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

Abstract

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. Pseudomonas 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. Staphylococcus 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. G. 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).

Galleria mellonella es un modelo animal utilizado extensamente como alternativa para investigar la virulencia y patogenicidad bacteriana in vivo. También es apropiado para estudiar la eficacia y toxicidad de compuestos. Las larvas tienen un tamaño manejable, son económicas de adquirir y reproducir, presentan un bajo riesgo biológico, y son más aceptadas éticamente. Además, tienen un sistema inmunológico innato muy similar al de los mamíferos. Utilizamos G. mellonella para desarrollar un modelo animal de infección y toxicidad estandarizado y reproducible. Pseudomonas aeruginosa, un patógeno oportunista, que infectando emplea ribonucleótido reductasa (RNR), catalizando la reducción de ribonucleótidos a desoxirribonucleótidos y proporcionando así las moléculas precursoras necesarias para la síntesis de ADN. Desarrollamos un vector sin promotor con bioluminiscencia, el cual se utilizó para construir fusiones con los promotores de los genes RNR. Además, optimizamos un protocolo de extracción de ARN bacteriano para facilitar el estudio de los niveles transcripcionales de genes in vivo. Debido a la multiresistencia emergente de Staphylococcus aureus, se probó la toxicidad y eficacia antimicrobiana de nuevos derivados del ácido oleanólico y maslínico en G. mellonella. De los catorce derivados probados, dos tenían menos toxicidad y más eficacia in vivo que in vitro. G. mellonella se usó para determinar la toxicidad de nanopartículas y estrategias terapéuticas. Mycolicibacterium brumae no fue tóxica para las larvas y los resultados se correlacionaron con los obtenidos con ratones. Las nanopartículas causaron efectos tóxicos en las larvas detectados por la medición de la dosis letal y la proliferación de hemocitos, entre otros indicadores. Debido a la amplia aplicabilidad de G. mellonella, se necesitan nuevas metodologías para maximizar su potencial. Además del protocolo de extracción de ARN previamente mencionado, también se optimizó otro de aclaramiento. Las larvas fueron inyectadas con nanopartículas, fijadas con paraformaldehído, permeabilizadas con metanol y aclaradas con alcohol bencílico y benzoato de bencilo.