Stenotrophomonas chitinases: The key to Batrachochytrium dendrobatidis inhibition?

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Mary Lally, Elle Barnes and Erin Carter, Biological Sciences, Fordham University, Bronx, NY, Elle Barnes, DOE Joint Genome Institute, Berkeley National Lab, Berkeley, CA, J.D. Lewis, Graduate School of Arts and Sciences - Biology, Fordham University, Armonk, NY

Background/Question/Methods
Batrachochytrium dendrobatidis (Bd), or the chytrid fungus, is a pathogen that has led to the decline of many amphibian species globally. However, bacteria living on some amphibians’ skin have been shown to slow Bd’s deadly spread. More specifically, Stenotrophomonas spp., found on the eastern redback salamander (Plethodon cinereus), have been shown to consistently and considerably inhibit Bd growth. Despite Stenotrophomonas spp. inhibitory ability, each isolate can slightly differ in its antifungal ability. These differences are thought to be related to chitinolytic ability. Since the chitinases of the Stentrophomonas spp., particularly S. rhizophila, have not been extensively studied, we investigated how chitinase genetic variation contributes to differences in Stenotrophomonas-related Bd-inhibition. We hypothesized that differences in antifungal ability would be related to changes in the Stenotrophomonas chitinases. To do this, we developed six pairs of degenerate primers to amplify the chitinase genes of ten isolates and sent for Sanger sequencing. Then, we used Geneious and MEGA software to analyze the differences amongst the isolates’ genes and amino acid sequences and compared variation to inhibitory ability.
Results/Conclusions
We amplified the complete chitinase gene of four Stenotrophomonas rhizophila and two S. maltophilia isolates in addition to partial sequences from four more S. rhizophila isolates. There was significant variation among the nucleotide and amino acid sequences. Compared to the most antifungal isolate’s chitinase, the nucleotide sequences had an average percent identity of 85.8%, and the amino acid sequences had an average of 86.4%. Additionally, we found evidence of partially-duplicated chitinases in the chromatograms. However, despite this variation in the isolates’ chitinase genes and amino acids, there was no clear correlation between percent inhibition and percent identity. Even though chitinase amino acid variation is not necessarily linked to changes in antifungal ability in Stenotrophomonas spp., the genus still possesses key inhibitory abilities that should be explored. In the future, ELISAs may reveal additional antifungal proteins released by the isolates and necessary for Bd inhibition, and the Stenotrophomonas spp. could be used as probiotics to slow Bd spread. By delving into the genomics of antifungal ability, we hope to add new options for the conservation of amphibians.