P3-09 Development and evaluation of the galleria mellonella (greater wax moth) model to study brucella host pathogen interaction

Abstract

Brucellosis is a zoonosis caused by bacteria of the genus Brucella. These gram-negative bacteria cause long-lasting infections, a process in which Brucella PAMP-modifications ( e.g. lipopolysaccharide [LPS]) hamper a prompt and effective activation of innate immunity. Laboratory models are essential to investigate Brucella virulence and although mice and, less frequently, guinea pigs have been used, ethical and practical considerations impede their use in high-throughput screening studies. Although lacking the complexity of mammalian immune system, the insect Galleria mellonella (greater wax moth) is increasingly being used as infection model as it conserves key aspects of innate immunity with mammals and has been useful in virulence analyses of relevant pathogens such as Klebsiella, Legionella, Mycobacteria and Listeria. To assess the potential of this model for the study of Brucella virulence, we evaluated G. mellonella larva survival upon infection with B. abortus 2308W wild-type and attenuated mutants (i.e. defective in the VirB Type-IV Secretion System [T4SS] or in the LPS-O- polysaccharide [O-PS]), and B. microti CCM4915. Then, we explored whether the survival profiles were related with a differential replication of brucellae by CFU-counting of whole-larva homogenates and fluorescence microscopy of primary- phagocyte isolates. Finally, we evaluated the ability of G. mellonella immunity to efficiently recognise Brucella by quantification of the pro-phenoloxidase system and melanisation activation after infection or LPS inoculation. As compared to K. pneumoniae 52145, B. abortus and B. microti induced a delayed and less severe mortality profile. Moreover, typical- brucellae did not trigger an early-melanisation response, consistent with the low immunostimulatory properties of Brucella LPS. Finally, we observed that Brucella virulence in Galleria is influenced by VirB and O-PS, since the corresponding mutants displayed even more marked delayed and less severe mortality profiles than the parental strain. Intriguingly, bacterial replication within larvae was affected by the lack of O-PS, but not of a functional T4SS. In light of these results, the G. mellonella model may represent an alternative tool for the study of Brucella interaction with innate immune, although the suitability of this model for the study of other aspects of Brucella pathogenesis, such as Brucella intracellular life, remains to be elucidated.