# Causes and Consequences of Hypermutability in Cryptococcus neoformans

> **NIH NIH F31** · DUKE UNIVERSITY · 2021 · $34,763

## Abstract

ABSTRACT
 To successfully survive and compete within their environmental niches, microorganisms must
stochastically acquire mutations or face evolutionary stagnation. Although increased mutation rates are often
deleterious in multicellular organisms, hypermutation can be beneficial for microbes in the context of a strong
selective pressure. To explore how hypermutation arises in nature and elucidate its consequences, we
employed a recently assembled collection of 387 sequenced clinical and environmental isolates of Cryptococcus
neoformans, a fungal pathogen responsible for approximately 15% of AIDS-related deaths annually. HIV-positive
individuals diagnosed with cryptococcal meningitis face unacceptably high mortality rates: up to 70% in low
income nations and 30% in North America. This high mortality is attributable to a dearth of antifungal treatment
options, so limited because of the conserved homology between many essential fungal and human proteins, and
to the high rates of resistance to antifungal drugs. Preliminary screening for the ability of each isolate to acquire
resistance to otherwise lethal concentrations of diverse antifungal agents has identified 30 hypermutator strains,
including two robust hypermutators. Characterization of the resistant colonies the two isolates produced revealed
that insertion of a single transposable element (TE) was largely responsible for de novo drug resistance. Long-
read whole genome sequencing (WGS) revealed that both hypermutator genomes encode >600 copies of this
TE and harbor a nonsense mutation in the first exon of an RNAi component known to be involved in TE silencing,
ZNF3. Quantitative trait loci mapping of F1 segregants from a genetic cross between one of the hypermutators
and the laboratory reference strain identified a single significant peak associated with hypermutation that
includes the mutant znf3 allele. Therefore, our central hypothesis is that hypermutability due to frequent
transposition in these isolates is attributable to the presence of a novel, functional TE in the C. neoformans
lineage as well as an RNAi defect. To determine the genetic and molecular basis of this elevated transposition
and define its impact on the fitness of these strains and their ability to acquire drug resistance in host-relevant
conditions, we propose two specific aims. In aim 1, genetic complementation, deletion, and reconstitution will
be used to define the roles of ZNF3 and the identified TE in hypermutation. Analysis of WGS of other isolates
in the collection will be conducted to identify suppressor mutations and characterize the evolutionary trajectory
of the identified hypermutator alleles. In aim 2, we will determine how these increased mutation rates and
transposition contribute to fitness and drug resistance in vitro through competition assays and Etests and in vivo
in Galleria mellonella and murine infection models. The combination of this powerful eukaryotic model
organism’s extensive history in th...

## Key facts

- **NIH application ID:** 10170252
- **Project number:** 5F31AI143136-03
- **Recipient organization:** DUKE UNIVERSITY
- **Principal Investigator:** Shelby Jordan Priest
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $34,763
- **Award type:** 5
- **Project period:** 2019-07-01 → 2022-05-08

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10170252

## Citation

> US National Institutes of Health, RePORTER application 10170252, Causes and Consequences of Hypermutability in Cryptococcus neoformans (5F31AI143136-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10170252. Licensed CC0.

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