Novel approaches to characterizing feline-associated dermatophytic fungi
Date
2022
Authors
Moskaluk, Alexandra Elizabeth, author
VandeWoude, Sue, advisor
Daniels, Josh, committee member
Schissler, Jennifer, committee member
Reynolds, Melissa, committee member
Journal Title
Journal ISSN
Volume Title
Abstract
Dermatophytes are highly infectious fungi that cause superficial infections in keratinized tissues in humans and animals. This group of fungi is defined by their ability to digest keratin and encompasses a wide range of species. Classification of many of these species has recently changed due to genetic analysis, potentially affecting clinical diagnosis and disease management. In Chapter One, we review dermatophyte classification including name changes for medically important species, current and potential diagnostic techniques for detecting dermatophytes, and an in-depth review of Microsporum canis, a prevalent zoonotic dermatophyte. M. canis commonly causes dermatophytosis in humans and cats, and is adapting to its primary host (domestic cats) as one of its mating types (MAT1-2) appears to be going extinct. Assessment of genetic variation among M. canis isolates in the United States has not been conducted. Further, M. canis mating type and assessment of disease severity associated with genotypic characteristics have not been rigorously evaluated. In Chapter Two, M. canis was isolated from 191 domestic cats across the US and characterized genotypes by evaluation of ITS sequence, MAT locus, and microsatellite loci analysis. The genes SSU1 and SUB3, which are associated with keratin adhesion and digestion, were sequenced from a subset of isolates to evaluate potential genetic associations with virulence. Analysis of microsatellite makers revealed three M. canis genetic clusters. Both clinic location and disease severity were significant predictors of microsatellite variants. 100% of the M. canis isolates were MAT1-1 mating gene type, indicating that MAT1-2 is very rare or extinct in the US and that asexual reproduction is the dominant form of replication. No genetic variation at SSU1 and SUB3 was observed. These findings pave the way for novel testing modalities for M. canis and provide insights about transmission and ecology of this ubiquitous and relatively uncharacterized agent. Chapter Three evaluated four dermatophytosis cases occurring in kittens collected from the study in Chapter Two that yielded fungi with colony morphology more similar to Arthroderma species than Microsporum. Morphologic and microscopic examinations were conducted, and gene segments for the ITS, β-tubulin, and translation elongation factor 1-alpha (TEF1) regions were sequenced from DNA extracted from these cultures. Sequences were aligned to other dermatophytes using maximum likelihood and neighbor-joining trees and were compared to previously described fungal species to assess nucleotide homology. We identified two previously undescribed fungal species, herein as Arthroderma lilyanum sp. nov. and Arthroderma mcgillisianum sp. nov. M. canis co-cultured in two of the four cases. Other physiologic tests supported this diagnosis. These species have significance as potential pathogens and should be considered as rule-outs for dermatophytosis in cats. The potential for infection of other species, including humans, should be considered. In Chapter Four, we investigated a critical adhesion protein (Sub3) utilized by M. canis during initial stages of infection, analyzing its production and expression under varying growth conditions. Additionally, as this protein must be expressed and produced for dermatophyte infections to occur, we developed and optimized a diagnostic antibody assay targeting this protein. While clinical samples of M. canis were found to have low Sub3 production, Sub3 levels were increased in culture when grown in baffled flasks and supplemented with either L-cysteine or cat hair. As Sub3 was also produced in cultures not supplemented with keratin or cysteine, this study demonstrated that Sub3 expression is not reliant on the present of keratin or its derivatives. These findings could help direct future metabolic studies of dermatophytes, particularly during the adherence phase of infections. Chapter Five explored two molecular approaches for developing diagnostic assays for dermatophytosis based on keratin metabolites: sulfite and S-sulfocysteine (SSC). Currently, fungal culture is still considered the "gold standard" for diagnosing dermatophytosis, however, modern molecular assays have overcome the main disadvantages of culture, allowing for tandem use with cultures. The first approach involved a starch and iodine indicator that reacts with sulfite and SSC, resulting in a visual color change. While this method had a low limit of detection, the indicator had many off-target reactions, leading to low specificity for dermatophyte metabolites. The second approach utilized tandem liquid chromatography with mass spectrometry, targeting SSC. Using the same cultures performed in Chapter Four, we were able to detect and quantify SSC from M. canis cultures grown with hair at days 15 and 18 post inoculation. These findings demonstrated that SSC is consumed/degraded by the fungi, particularly during early growth stages. Collectively, this work provides future directions for genetic and metabolic studies of dermatophytes and how to leverage unique characteristics of dermatophytes for developing novel diagnostic assays. We conclude that M. canis genetics influence clinical disease presentation and further whole genome studies could help elucidate key genetic regions involved in dermatophyte pathogenesis. Furthermore, as M. canis continues to adapt to its primary host of cats, having a rapid, accurate diagnostic assay will become even more critical, particularly in high-density populations.