Development of Quantitative Proteomics Using iTRAQ Based on the Immunological Response of Galleria mellonella Larvae Challenged with Fusarium oxysporum Microconidia

Abstract

Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 104 or 106 microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 104 microconidia/mL at 37°C over expressed many more proteins than other treatments.

Galleria mellonella: An invertebrate model to study pathogenicity in correctly defined fungal species

“…Comparing the humoral response to different invading pathogens, gram negative, and gram positive bacteria, Candida, and Fusarium, revealed that the kinetics and grade of up-regulation of lysozyme and different antifungal peptides varies, depending on the class of the infecting agent, indicating that the G. mellonella immune system is able to distinguish between classes of pathogens (Mak et al 2010). Recently, a multiplex proteomic analysis, iTRAQ, was applied to the Galleria model, investigating changes in the expression level of proteins and peptides of larvae challenged with Fusarium conidia (Munoz-Gomez et al 2014). Validations of protein expression by RT-PCR showed that data obtained with iTRAQ were consistent, making it a useful tool for further investigations.…”

“…The cellular components, haemocytes, are able to phagocytose and kill pathogens by producing reactive oxygen species and lytic enzymes and can be seen as the invertebrate equivalent of neutrophils (Bergin et al 2005). The humoral response to infecting microbes comprehends antimicrobial peptides which were shown to be induced by fungal pathogens, and furthermore differ from those induced by bacterial pathogens (Bergin et al 2006;Munoz-Gomez et al 2014). This explains the comparable data obtained in Galleria virulence studies and murine models (Brennan et al 2002;Slater et al 2011).…”

Immunity of the greater wax mothGalleria mellonella

“…Then prepupae and pupae are formed and, after additional 2 weeks, adult moths appear. This bee moth has been a good model to study insect immune response and virulence factors of many pathogens, including human pathogens such as Pseudomonas aeruginosa , Enterococcus faecalis , Staphylococcus aureus , Candida albicans , Fusarium oxysporum , and Aspergillus fumigatus (Gibreel & Upton, ; Gomez‐Lopez et al ., ; Koch et al ., ; Munoz‐Gomez et al ., ; Maekawa et al ., ; Vaz et al ., ). Their virulence factors can be studied first on the insect model, which is easier, cheaper, and more ethically acceptable, before testing on mammalian organisms (Junquirella, ; Arvanitis et al ., ; Cook & McArthur, ).…”

Establishment and Validation of Galleria mellonella as a Novel Model Organism To Study Mycobacterium abscessus Infection, Pathogenesis, and Treatment

“…Larvae survive in wide range of temperatures (from 25 to 37°C), allowing the simulation of various physiologic conditions. This allows research of a wide range of pathogens, including fungal (Fusarium oxysporum [21,22], Aspergillus fumigatus [23], and Candida albicans [24,25]) and bacterial, such as Staphylococcus aureus (26), Proteus vulgaris (22), Serratia marcescens (27), Pseudomonas aeruginosa (28), Listeria monocytogenes (29), and Enterococcus faecalis (30). In our study, we establish the G. mellonella model for M. abscessus infection.…”