Scientists at Duke University say they have sequenced the entire genome and all the RNA products of the most important pathogenic lineage of Cryptococcus neoformans, a strain called H99.

Their study (“Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation”), which appears in PLOS Genetics, also described 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.

C. neoformans is a fungus responsible for a million cases of pneumonia and meningitis every year. The Duke findings provide a playbook that can be used to understand how the pathogen causes disease and to develop methods to keep it from evolving into even deadlier strains, according to the researchers.

“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,” explained study coauthor John Perfect, M.D., a professor of medicine at Duke who first isolated H99 from a patient with cryptococcal meningitis 36 years ago.

Fred Dietrich, Ph.D., senior study author and associate professor of molecular genetics and microbiology at the Duke University School of Medicine, and his colleagues decided that the best way to investigate how the virulence of this pathogen could change over time was to develop a carefully annotated genomic map of the H99 strain, both in its original state as well as after it had been cultured. In an effort that took ten years and dozens of collaborators, the researchers sequenced the original H99 and nine other cultured variants, analyzing both the genome as well as the transcriptome.

The researchers found 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.

“We sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation,” wrote the investigators. “This genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.”

“Our results provide the groundwork needed to understand how this organism causes disease, because the next step will involve mutating every gene one by one to see which ones are required for pathogenesis,” added Joseph Heitman, M.D., Ph.D., senior study author and professor and chair of molecular genetics and microbiology at Duke.

The results will not only help researchers study this particular organism, but can also serve as a starting point for studies on other strains of C. neoformans, he added.

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