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.

Ubiquitin proteases remove ubiquitin monomers or polymers to modify the stability or activity of proteins and thereby serve as key regulators of signal transduction. Here, we describe the function of the Drosophila ubiquitin-specific protease 36 (dUSP36) in negative regulation of the immune deficiency (IMD) pathway controlled by the IMD protein. Overexpression of catalytically active dUSP36 ubiquitin protease suppresses fly immunity against Gram-negative pathogens. Conversely, silencing dUsp36 provokes IMD-dependent constitutive activation of IMD-downstream Jun kinase and NF-kappaB signaling pathways but not of the Toll pathway. This deregulation is lost in axenic flies, indicating that dUSP36 prevents constitutive immune signal activation by commensal bacteria. dUSP36 interacts with IMD and prevents K63-polyubiquitinated IMD accumulation while promoting IMD degradation in vivo. Blocking the proteasome in dUsp36-expressing S2 cells increases K48-polyubiquitinated IMD and prevents its degradation. Our findings identify dUSP36 as a repressor whose IMD deubiquitination activity prevents nonspecific activation of innate immune signaling.

SummaryWe show here that transgenic Drosophila can be used to decipher the effect of a bacterial toxin on innate immunity and demonstrate the contribution of blood cells in fly resistance to bacterial infection. ExoS is a Pseudomonas aeruginosa exotoxin directly translocated into the host cell cytoplasm through the type III secretion system found in many Gram-negative bacteria. It contains a N-terminal GTPase activating (GAP) domain that prevents cytoskeleton reorganization by Rho family of GTPases in cell culture. Directed expression of the ExoS GAP domain (ExoSGAP) during fly eye morphogenesis inhibited Rac1-, Cdc42-and Rho-dependent signalling, demonstrating for the first time its activity on RhoGTPases in a whole organism. We further showed that fly resistance to P. aeruginosa infections was altered when ExoSGAP was expressed either ubiquitously or in haemocytes, but not when expressed into the fat body, the major source of NF-k k k k B-dependent anti-microbial peptide synthesis. Fly sensitivity to infection was also observed with Gram-positive Staphylococcus aureus strain and was associated to a reduced phagocytosis capacity of ExoSGAP-expressing haemocytes. Our results highlight the major contribution of cellular immunity during the first hours after Drosophila infection by P. aeruginosa , an opportunist pathogen affecting patients with pathologies associated to a reduced leukocyte number.

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SummaryThe human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa, and identified mutants with attenuated virulence towards Dictyostelium. These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa.

SummaryNonaspanins are characterised by a large N-terminal extracellular domain and nine putative transmembrane domains. This evolutionarily conserved family comprises three members in Dictyostelium discoideum (Phg1A, Phg1B and Phg1C) and Drosophila melanogaster, and four in mammals (TM9SF1-TM9SF4), the function of which is essentially unknown. Genetic studies in Dictyostelium demonstrated that Phg1A is required for cell adhesion and phagocytosis. We created Phg1A/TM9SF4-null mutant flies and showed that they were sensitive to pathogenic Gram-negative, but not Gram-positive, bacteria. This increased sensitivity was not due to impaired Toll or Imd signalling, but rather to a defective cellular immune response. TM9SF4-null larval macrophages phagocytosed Gram-negative E. coli inefficiently, although Gram-positive S. aureus were phagocytosed normally. Mutant larvae also had a decreased wasp egg encapsulation rate, a process requiring haemocyte-dependent adhesion to parasitoids. Defective cellular immunity was coupled to morphological and adhesion defects in mutant larval haemocytes, which had an abnormal actin cytoskeleton. TM9SF4, and its closest paralogue TM9SF2, were both required for bacterial internalisation in S2 cells, where they displayed partial redundancy. Our study highlights the contribution of phagocytes to host defence in an organism possessing a complex innate immune response and suggests an evolutionarily conserved function of TM9SF4 in eukaryotic phagocytes.

MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) transcription factors that activate muscle-specific genes. The basic helix I sequence of the bHLH motif contains a consensus sequence for protein kinase C (PKC) substrates. We show here that MyoD is indeed phosphorylated by PKC in vitro on Thr 115 within the basic part of the bHLH motif. By analogy with calmodulin-target peptide models, we also identified within the consensus basic helix I motif of myogenic proteins a conserved putative calmodulin/S100-binding domain. Calcium-dependent interaction between MyoD with calmodulin and the abundant muscle S100a(alpha alpha) proteins was demonstrated by affinity chromatography and cross-linking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100a was found to be more efficient than calmodulin in antagonizing DNA binding to MyoD. We next developed a rapid purification method for bacterial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of that peptide by PKC and its interactions with calmodulin and S100a. We confirmed the phosphorylation of the threonine residue 115 in the MyoD-bHLH by PKC with a Km of 0.8 microM. Calmodulin and S100a binding inhibited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent interaction between calcium binding proteins and the MyoD-bHLH was identified by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin with a dissociation constant around 20 nM. S100a inhibited stoichiometrically the binding of the bHLH peptide for labeled calmodulin, suggesting an affinity of S100a for the bHLH peptide at least 1 order of magnitude higher than calmodulin. In favor of an in vivo interaction between S100a and MyoD, we report that S100a- and MyoD-like immunoreactivities colocalize in H9c2 cells, and that a significant amount of MyoD-like immunoreactivity is recovered in the S100a immunoprecipitate from crude H9c2 cell extract in the presence of calcium. We propose that myogenic proteins represent a new family of calmodulin/S100-binding PKC substrates and that calmodulin/S100a could participate in the regulation of the bHLH myogenic protein activities.

Pathogen recognition and engulfment by phagocytic cells of the blood cell lineage constitute the first line of defense against invading pathogens. This cellular immune response is conserved throughout evolution and depends strictly on cytoskeletal changes regulated by the RhoGTPases family. Many pathogens have developed toxins modifying RhoGTPases activity to their own benefit. In particular, the Exoenzyme S (ExoS) toxin of the Gram-negative bacteria Pseudomonas aeruginosa is directly injected into the host cell cytoplasm and contains a GAP domain (ExoSGAP) targeting RhoGTPases. Searching for the contribution of each RhoGTPases, Rho1, Rac1, Rac2, Mtl (Mig2-like) and Cdc42 to fly resistance to P. aeruginosa infections, we found that Rac2 is required to resist to P. aeruginosa and to other Gram-negative or Gram-positive bacteria. The Rac2 immune-deficient phenotype is attributable to defective engulfment of pathogens since Rac2 -mutant macrophages exhibited strong reduction in the phagocytosis level of both Gram-negative and Gram-positive bacterial particles whereas systemic immune signaling pathways, including Toll, Immune deficiency and Jun kinases, were not affected. Co-expression of Rac2 and ExoSGAP rescued the increased sensitivity to P. aeruginosa observed in ExoSGAP-expressing flies suggesting that Rac2 is the main host factor whose function is inhibited by the GAP domain of the ExoS toxin.