Date of Award

4-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbiology, Immunology, and Biochemistry

Research Advisor

P. David Rogers, PharmD, PhD

Committee

Glen E. Palmer, PhD; Brian M. Peters, PhD; Todd B. Reynolds, PhD; Michael A. Whitt, PhD

Keywords

antimicrobial resistance, Candida parapsilosis, fluconazole, triazole

Abstract

Invasive candidiasis is a severe fungal infection associated with significant morbidity and mortality, particularly among the critically ill and immunocompromised. Candida parapsilosis is the most common non-albicans species causing invasive Candida infections in pediatric and neonatal populations worldwide and is particularly common in the countries of South America, Western Asia, Mediterranean Europe, and Southern Africa. For many of these countries, fluconazole and other triazoles are the first line antifungal agents used for effective treatment of invasive Candida infection. Until recently, rates of fluconazole resistance among C. parapsilosis isolates were relatively low, therefore the determination of clinically relevant resistance mechanisms in C. parapsilosis isolates were primarily presumed from the observations of Candida albicans. Gain-of-function polymorphisms in MRR1 and TAC1 have been shown to elevate the expression of MDR1 and CDR1/CDR2 respectively and directly contribute to fluconazole resistance in Candida albicans. Our lab previously identified three resistant isolates with upregulated CpMDR1 expression that contained CpMRR1 mutations, while CpTAC1 mutations were found in three isolates with upregulated CpCDR1 expression. Deletion of CpMDR1 or CpCDR1 from strains containing the nonsynonymous CpMRR1 or CpTAC1 polymorphisms had little to no impact on fluconazole minimum inhibitory concentrations (MIC), suggesting the presence of uncharacterized resistance effectors. This dissertation reviews the emergence of resistance, presents investigations of three major mediators of fluconazole resistance, and characterizes a collection of clinical isolates to better identify and understand how fluconazole resistance in C. parapsilosis. A recently developed CRISPR-Cas9 system was used to edit CpMRR1 alleles in the clinical isolate backgrounds and allowed for characterization of the single nucleotide polymorphisms (SNPs) leading to the substitutions A854V, I283R, and R479K and gain-of-function in CpMrr1. Antifungal susceptibility testing demonstrated that gain-of-functions (GOF) in CpMrr1 increased fluconazole MIC 128-fold when placed into a susceptible background while correction of CpMRR1 SNPs to the wildtype nucleotides decreased fluconazole MICs  32-fold. Transcriptional profiling revealed the previously identified CpMDR1, the novel major facilitator superfamily (MFS) transporter, herein named CpMDR1B, and an ATP-binding cassette (ABC) transporter, herein designated CpCDR1B, were all upregulated by CpMrr1 GOF. Our development of a promotor replacement method for C. parapsilosis and implementation of a barcoded gene disruption system, demonstrated the direct contribution of CpCDR1B and CpMDR1B expression on fluconazole susceptibility and confirmed expression of CpMDR1 was not a primary driver of CpMrr1-mediated resistance. Subsequent investigation of putative GOF mutations in CpTAC1 showed correction of a SNP leading to the G650E substitution in a resistant clinical isolate decreased fluconazole MICs by 32-fold. Transcriptional profiling showed elevated expression for three ABC transporters, CpCDR1, CpCDR1B, and a gene identified here as CpCDR1C. Utilizing the overexpression and disruption systems, we demonstrated the direct effects of CpCDR1, CpCDR1B and CpCDR1C on triazole MICs. The single base editing system was also used to place the SNP leading to the Y132F substitution into the triazole drug target CpErg11 of susceptible isolates. Antifungal susceptibility testing demonstrated this frequently cited driver of resistance was insufficient in-and-of itself to elicit high-level fluconazole resistance in C. parapsilosis. Finally, next generation sequencing was used to genotypically and phenotypically characterize the entire clinical isolate collection to identify key and potentially novel markers of fluconazole resistance in C. parapsilosis. Phylogenic analysis revealed distinctive clusters of isolates with similar resistance mechanisms while implying fluconazole resistant C. parapsilosis both with and without the CpErg11 substitution, Y132F were capable of persisting in healthcare facilities. Additionally, eight isolates with clinical fluconazole resistance demonstrated distinct patterns of upregulated MFS and ABC transporters compared to the susceptible isolates while the presence of wildtype CpMRR1, CpTAC1, CpERG11, CpUPC2 and CpERG3 suggests novel mediators of fluconazole resistance within C. parapsilosis. The absence of meaningful CpERG11 upregulation among resistant clinical isolates alongside distinct expression patterns for both of MFS and ABC transporters emphasizes the importance of looking beyond CpErg11 when investigating clinical resistance in C. parapsilosis. Understanding how resistance is regulated, develops, and even persists among clinical isolates is fundamental to the preservation of triazoles as effective treatments for invasive C. parapsilosis infection.

Declaration of Authorship

Declaration of Authorship is included in the supplemental files.

ORCID

https://orcid.org/0000-0002-1695-9561

DOI

10.21007/etd.cghs.2023.0620

Additional Files
2023-007-Doorley-DOA.pdf (257 kB)
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