C. elegans as an in vivo model to decipher microbial virulence Centre d Immunologie de Marseille-Luminy
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
http://www.wormatlas.org/
http://www.wormatlas.org/
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 http://www Devgen nl
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.
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
Assays to test for infectivity or toxicity Is the microorganism intoxicating or infecting C. elegans?
Survival of C. elegans on different bacteria 100 90 80 70 60 50 40 E. coli OP50 E. coli 1106 Bacillus megaterium 30 20 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (days Couillault & Ewbank (2002) Infect & Immun.
Infection of C. elegans by different bacteria 100 Worms alive (%) 90 80 70 60 50 40 30 E. coli OP50 Photorhabdus luminescens Hb Xenorhabdus nematophila A24 Aeromonas hydrophila AH6 Aeromonas hydrophila AH10 Shewanella massalia Shewanella frigidimarina 20 10 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (days) Toxicity or infectivity? Couillault & Ewbank, Infect & Immun. (2002)
Infectivity or toxicity? 100 90 % worms alive 80 70 60 50 40 30 20 10 E. coli OP50 Aeromonas hydrophila AH6 Agrobacterium tumefaciens Photorhabdus luminescens Hb Xenorhabdus nematophila A24 Erwinia carotovora 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Time (days) Couillault & Ewbank (2002) Infect & Immun.
Infectivity or toxicity? + Filters Survival?
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
In vivo observation of the infection
E. coli does not enter the intestine Short contact with fluorescent E. coli Intact bacteria Autofluorescent gut granules Kurz et al. (2003) Embo J
S. marcescens enters the intestinal lumen Short contact with fluorescent S. marcescens Kurz et al. (2003) Embo J
Intestinal colonization E. coli S. marcescens Kurz et al. (2003) Embo J
Assays to decipher microbial virulence Use of nematodes to identify microbial virulence genes required for infection in mammals
C. elegans to identify universal virulence factors Unbiaised forward genetic screens for attenuated bacterial mutants
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
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.
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
Assays to rapidly detect infectivity or toxicity
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
Antifungal innate immunity
D. coniospora kills worms 100 % worms alive 50 0 0 12 24 36 48 60 Time (Hours)
Antimicrobial peptide genes are induced 100 Microarrays % worms alive 50 0 0 12 24 36 48 60 Time (Hours) Antimicrobial peptide genes nlps Couillault et al. 2004
A reporter gene to detect an immune response Pathogen transgenic strain pcol-12 RFP constitutive pnlp-29 GFP inducible Non-infected Infected
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
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
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