Dictyostelium discoideum: a model host to measure bacterial virulence

Froquet, Romain Bruno; Lelong, Emmanuelle; Marchetti, Anna; Cosson, Pierre

doi:10.1038/nprot.2008.212

Cited by 95 publications

(116 citation statements)

References 15 publications

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“…Virulence assays were performed essentially as previously described (1,12,26). Briefly, 50 l of an overnight culture of P. entomophila was deposited into an individual well of a 24-well plate containing 2 ml of SM-Agar (peptone at 10 g/liter, yeast extract at 1 g/liter, KH 2 PO 4 at 2.2 g/liter, K 2 HPO 4 at 1 g/liter, MgSO 4 ⅐ 7H 2 O at 1 g/liter, agar at 20 g/liter) and dried.…”

Section: Methodsmentioning

confidence: 99%

Association of Hemolytic Activity ofPseudomonas entomophila, a Versatile Soil Bacterium, with Cyclic Lipopeptide Production

Vallet-Gély

Новиков

Augusto

et al. 2010

Appl Environ Microbiol

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124

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Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C 10 OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.

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Section: Methodsmentioning

confidence: 99%

Association of Hemolytic Activity ofPseudomonas entomophila, a Versatile Soil Bacterium, with Cyclic Lipopeptide Production

Vallet-Gély

Новиков

Augusto

et al. 2010

Appl Environ Microbiol

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124

130

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“…Notably, three K. pneumoniae strains with reduced virulence in mammalian models (47, 48) had LD 50 s significantly higher than those of K. pneumoniae 52145 and MGH78578 (P Ͻ 0.05 by the one-sample t test) ( Table 2). Moreover, a nonpathogenic laboratory-adapted strain of Klebsiella had one of the highest LD 50 s. This strain is used as a control strain in infections of nonmammalian hosts (49).…”

Section: K Pneumoniae Infection Causes Death Of G Mellonella Larvaementioning

confidence: 99%

Modeling Klebsiella pneumoniae Pathogenesis by Infection of the Wax Moth Galleria mellonella

et al. 2013

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The implementation of infection models that approximate human disease is essential for understanding pathogenesis at the molecular level and for testing new therapies before they are entered into clinical stages. Insects are increasingly being used as surrogate hosts because they share, with mammals, essential aspects of the innate immune response to infections. We examined whether the larva of the wax moth Galleria mellonella could be used as a host model to conceptually approximate Klebsiella pneumoniae-triggered pneumonia. We report that the G. mellonella model is capable of distinguishing between pathogenic and nonpathogenic Klebsiella strains. Moreover, K. pneumoniae infection of G. mellonella models some of the known features of Klebsiella-induced pneumonia, i.e., cell death associated with bacterial replication, avoidance of phagocytosis by phagocytes, and the attenuation of host defense responses, chiefly the production of antimicrobial factors. Similar to the case for the mouse pneumonia model, activation of innate responses improved G. mellonella survival against subsequent Klebsiella challenge. Virulence factors necessary in the mouse pneumonia model were also implicated in the Galleria model. We found that mutants lacking capsule polysaccharide, lipid A decorations, or the outer membrane proteins OmpA and OmpK36 were attenuated in Galleria. All mutants activated G. mellonella defensive responses. The Galleria model also allowed us to monitor Klebsiella gene expression. The expression levels of cps and the loci implicated in lipid A remodeling peaked during the first hours postinfection, in a PhoPQ-and PmrAB-governed process. Taken together, these results support the utility of G. mellonella as a surrogate host for assessing infections with K. pneumoniae. In 1890, Robert Koch formulated Koch's postulates as general guidelines for identifying disease-causing organisms. One century later, Stanley Falkow established the molecular version of Koch's postulates, this time to guide the identification of microbial genes encoding virulence factors. One of the key points of the molecular postulates is to test the virulence of a microorganism with an inactivated candidate virulence gene in an appropriate animal model. Therefore, the use of animal models to identify the virulence factors of human pathogens is indispensable. Currently, identification and characterization of novel virulence factors rely largely on assessing mutant bacteria for growth in the organs of infected mice. The dependence on mouse infection models limits large-scale analysis of virulence due to the large number of animals needed to obtain statistically significant results.To circumvent these issues, the search for alternative host models is ongoing. Ideally, these alternative models should be easy to maintain and infect, should be amenable to genetic manipulation, and should model aspects of vertebrate defenses upon infection, chiefly the immune response. The immune defense consists of two main parts, an innate and an adaptive response, wit...

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“…D. discoideum growth on bacteria lawn was tested as reported (Froquet et al, 2009). Chemotaxis was assayed using a semi-quantitative assay (Wallace and Frazier, 1979).…”

Section: Antibodies and Immunofluorescence Microscopymentioning

confidence: 99%

Defective lysosome maturation and Legionella pneumophila replication in Dictyostelium ArfGAP ACAP-A mutant cells

Baïlo

Cosson²,

Charette

et al. 2014

Journal of Cell Science

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Dictyostelium discoideum ACAP-A is an Arf GTPase-activating protein (GAP) involved in cytokinesis, cell migration and actin cytoskeleton dynamics. In mammalian cells, ACAP family members regulate endocytic protein trafficking. Here, we explored the function of ACAP-A in the endocytic pathway of D. discoideum. In the absence of ACAP-A, the efficiency of fusion between postlysosomes and the plasma membrane was reduced, resulting in the accumulation of post-lysosomes. Moreover, internalized fluidphase markers showed extended intracellular transit times, and the transfer kinetics of phagocyted particles from lysosomes to postlysosomes was reduced. Neutralization of lysosomal pH, one essential step in lysosome maturation, was also delayed. Whereas expression of ACAP-A-GFP in acapA 2 cells restored normal particle transport kinetics, a mutant ACAP-A protein with no GAP activity towards the small GTPase ArfA failed to complement this defect. Taken together, these data support a role for ACAP-A in maturation of lysosomes into post-lysosomes through an ArfAdependent mechanism. In addition, we reveal that ACAP-A is required for efficient intracellular growth of Legionella pneumophila, a pathogen known to subvert the endocytic host cell machinery for replication. This further emphasizes the role of ACAP-A in the endocytic pathway.

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Dictyostelium discoideum: a model host to measure bacterial virulence

Cited by 95 publications

References 15 publications

Association of Hemolytic Activity ofPseudomonas entomophila, a Versatile Soil Bacterium, with Cyclic Lipopeptide Production

Association of Hemolytic Activity ofPseudomonas entomophila, a Versatile Soil Bacterium, with Cyclic Lipopeptide Production

Modeling Klebsiella pneumoniae Pathogenesis by Infection of the Wax Moth Galleria mellonella

Defective lysosome maturation and Legionella pneumophila replication in Dictyostelium ArfGAP ACAP-A mutant cells

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