Deciphering the utility of Galleria mellonella as an infection and toxicity in vivo model

Galleria mellonella (greater wax moth) is a popular animal model that has been extensively used as an alternative in vivo model for investigating the virulence and pathogenicity of different bacteria. G. mellonella has also been shown to be a suitable model for studying the efficacy and toxicity of various compounds. Recently, this model has been gaining popularity as the larvae are conveniently sized for manipulation, they do not need constant feeding, they are inexpensive to purchase and to breed, they do not require much space or special infrastructure, they present a low biohazard risk, and they are more ethically accepted. More importantly, G. mellonella has an innate immune system very similar to the one found in mammals. In this thesis, G. mellonella was used to develop a standardized and reproducible animal model of infection and toxicity. \nPseudomonas aeruginosa is an opportunistic pathogen that has gained great medical importance as it causes serious illnesses in humans and it can be resistant to many antibiotics. During infection, ribonucleotide reductases (RNR) play an essential role as they catalyze the reduction of ribonucleotides to deoxyribonucleotides, thus providing the precursor molecules needed for DNA synthesis. Since G. mellonella has been proven to be a suitable model for P. aeruginosa infections, we developed a promoter probe vector with bioluminescence expression to enhance the study and monitoring of a P. aeruginosa in vivo infection. This vector was used to construct different RNR gene promoter fusions as proof of concept. Additionally, we optimized a total bacterial RNA extraction protocol to facilitate the study of transcriptional gene levels during in vivo infections. \nStaphylococcus aureus is also considered an opportunistic pathogen. This bacterium is also capable of forming biofilms and it is considered an important cause of biofilm formation in catheters and prostheses. Due to the misuse and overuse of antimicrobials, multi-resistant bacteria are rapidly appearing so there is a critical need for new antimicrobials. The toxicity and antimicrobial efficacy against S. aureus of novel oleanolic and maslinic acid derivatives were determined using G. mellonella. Out of the 14 derivatives tested, 2 were found to have improved toxicity and efficacy in vivo when compared to the in vitro results. \nG. mellonella was also used to test the toxicity of other therapeutical strategies and nanoparticles (NPs). Mycolicibacterium brumae was not toxic to G. mellonella larvae, and the results correlated with the results obtained with mice. The different NPs caused a variety of acute toxicity effects that were detected by an array of indicators within the larvae, such as lethal dose calculation, hemocyte proliferation, NP distribution, behavioral changes, and histological alterations. Due to the broad applicability of the G. mellonella model, new methodologies are warranted to exploit its full potential. Besides the optimized RNA extraction protocol already mentioned, an optical clearing protocol was also optimized in this work. As a proof of concept for our larvae clearance protocol, fluorescent rhodamine NPs were injected into larvae that were then fixed with paraformaldehyde, permeabilized with increasing concentrations of methanol, and cleared with BABB (Benzyl Alcohol and Benzyl Benzoate).