The team used a genetic technique called transposon mutagenesis sequencing, or TN-seq, where they damaged C. neoformans’ genome by bombarding millions of cells with small DNA segments called transposons.

“The analogy we use to explain TN-seq dates back to WWII,” said Billmyre. “Fighter planes returning to hangars were mapped for bullet damage to devise ways to strengthen them. However, areas of planes lacking damage were not necessarily better reinforced, but rather were never mapped because they never returned, a phenomenon called survivorship bias.”

Transposons landing within essential genes cause the fungal cells to die. By sequencing the DNA of the surviving cells, researchers can map which genes are vital for survival and which are not. Zanders explained: “The TN-seq approach mirrors this survivorship bias with transposon-ridden fungi. When we look genome-wide at all the places with and without damage, we can infer that if you damage a required region of the genome, the organism will die.”

TN-seq has been used widely in bacteria and in more established fungal species like baker's yeast. This is the first time the approach was adapted for C. neoformans. It allowed the team to create a mutant library for C. neoformans—with millions of transposon-induced mutations including those in DNA that regulate essential genes. The researchers could then ask even more nuanced questions, such as which genes contribute not only to survival but also to resistance of antifungal drugs.

“Traditional methods involve deleting one gene at a time, but TN-seq lets us make deletions for the entire genome, allowing us to rapidly identify the repertoire of essential genes in Cryptococcus,” said Billmyre. “In addition, we were also able to use the tool to test both essential and non-essential genes that confer resistance to the most common antifungal, fluconazole.”

Billmyre was recently awarded the prestigious NIH New Innovator Award to examine how fungi evolve to grow at high temperatures, which is key to understanding pathogenicity.

"My lab is now trying to understand the network of genes that enable fungal pathogens to grow at human body temperature,” said Billmyre. “This can inform us of what might happen in the future if increases in global temperature cause different species of fungi to acquire pathogenic properties.”

Additional authors include Caroline Craig, Joshua Lyon, Claire Reichardt, Amy Kuhn, and Michael Eickbush. 

This work was funded by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) (awards: DP2GM132936, R35GM151982), the National Institute of Allergy and Infectious Diseases of the NIH (award: DP2AI184725), and with institutional support from the University of Georgia and the Stowers Institute for Medical Research. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. 

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