Author ORCID Identifier

https://orcid.org/0000-0001-9909-4702

Date of Award

Summer 6-21-2024

Document Type

Thesis (Ph.D.)

Department or Program

Microbiology and Immunology

First Advisor

Deborah Hogan

Abstract

Chronic fungal infections are highly recalcitrant to treatment; we postulated that as populations persist, increasing genetic diversity is reflected in phenotypic heterogeneity, contributing to treatment inefficacies. The study of evolutionary patterns is underrepresented in chronic fungal infections, and to supplement this body of knowledge, we leveraged isolates acquired from four individuals with chronic fungal-dominated cystic fibrosis infections. We evaluated in-host evolution through a whole-genome sequencing approach, comparing multiple isolates obtained from each subject's sputum and bronchoalveolar lavage fluid. Our analysis found non-synonymous mutations that arose in parallel across the independent infections in the gene MRS4, which encodes a mitochondrial inner membrane iron transporter.

We used CRISPR-Cas9 genome editing for allele swaps to demonstrate that MRS4 alleles found in vivo encoded variants with decreased activity relative to the reference allele. RNAseq analysis further supported that MRS4 variants had acquired mutations that reduced function and affected broad iron storage and regulation changes. We posit that loss of Mrs4 activity was positively selected during chronic lung infections for the enhanced ability to store intracellular iron, potentially in response to nutritional iron restriction, which is considered to be a common feature of the CF lung.

During our investigation, we unexpectedly observed an unreported phenotype of MRS4 mutation, the degradation of an extracellular pH dye, bromocresol purple (BCP). We investigated candidate genes for degradation activity using GRACE conditional expression strains and determined the ferric reductase-like CFL2 as necessary and sufficient, disproving our initial hypotheses that BCP degradation was mediated by laccase or sulfonate dioxygenase activity. We similarly observed heterogeneity in degradation activity in clinical isolates of mixed species Candida lung infections collected over eight years, and preliminary results indicated that BCP degradation can be used to distinguish isolates with a hyperactive iron acquisition response and potentially even identify different mutations acquired in genes such as NRG1.

This work elucidates a new conserved target for fungal evolution, has implications for future antifungal drug development, unravels the regulation and effector of biodegradation activity that may influence the way that Candida species interact with the infection environment, and will enhance our understanding of the selective pressures within the human lung.

Original Citation

Chapter 2 was originally published as Murante D, Demers EG, Kurbessoian T, Ruzic M, Ashare A, Stajich JE, Hogan DA. 2023. Mrs4 loss of function in fungi during adaptation to the cystic fibrosis lung. mBio 14:e0117123

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