia and meningitis every year, is not a problem for most people unless they are immune-suppressed, according to Dr. John Perfect, a professor of medicine at Duke University who first isolated the H99 strain of the organism from a patient with cryptococcal meningitis 36 years ago. However, the pathogen has caused significant mortality and morbidity, especially in sub-Saharan Africa where HIV remains largely unchecked in many areas.

Perfect and other researchers have sequenced the entire genome and all the RNA products of the H99 strain, the most important pathogenic lineage of C. neoformans. The results, which appear April 17 in PLOS Genetics under the title “Analysis of the genome and transcriptome of Cryptococcus neoformans var. grubii reveals complex RNA expression and microevolution leading to virulence attenuation,” also describe a number of genetic changes that can occur after laboratory handling of H99 that make it more susceptible to stress, hamper its ability to sexually reproduce and render it less virulent.

The study provides a playbook that can be used to understand how the pathogen causes disease and develop methods to keep it from evolving into even deadlier strains. According to Perfect, “We are beginning to get a grasp on what makes this organism tick. By having a carefully annotated genome of H99, we can investigate how this and similar organisms can change and mutate and begin to understand why they aren’t easily killed by antifungal medications.”

The pathogen primarily infects individuals with compromised immune systems, such as patients undergoing transplant or those afflicted with HIV/AIDS. Researchers have spent many years conducting genetic, molecular and virulence studies on the H99 strain C. neoformans, noting that over time, the strain became less and less virulent as they grew it in the laboratory.

To investigate how the virulence of this pathogen could change over time, the Duke researchers developed a carefully annotated genomic map of the H99 strain, both in its original state and after it had been cultured. In an effort that took 10 years and dozens of collaborators, the researchers sequenced the original H99 and nine other cultured variants, analyzing both the genome, the genetic code written in the DNA, and the transcriptome, the RNA molecules that occupy the second step in the flow of genetic information from DNA to RNA to protein. They discovered that the organism possessed a number of molecular tricks—such as the ability to produce genetic messages from both strands of DNA—that enable it to adapt and survive in changing conditions. The researchers discovered that the original and cultured strains were surprisingly similar to each other.

“Cryptococcus neoformans has to cope with a large number of different stresses and probably needs a very flexible metabolism. It is tempting to hypothesize that its complex RNA metabolism provides a mechanism to achieve such flexibility,” says Dr. Guilhem Janbon, lead study author and faculty in molecular mycology at the Pasteur Institute.

After scanning the 20 million A’s, C’s, T’s and G’s that make up the pathogen’s genetic code, they found only 11 single nucleotide variants and 11 insertions or deletions that could explain why cultured strains behaved differently.

Eventually, the researchers will study other strains of C. neoformans. They will also try to use the research to find the path to drugs that will kill the organism faster. Some of the currently available drugs are highly toxic, but sometimes drug combinations have to be used, according to Perfect.

To combat the effects of the pathogen, Perfect believes that researchers have to “push new drug development,” and there is NIH funding for that purpose at Duke. He also believes that new diagnostic tests, including one that performs a lateral flow assay with a dipstick in blood or urine, could provide earlier point-of-care screening for high-risk patients in areas where there is minimal medical expertise.

The research was supported by grants from the National Institutes of Health and a grant from the French National Research Agency. “It was a true multidisciplinary study involving bioinformatics, pathogenesis, clinical medicine and other disciplines, showing the power of how people can work together to understand microevolution,” Perfect notes.