Bacteria, We Can See Your Halo: Chitinases in the Amphibian Skin Microbiome

Abstract

Microbial communities can play an important role in protecting hosts against pathogens, but the mechanisms underlying microbiome-mediated host defense are still not fully understood. Amphibian skin microbiomes are an important system for studying these interactions because some bacteria found on amphibian skin can inhibit the growth of the fungal pathogen Batrachochytrium dendrobatidis (Bd), which has contributed to global amphibian declines. My dissertation research focused on expanding our understanding of the mechanisms involved in microbiome-mediated host defense, with a primary focus on the role of bacterial chitinase genes within the amphibian skin microbiome. My dissertation builds on previous work conducted in the Belden lab using a collection of 666 bacterial isolates cultured from skin swabs of four amphibian species in Virginia. Prior work demonstrated that dominant members of the amphibian skin microbiome were more likely to inhibit Bd growth and identified several candidate genetic pathways potentially associated with antifungal activity, including chitinase genes. These findings provided the foundation for investigating the functional importance of bacterial chitinases in amphibian host defense. In chapter two, I investigated the prevalence, diversity, and functional activity of chitinase genes in amphibian skin bacterial isolates. I established an in vitro plate assay to test for chitin degradation in 27 isolates with annotated genomes and combined these functional assays with genomic analyses to characterize chitinase diversity. I also used PCR to screen 124 additional amphibian skin isolates for the presence of chiA genes to better understand how widespread these genes are within the amphibian skin microbiome. Although multiple bacterial genera contained predicted catalytic chitinase domains, only Janthinobacterium and Streptomyces isolates demonstrated observable chitin hydrolysis. In addition, neither Bd inhibition ability nor functional chitin degradation predicted the presence of chitinase genes, suggesting that genomic predictions alone may not accurately reflect antifungal activity. One important finding from this chapter was that Streptomyces isolates contained the greatest diversity and abundance of chitinase genes. In chapter three, I expanded this work through a comparative genomics study of amphibian skin Streptomyces isolates collected from Panama. Since Streptomyces are medically important bacteria and major producers of natural products, this project also had broader applications beyond amphibian systems. Across 11 unique isolates, only three demonstrated functional chitin hydrolysis, and no relationship was found between catalytic domain copy number and chitin degradation. Comparative genomic analyses also identified 17 biosynthetic gene clusters with the potential to produce siderophores, antibiotics, and antifungal compounds. In my final chapter, I broadened my research focus beyond amphibian systems to investigate relationships between mucosal microbiomes and disease status in shelter-housed kittens with upper respiratory infections. Using bacterial amplicon sequencing, I characterized ocular and throat microbiomes in kittens of varying health statuses including healthy, sick,. Healthy kittens had similar bacterial richness and dominant genera across ocular and throat samples, while samples from sick kittens were dominated by members of the families Mycoplasmataceae. Overall, my dissertation integrates microbiology, comparative genomics, and ecology to better understand the host microbiome and its potential roles in disease dynamics. This work highlights the challenge of linking microbial genomic traits with functional activity and demonstrates that predicted antimicrobial genes do not necessarily correspond with observed antifungal function.