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Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

ABSTRACT: Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites.

SUBMITTER: Goupil LS

PROVIDER: S-EPMC4976874 | biostudies-literature | 2016 Aug

REPOSITORIES: biostudies-literature

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Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

Goupil Louise S LS Ivry Sam L SL Hsieh Ivy I Suzuki Brian M BM Craik Charles S CS O'Donoghue Anthony J AJ McKerrow James H JH

PLoS neglected tropical diseases 20160808 8

Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA int ...[more]

PMID: 27501047

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Project description:Obligate bacterial symbionts alter the diets of host animals in numerous ways, but the ecological roles of facultative bacterial residents that colonize insect guts remain unclear. Carabid beetles are a common group of beneficial insects appreciated for their ability to consume insect prey and seeds, but the contributions of microbes to diet diversification in this and similar groups of facultative granivores are largely unknown.Using 16S rRNA gene clone libraries and terminal restriction fragment (tRF) length polymorphism analyses of these genes, we examined the bacterial communities within the guts of facultatively granivorous, adult Harpalus pensylvanicus (Carabidae), fed one of five dietary treatments: 1) an untreated Field population, 2) Seeds with antibiotics (seeds were from Chenopodium album), 3) Seeds without antibiotics, 4) Prey with antibiotics (prey were Acheta domesticus eggs), and 5) Prey without antibiotics. The number of seeds and prey consumed by each beetle were recorded following treatment. Harpalus pensylvanicus possessed a fairly simple gut community of approximately 3-4 bacterial operational taxonomic units (OTU) per beetle that were affiliated with the Gammaproteobacteria, Bacilli, Alphaproteobacteria, and Mollicutes. Bacterial communities of the host varied among the diet and antibiotic treatments. The field population and beetles fed seeds without antibiotics had the closest matching bacterial communities, and the communities in the beetles fed antibiotics were more closely related to each other than to those of the beetles that did not receive antibiotics. Antibiotics reduced and altered the bacterial communities found in the beetle guts. Moreover, beetles fed antibiotics ate fewer seeds, and those beetles that harbored the bacterium Enterococcus faecalis consumed more seeds on average than those lacking this symbiont.We conclude that the relationships between the bacterium E. faecalis and this factultative granivore's ability to consume seeds merit further investigation, and that facultative associations with symbiotic bacteria have important implications for the nutritional ecology of their hosts.

Dataset Information

Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

Publications

Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

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