C. elegans as an in vivo model to decipher microbial virulence. Centre d Immunologie de Marseille-Luminy

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1 C. elegans as an in vivo model to decipher microbial virulence Centre d Immunologie de Marseille-Luminy

2 C. elegans : a model organism Mechanisms of apoptosis, RNA interference Neuronal function and development Intracellular signaling pathways Stress, pathogenesis (Huntington, muscular dystrophy ) Host-pathogen interactions

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5 Why C. elegans? Small size 1mm Thousands of animals on a Petri dish Transparent Reporter gene expression Study bacterial infection in vivo Rapid generation time From egg to gravid adult in 2 days 300 eggs laid in 3 days Clonal All the worms in a population can be genetically identical 0.1 mm image taken from Devgen nl

6 Why C. elegans to study host-pathogen interactions? Some Pathogens have a broad range of hosts. Some of their virulence factors might be used whatever the host is. Universal virulence factors. Science (1995) Ausubel Lab Using C. elegans as a host can allow the identification of these virulence factors.

7 Known pathogens of C. elegans Gram-positive bacteria: Bacillus thuringiensis Enterococcus faecalis Listeria monocytogenes Microbacterium nematophilum Staphylococcus aureus Streptococcus pneumoniae Gram-negative bacteria: Fungi: Arthrobotrys sp. Candida albicans Cryptococcus neoformans Drechmeria coniospora Duddingtonia flagrans Paecilomyces lilacinus Acinetobacter baumannii Aeromonas hydrophila Agrobacterium tumefaciens Burkholderia cepacia B. thailandensis B. pseudomallei Erwinia christamthemi E. carotovora carotovora Escherichia coli (EPEPC & EHEC) Pseudomonas aeruginosa P. fluorescens Salmonella typhimurium Shewanella frigidimarina S. massalia Serratia marcescens Vibrio cholerae Yersinia spp. VIRUSES VSV, FHV

8 Assays to test for infectivity or toxicity Is the microorganism intoxicating or infecting C. elegans?

9 Survival of C. elegans on different bacteria E. coli OP50 E. coli 1106 Bacillus megaterium Time (days Couillault & Ewbank (2002) Infect & Immun.

10 Infection of C. elegans by different bacteria 100 Worms alive (%) E. coli OP50 Photorhabdus luminescens Hb Xenorhabdus nematophila A24 Aeromonas hydrophila AH6 Aeromonas hydrophila AH10 Shewanella massalia Shewanella frigidimarina Time (days) Toxicity or infectivity? Couillault & Ewbank, Infect & Immun. (2002)

11 Infectivity or toxicity? % worms alive E. coli OP50 Aeromonas hydrophila AH6 Agrobacterium tumefaciens Photorhabdus luminescens Hb Xenorhabdus nematophila A24 Erwinia carotovora Time (days) Couillault & Ewbank (2002) Infect & Immun.

12 Infectivity or toxicity? + Filters Survival?

13 Infectivity or toxicity? P. aeruginosa PA01 Darby et al., (1999) PNAS Toxin secreted in the media P. aeruginosa PA14 Mahajan-Miklos et al., Cell (1999) Tan et al., PNAS (1999) Infection S. marcescens Db11 Kurz et al., (2003) Embo J

14 In vivo observation of the infection

15 E. coli does not enter the intestine Short contact with fluorescent E. coli Intact bacteria Autofluorescent gut granules Kurz et al. (2003) Embo J

16 S. marcescens enters the intestinal lumen Short contact with fluorescent S. marcescens Kurz et al. (2003) Embo J

17 Intestinal colonization E. coli S. marcescens Kurz et al. (2003) Embo J

18 Assays to decipher microbial virulence Use of nematodes to identify microbial virulence genes required for infection in mammals

19 C. elegans to identify universal virulence factors Unbiaised forward genetic screens for attenuated bacterial mutants

20 Screening protocol Bank of bacterial Mutants (transposon) Synchronized worms n individual clones 96, 24 or 6 well plates Infection Identification of less virulent bacterial clones

21 PA14 Universal virulence factors 2000 mutants screened in C. elegans 8 mutants Decreased virulence in: Arabidopsis 6 7 Wax moth 6 Mahajan-Miklos et al. (2000) Mol. Microbiol.

22 Serratia marcescens Universal virulence factors 2000 mutants screened in C. elegans 23 mutants Decreased virulence in: Drosophila 10 Human cells 3 1/3 Kurz et al. (2003) Embo J

23 Assays to rapidly detect infectivity or toxicity

24 Use of the worm as a biosensor for microbial pathogenicity Already done by several groups to assess soil pollution Identification of genes induced under a specific treatment Construction of transgenic nematodes with fluorescent reporters No heavy metals Heavy metals

25 Antifungal innate immunity

26 D. coniospora kills worms 100 % worms alive Time (Hours)

27 Antimicrobial peptide genes are induced 100 Microarrays % worms alive Time (Hours) Antimicrobial peptide genes nlps Couillault et al. 2004

28 A reporter gene to detect an immune response Pathogen transgenic strain pcol-12 RFP constitutive pnlp-29 GFP inducible Non-infected Infected

29 Why C. elegans to assess potential toxicity in humans? Cost, speed reproducibility In vivo assays with a multicellular organism Natural infection Tools available to decipher molecular mechanisms Ethical issues

30 Drawbacks and conclusions Many cases where infectivity in nematodes means infectivity in mammals (Serratia, Pseudomonas, Salmonella, ) But, cases with nematode-specific infectivity Drechmeria, Microbacterium, Mammalian-specific pathogens will be missed So, infectivity in nematodes is not a good sign for mammals, but a lack of infectivity doesn t mean it s innocuous for vertebrates

31 Equipe FRM Jonathan Ewbank s lab C. Couillault J. Belougne S. Cypowyj L. Kurz K.Z. Lee I. Engelmann N. Pujol B. Squiban K. Ziegler O. Zugasti Tohey Matsuyama Cori Bargmann Niigata New York Elizabeth Pradel Centre d Immunologie de Marseille-Luminy