Macroautophagy is a highly conserved intracellular bulk degradation system of all eukaryotic cells. It is governed by a large number of autophagy proteins (ATGs) and is crucial for many cellular processes. Here, we describe the phenotypes of Dictyostelium discoideum ATG16− and ATG9−/16− cells and compare them to the previously reported ATG9− mutant. ATG16 deficiency caused an increase in the expression of several core autophagy genes, among them atg9 and the two atg8 paralogues. The single and double ATG9 and ATG16 knock-out mutants had complex phenotypes and displayed severe and comparable defects in pinocytosis and phagocytosis. Uptake of Legionella pneumophila was reduced. In addition, ATG9− and ATG16− cells had dramatic defects in autophagy, development and proteasomal activity which were much more severe in the ATG9−/16− double mutant. Mutant cells showed an increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates which partially co-localized with ATG16-GFP in ATG9−/16− cells. The more severe autophagic, developmental and proteasomal phenotypes of ATG9−/16− cells imply that ATG9 and ATG16 probably function in parallel in autophagy and have in addition autophagy-independent functions in further cellular processes.

Small molecule signaling promotes the communication between bacteria as well as between bacteria and eukaryotes. The opportunistic pathogenic bacterium Legionella pneumophila employs LAI-1 (3-hydroxypentadecane-4-one) for bacterial cell-cell communication. LAI-1 is produced and detected by the Lqs (Legionella quorum sensing) system, which regulates a variety of processes including natural competence for DNA uptake and pathogen-host cell interactions. In this study, we analyze the role of LAI-1 in inter-kingdom signaling. L. pneumophila lacking the autoinducer synthase LqsA no longer impeded the migration of infected cells, and the defect was complemented by plasmid-borne lqsA. Synthetic LAI-1 dose-dependently inhibited cell migration, without affecting bacterial uptake or cytotoxicity. The forward migration index but not the velocity of LAI-1-treated cells was reduced, and the cell cytoskeleton appeared destabilized. LAI-1-dependent inhibition of cell migration involved the scaffold protein IQGAP1, the small GTPase Cdc42 as well as the Cdc42-specific guanine nucleotide exchange factor ARHGEF9, but not other modulators of Cdc42, or RhoA, Rac1 or Ran GTPase. Upon treatment with LAI-1, Cdc42 was inactivated and IQGAP1 redistributed to the cell cortex regardless of whether Cdc42 was present or not. Furthermore, LAI-1 reversed the inhibition of cell migration by L. pneumophila, suggesting that the compound and the bacteria antagonistically target host signaling pathway(s). Collectively, the results indicate that the L. pneumophila quorum sensing compound LAI-1 modulates migration of eukaryotic cells through a signaling pathway involving IQGAP1, Cdc42 and ARHGEF9.

SummaryIn unicellular amoebae, such as Dictyostelium discoideum, bacterial phagocytosis is a food hunting device, while in higher organisms it is the first defence barrier against microbial infection. In both cases, pathogenic bacteria exploit phagocytosis to enter the cell and multiply intracellularly. Salmonella typhimurium, the agent of food-borne gastroenteritis, is phagocytosed by both macrophages and Dictyostelium cells. By using cell biological assays and global transcriptional analysis with DNA microarrays covering the Dictyostelium genome, we show here that S. typhimurium is pathogenic for Dictyostelium cells. Depending on the degree of virulence, which in turn depended on bacterial growth conditions, Salmonella could kill Dictyostelium cells or inhibit their growth and development. In the early phase of infection in nonnutrient buffer, the ingested bacteria escaped degradation, induced a starvation-like transcriptional response but inhibited selectively genes required for chemotaxis and aggregation. This way differentiation of the host cells into spore and stalk cells was blocked or delayed, which in turn is likely to be favourable for the establishment of a replicative niche for Salmonella. Inhibition of the aggregation competence and chemotactic streaming of aggregation-competent cells in the presence of Salmonella suggests interference with cAMP signalling.

Protein turnover and quality control by the proteasome is of paramount importance for cell homeostasis. Dysfunction of the proteasome is associated with aging processes and human diseases such as neurodegeneration, cardiomyopathy, and cancer. The regulation, i.e. activation and inhibition of this fundamentally important protein degradation system, is still widely unexplored. We demonstrate here that the evolutionarily highly conserved type II triple-A ATPase VCP and the proteasome inhibitor PSMF1/PI31 interact directly, and antagonistically regulate proteasomal activity. Our data provide novel insights into the regulation of proteasomal activity.