SummaryThe amoeba Dictyostelium discoideum shares many traits with mammalian macrophages, in particular the ability to phagocytose and kill bacteria. In response, pathogenic bacteria use conserved mechanisms to fight amoebae and mammalian phagocytes. Here we developed an assay using Dictyostelium to monitor phagocyte-bacteria interactions. Genetic analysis revealed that the virulence of Klebsiella pneumoniae measured by this test is very similar to that observed in a mouse pneumonia model. Using this assay, two new host resistance genes ( PHG1 and KIL1 ) were identified and shown to be involved in intracellular killing of K. pneumoniae by phagocytes. Phg1 is a member of the 9TM family of proteins, and Kil1 is a sulphotransferase. The loss of PHG1 resulted in Dictyostelium susceptibility to a small subset of bacterial species including K. pneumoniae . Remarkably, Drosophila mutants deficient for PHG1 also exhibited a specific susceptibility to K. pneumoniae infections. Systematic analysis of several additional Dictyostelium mutants created a two-dimensional virulence array, where the complex interactions between host and bacteria are visualized.

Dictyostelium amoebae have been used as a host model to measure virulence of a wide range of bacterial pathogens. The simple protocol described here takes advantage of the ability of Dictyostelium to grow and form plaques on a lawn of nonpathogenic bacteria but not on virulent bacteria. This assay can be modulated to measure the virulence of different bacterial pathogens. By adjusting various parameters such as cell numbers or media, a more quantitative measure of bacterial virulence can also be obtained. The entire procedure takes about 5 h to compete, and up to 2 further weeks may be required for plaques to form on the bacterial lawn.

The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.

ETOC: TM9/Phg1 proteins are essential for cellular adhesion in many systems, from Dictyostelium to human cells, yet their exact role remains unknown. We demonstrate that TM9 proteins participate in adhesion in Dictyostelium cells by controlling the surface levels of SibA adhesion molecules, notably by influencing their sorting in the endocytic pathway.

Molecular mechanisms ensuring cellular adhesion have been studied in detail in Dictyostelium amoebae, but little is known about the regulation of cellular adhesion in these cells. Here, we show that cellular adhesion is regulated in Dictyostelium, notably by the concentration of a cellular secreted factor accumulating in the medium. This constitutes a quorum-sensing mechanism allowing coordinated regulation of cellular adhesion in a Dictyostelium population. In order to understand the mechanism underlying this regulation, we analyzed the expression of recently identified Dictyostelium adhesion molecules (Sib proteins) that present features also found in mammalian integrins. sibA and sibC are both expressed in vegetative Dictyostelium cells, but the expression of sibC is repressed strongly in conditions where cellular adhesion decreases. Analysis of sibA and sibC mutant cells further suggests that variations in the expression levels of sibC account largely for changes in cellular adhesion in response to environmental cues.

Bacterial virulence can only be assessed by confronting bacteria with a host. Here, we present a new simple assay to evaluate Aeromonas virulence, making use of Dictyostelium amoebae as an alternative host model. This assay can be modulated to assess virulence of very different Aeromonas species.

Multicellular development of Dictyostelium is induced by starvation and is crucial for its long-term survival. Coronin A mediates the transition from growth to development of the cells and initiates the cAMP-dependent relay by regulating the response to secreted cell density and nutrient deprivation factors.

TM9 proteins constitute a well defined family, characterized by the presence of a large variable extracellular domain and nine putative transmembrane domains. This family is highly conserved throughout evolution and comprises three members in Dictyostelium discoideum and Saccharomyces cerevisiae and four in humans and mice. In Dictyostelium, previous analysis demonstrated that TM9 proteins are implicated in cellular adhesion. In this study, we generated TM9 mutants in S. cerevisiae and analyzed their phenotype with particular attention to cellular adhesion. S. cerevisiae strains lacking any one of the three TM9 proteins were severely suppressed for adhesive growth and filamentous growth under conditions of nitrogen starvation. In these mutants, expression of the FLO11-lacZ reporter gene was strongly reduced, whereas expression of FRE(Ty1)-lacZ was not, suggesting that TM9 proteins are implicated at a late stage of nutrient-controlled signaling pathways. We also reexamined the phenotype of Dictyostelium TM9 mutant cells, focusing on nutrient-controlled cellular functions. Although the initiation of multicellular development and autophagy was unaltered in Dictyostelium TM9 mutants, nutrientcontrolled secretion of lysosomal enzymes was dysregulated in these cells. These results suggest that in both yeast and amoebae, TM9 proteins participate in the control of specific cellular functions in response to changing nutrient conditions.