Abstract
BackgroundHelicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world’s population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection.ResultsIn the present study, we examined the effectiveness of the larvae of the wax moth Galleria mellonella as a new model for H. pylori infection. G. mellonella larvae were inoculated with bacterial suspensions or broth culture filtrates from either different wild-type H. pylori strains or their mutants defective in specific virulence determinants, such as VacA, CagA, CagE, the whole pathogenicity island (PAI) cag, urease, and gamma-glutamyl transpeptidase (GGT). We also tested purified VacA cytotoxin. Survival curves were plotted using the Kaplan-Meier method and LD50 lethal doses were calculated. Viable bacteria in the hemocoel were counted at different time points post-infection, while apoptosis in larval hemocytes was evaluated by annexin V staining. We found that wild-type and mutant H. pylori strains were able to survive and replicate in G. mellonella larvae which underwent death rapidly after infection. H. pylori mutant strains defective in either VacA, or CagA, or CagE, or cag PAI, or urease, but not GGT-defective mutants, were less virulent than the respective parental strain. Broth culture filtrates from wild-type strains G27 and 60190 and their mutants replicated the effects observed using their respective bacterial suspension. Also, purified VacA cytotoxin was able to kill the larvae. The killing of larvae always correlated with the induction of apoptosis in hemocytes.ConclusionsG. mellonella larvae are susceptible to H. pylori infection and may represent an easy to use in vivo model to identify virulence factors and pathogenic mechanisms of H. pylori. The experimental model described can be useful to screen a large number of clinical H. pylori strain and to correlate virulence of H. pylori strains with patients’ disease status.
Highlights
Helicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world’s population is colonized by the bacterium
H. pylori infection causes death of G. mellonella larvae We examined the susceptibility of G. mellonella to wild-type H. pylori strains G27, 60190 and M5, which are widely used for molecular pathogenesis studies
That killing of larvae is dependent on the expression of a functional cag pathogenicity island (PAI) and VacA cytotoxin is in accordance with previous data obtained in in vitro models showing that H. pylori-dependent epithelial cell damage and apoptosis of monocytes is dependent on VacA and cag PAI determinants [14]
Summary
Helicobacter pylori is the first bacterium formally recognized as a carcinogen and is one of the most successful human pathogens, as over half of the world’s population is colonized by the bacterium. H. pylori-induced gastroduodenal disease depends on the inflammatory response of the host and on the production of specific bacterial virulence factors, such as urease, the vacuolating cytotoxin VacA, gamma-glutamyl transpeptidase (GGT), and a 40-kbp pathogenicity island (cag PAI) encoding the 120–145 kDa immunodominant protein cytotoxin-associated gene A (CagA) as well as a type IV secretion system that injects CagA into the host cell [1,2,3,4,5,6,7,8,9]. Rodent models of wild-type mice, knock-out or transgenic mice and mongolian gerbils have been used to reproduce H. pylori persistent infection and disease [16,17,18] These mammalian models are very expensive and time-consuming because they require specific animal facilities not widely accessible to all research groups, a large number of animals in order to obtain statistically significant results, and a formal approval by the local Ethics Committee
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