# Deciphering the significance of pathogen genetic variation and evolution

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2024 · $726,574

## Abstract

PROJECT SUMMARY
Using a combination of genetics and experimental evolution, our team seeks to understand complex epistasis
with respect to genetic background in the human pathogen Cryptococcus neoformans. This yeast is an ideal
platform for such investigations due to 1) its facile genetics, 2) the availability of hundreds of phylogenetically
well-separated genomes, and 3) mouse infection models that recapitulate key aspects of human disease. Most
studies have been performed with a reference strain, the clinical isolate H99, or in congenic derivatives (KN99a
and KN99). In unpublished work, our lab has generated a gene deletion strain collection in the KN99
background and profiled it in mice and in over 100 diverse in vitro conditions. A reference strain is important for
the field to be able to compare findings, but it has a major weakness: across genetic backgrounds, it is unknown
whether the same genes are 1) essential for viability, 2) mediate the responses to small molecule challenges,
and 3) are required for fitness in the mammalian host. In addition to being of fundamental scientific importance,
answering these questions, which all relate to the larger question of complex epistasis, is important for
developing effective therapies across genetic backgrounds and understanding drug resistance. Population
genomic analyses have revealed that C. neoformans exists in roughly four major clades that diverged ~5 million
years ago and show evidence of speciation. Over a million SNPs have been identified in a set of nearly 400
strains. Members of each clade have been identified in human infections, and there is evidence for clade-
specific traits. Anecdotal studies have provided examples of strain-specific phenotypes, raising the question of
what genotype-phenotype relationships are general vs. dependent on genetic background and the underlying
mechanisms. Powerful new CRISPR/Cas9-based tools that we have recently developed and other
technological advances now make it feasible to address this fundamental question. In this work, we will
determine how Cryptococcus neoformans genetic background impacts the fundamental traits of gene
essentiality (Aim 1), the role of genes in fitness in the mammalian host (Aim 2), and the fitness roles of genes
under diverse environments (Aim 3). We will pursue mechanisms of the latter through experimental evolution
(Aim 3).

## Key facts

- **NIH application ID:** 10944871
- **Project number:** 1R01AI185204-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Hiten D Madhani
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $726,574
- **Award type:** 1
- **Project period:** 2024-07-08 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10944871

## Citation

> US National Institutes of Health, RePORTER application 10944871, Deciphering the significance of pathogen genetic variation and evolution (1R01AI185204-01). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10944871. Licensed CC0.

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