Investigation of thyroid hormone-dependent molecular changes in the bullfrog back skin with special emphasis on the innate immune system




Corrie, Lorissa

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As the aquatic tadpole undergoes metamorphosis to become a terrestrial juvenile frog, its innate immune system must adapt to its new environment. Skin is a primary line of defense in the innate immune system since it acts as an important physical, microbial, and chemical barrier that is constantly in contact with a microbially diverse environment throughout its life cycle. In amphibians, skin undergoes complex remodelling during postembryonic development. Metamorphosis is regulated by thyroid hormone (TH) initiated gene expression that leads to the start of the metamorphic programming. This can be induced by the addition of exogenous TH. While different tissues respond to TH in distinctive ways during metamorphosis, how these tissues respond to TH is poorly understood. Temperature modulation, which regulates metamorphic timing, is a unique way to uncover early TH-induced transcriptomic events related to a molecular memory. Using RNA-sequencing analysis, American bullfrog (Rana [Lithobates] catesbeiana) back skin transcripts were profiled during natural and temperature-modulated induced metamorphosis. During natural metamorphosis, significant differential expression was observed in over 6,500 transcripts. Premetamorphic tadpoles maintained at 5oC, a temperature that is non-permissive for inducing morphological changes, showed 83 differentially expressed transcripts within 48 h after TH administration. Of note is the induction of thibz that has previously been identified as a molecular memory component in other tissues. Over 3,600 differentially expressed transcripts were detected in TH-treated tadpoles compared to the controls at permissive temperature (24oC) after 48 h or when tadpoles were held at 5oC and then shifted to 24oC. We identified several innate immune system components: keratins, mucins, and antimicrobial peptides (AMPs) whose transcript levels changed during natural and TH-induced metamorphosis. A bioinformatics AMP identification pipeline, rAMPage, was applied to these RNA-seq data sets to identify 489 novel AMP candidates. AMPs have direct acting and immune stimulatory antimicrobial abilities and are promising antibiotic alternatives in public health and agricultural sectors. We synthesized and tested un-amidated and amidated C-terminal versions of 111 top-scoring novel AMP candidates for their antimicrobial activity and cell toxicity with a focus on agriculturally relevant pathogens (avian pathogenic Escherichia coli (APEC) and Salmonella enterica serovar Enteritidis (SE)). We found that 28 peptides were moderately or highly active (Minimum inhibitory concentration  32 μg/mL) against APEC and six against SE, most of which displayed low cell toxicity. Active AMPs were physicochemically classified as positively charged, alpha helices with amphipathic characteristics. These AMPs are candidates for further testing as therapeutics to prevent or treat disease in poultry. While the present work is focused on the identification of amphibian AMPs, it has far-reaching applications, and the approach can be used to identify AMPs as an antibiotic alternative from any genomic resource.



amphibian, antimicrobial peptide, thyroid hormone, innate immune system, RNA-sequencing, transcriptomics, antibiotic resistance