New tools for maggot debridement therapy research: From the establishment of qRT-PCR to the characterization of Lucilia sericata Urate Oxidase

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BaumannAndre_2017_04_25.pdf (8.75 MB)

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2017

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DOI:
http://dx.doi.org/10.22029/jlupub-10410

Abstract

Maggot debridement therapy (MDT) is an U.S. Food and Drug Administration (FDA) as well as European Medicines Agency (EMA) approved treatment for various chronic and recalcitrant wounds. MDT was successfully applied in numerous case studies (Sherman 2002; Sherman 2003) and shown to have beneficial effects on wound debridement, disinfection and hastened healing processes (Sherman 2014). Larval secretions and excretions (E/S) represent a complex mixture of enzymes, AMPs and small molecules and as such serves as a rich source of new therapeutics (Sherman 2014). Unfortunately, the complexity of this mixture makes it very hard to decipher the exact roles and benefits of individual components in the wound healing process and requires more detailed studies (Davydov 2011).This thesis is based on two parts. The first part, published in PLOS ONE (Baumann et al. 2015), focused on the establishment of a qRT-PCR approach by investigating tissue specific expression stability of 10 candidate reference genes in immune challenge experiments. Quantitative RT-PCR serves as a golden standard in the field of gene expression (Bustin et al. 2009; Nolan et al. 2006) and its establishment in L. sericata serves as an important tool for identification and validation of genes involved in maggot therapy. My study shows that the combination of three reference genes RPLP0, EF1alpha and RPS3 provides reliable normalization of L. sericata expression data among all tested samples, thus allowing for the first time the precise normalization of gene expression in larval tissues involved in production of E/S. The methods of tissue sample preparation and gene expression analysis established during this thesis also contributed to Beckert et al. (2015) and Beckert et al. (2016). Reference genes were applied in Pöppel et al. (2016) and Franta et al. (2016).Second part of this thesis, submitted to Insect Biochemistry and Molecular Biology (Baumann et al. 2016), focuses on recombinant production and characterization of L. sericata Urate Oxidase (UO) an enzyme that exclusively creates allantoin, which contributes to the wound healing process (Araujo et al. 2010; DiSalvo 2002). I was the first to produce and characterize an insect UO. Recombinant UO was produced using E. coli expression system, purified via IMAC (denaturing chromatography), refolded and tested for its pH optimum (in the alkaline), temperature optimum (20-25 °C), competitive inhibition (typical for UO), cofactors (cofactor independent) and stability (short shelf life). Furthermore, I monitored the presence of L. sericata UO on mRNA as well as protein level in various L. sericata tissues and showed that both UO gene as well as native UO localize predominately inside Malpighian tube cells sharing strictly cytosolic localization. Based on these data it can be assumed that allantoin is produced by UO to remove uric acid from the insect hemolymph by the Malpighian tubes and is excreted via the hindgut as nitrogenous waste product. Those findings support the hypothesis that not only actively secreted molecules, but also excretion products contribute to the beneficial effects of MDT.

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