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Open Access Methodology article

Use of larvae of the wax moth Galleria mellonella as an in vivo model to study the virulence of Helicobacter pylori

Maria Giannouli12, Anna Teresa Palatucci34, Valentina Rubino3, Giuseppina Ruggiero3, Marco Romano5, Maria Triassi2, Vittorio Ricci1* and Raffaele Zarrilli26*

Author Affiliations

1 Department of Molecular Medicine, Human Physiology Section, University of Pavia Medical School, Pavia, Italy

2 Department of Public Health, Hygiene Section, University of Naples “Federico II”, Naples, Italy

3 Department of Translational Medical Sciences, University of Naples “Federico II”, Naples, Italy

4 PhD School of Science, University of Basilicata, Potenza, Italy

5 Department of Clinical and Experimental Medicine, Chair of Gastroenterology, Second University of Naples, Naples, Italy

6 CEINGE Biotecnologie Avanzate, Naples, Italy

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BMC Microbiology 2014, 14:228  doi:10.1186/s12866-014-0228-0

Published: 27 August 2014

Abstract

Background

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. The study of Helicobacter pylori pathogenic action would greatly benefit by easy-to-use models of infection.

Results

In 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.

Conclusions

G. 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.

Keywords:
Helicobacter pylori; Virulence factors; Galleria mellonella; Infection; Apoptosis