Barbara Pergolizzi

Function and Mechanism of Action of Dictyostelium Nramp1 (Slc11a1) in Bacterial Infection

Dictyostelium amoebae are professional phagocytes, which ingest bacteria as the principal source of food. We have cloned the Dictyostelium homologue of human natural resistance‐associated membrane protein 1 (Nramp1) [solute carrier family 11 member 1 (Slc11a1)], an endo‐lysosomal membrane protein that confers on macrophages resistance to infection by a variety of intracellular bacteria and protozoa. The Dictyostelium Nramp1 gene encodes a protein of 53 kDa with 11 putative transmembrane domains. The Nramp1 gene is transcribed during the growth‐phase and downregulated to barely detectable levels upon starvation. To gain insights into their intracellular localization, we fused Nramp1 or the vatB subunit of the V‐H+ATPase with green fluorescent protein and expressed in cells. Green fluorescent protein‐vatB was inserted in membranes of all acidic compartments and the contractile vacuole network and decorated macropinosomes and phagosomes. Green fluorescent protein‐Nramp1 decorated macropinosomes and phagosomes, in addition to intracellular vesicular compartments positive for endosomal SNARE protein Vti1 or vacuolin, a marker of the exocytic pathway. Nramp1 disruption generated mutants that were more permissive hosts than wild‐type cells for intracellular growth of Legionella pneumophila and Micobacterium avium. Nramp1 overexpression protected cells from L. pneumophila infection. Evidence is provided that Nramp1 transports metal cations out of the phagolysosome in an ATP‐dependent process and that L. pneumophila and M. avium use different mechanisms to neutralize Nramp1 activity.

Genome-wide transcriptional changes induced by phagocytosis or growth on bacteria in Dictyostelium

BackgroundPhagocytosis plays a major role in the defense of higher organisms against microbial infection and provides also the basis for antigen processing in the immune response. Cells of the model organism Dictyostelium are professional phagocytes that exploit phagocytosis of bacteria as the preferred way to ingest food, besides killing pathogens. We have investigated Dictyostelium differential gene expression during phagocytosis of non-pathogenic bacteria, using DNA microarrays, in order to identify molecular functions and novel genes involved in phagocytosis.ResultsThe gene expression profiles of cells incubated for a brief time with bacteria were compared with cells either incubated in axenic medium or growing on bacteria. Transcriptional changes during exponential growth in axenic medium or on bacteria were also compared. We recognized 443 and 59 genes that are differentially regulated by phagocytosis or by the different growth conditions (growth on bacteria vs. axenic medium), respectively, and 102 genes regulated by both processes. Roughly one third of the genes are up-regulated compared to macropinocytosis and axenic growth. Functional annotation of differentially regulated genes with different tools revealed that phagocytosis induces profound changes in carbohydrate, aminoacid and lipid metabolism, and in cytoskeletal components. Genes regulating translation and mitochondrial biogenesis are mostly up-regulated. Genes involved in sterol biosynthesis are selectively up-regulated, suggesting a shift in membrane lipid composition linked to phagocytosis. Very few changes were detected in genes required for vesicle fission/fusion, indicating that the intracellular traffic machinery is mostly in common between phagocytosis and macropinocytosis. A few putative receptors, including GPCR family 3 proteins, scaffolding and adhesion proteins, components of signal transduction and transcription factors have been identified, which could be part of a signalling complex regulating phagocytosis and adaptational downstream responses.ConclusionThe results highlight differences between phagocytosis and macropinocytosis, and provide the basis for targeted functional analysis of new candidate genes and for comparison studies with transcriptomes during infection with pathogenic bacteria.

SrfB, a member of the Serum Response Factor family of transcription factors, regulates starvation response and early development in Dictyostelium

The Serum Response Factor (SRF) is an important regulator of cell proliferation and differentiation. Dictyostelium discoideum srfB gene codes for an SRF homologue and is expressed in vegetative cells and during development under the control of three alternative promoters, which show different cell-type specific patterns of expression. The two more proximal promoters directed gene transcription in prestalk AB, stalk and lower-cup cells. The generation of a strain where the srfB gene has been interrupted (srfB−) has shown that this gene is required for regulation of actin–cytoskeleton-related functions, such as cytokinesis and macropinocytosis. The mutant failed to develop well in suspension, but could be rescued by cAMP pulsing, suggesting a defect in cAMP signaling. srfB− cells showed impaired chemotaxis to cAMP and defective lateral pseudopodium inhibition. Nevertheless, srfB− cells aggregated on agar plates and nitrocellulose filters 2 h earlier than wild type cells, and completed development, showing an increased tendency to form slug structures. Analysis of wild type and srfB− strains detected significant differences in the regulation of gene expression upon starvation. Genes coding for lysosomal and ribosomal proteins, developmentally-regulated genes, and some genes coding for proteins involved in cytoskeleton regulation were deregulated during the first stages of development.

A new HECT ubiquitin ligase regulating chemotaxis and development in Dictyostelium discoideum

ABSTRACT Cyclic AMP (cAMP) binding to G-protein-coupled receptors (GPCRs) orchestrates chemotaxis and development in Dictyostelium. By activating the RasC–TORC2–PKB (PKB is also known as AKT in mammals) module, cAMP regulates cell polarization during chemotaxis. TORC2 also mediates GPCR-dependent stimulation of adenylyl cyclase A (ACA), enhancing cAMP relay and developmental gene expression. Thus, mutants defective in the TORC2 Pia subunit (also known as Rictor in mammals) are impaired in chemotaxis and development. Near-saturation mutagenesis of a Pia mutant by random gene disruption led to selection of two suppressor mutants in which spontaneous chemotaxis and development were restored. PKB phosphorylation and chemotactic cell polarization were rescued, whereas Pia-dependent ACA stimulation was not restored but bypassed, leading to cAMP-dependent developmental gene expression. Knocking out the gene encoding the adenylylcyclase B (ACB) in the parental strain showed ACB to be essential for this process. The gene tagged in the suppressor mutants encodes a newly unidentified HECT ubiquitin ligase that is homologous to mammalian HERC1, but harbours a pleckstrin homology domain. Expression of the isolated wild-type HECT domain, but not a mutant HECT C5185S form, from this protein was sufficient to reconstitute the parental phenotype. The new ubiquitin ligase appears to regulate cell sensitivity to cAMP signalling and TORC2-dependent PKB phosphorylation. Highlighted Article: Disrupting a new HECT ubiquitin ligase restores chemotaxis and development in a Dictyostelium mutant deficient in the Pia subunit of the TORC2 complex (Rictor in mammals), which regulates cAMP relay and cell migration.

Ras proteins signaling in the early metazoan Dictyostelium discoideum.

Since the discovery of Ras, Ras-mediated transforming activity has been the major investigative area of interest. Soon thereafter it has emerged that Ras family members regulate different biological processes, other than cell growth, like development and fine-tune the balance between cell death and survival. The lower metazoan Dictyostelium discoideum is a powerful and genetically accessible model organism that has been used to elucidate the roles played by different Ras members in some biological processes, such as cell motility and development. In the following chapter we describe some very basic techniques aiming to identify novel Ras signaling components, throughout insertional mutagenesis screening, and to investigate their role(s) in development and chemotaxis processes.

G-Protein Dependent Signal Transduction and Ubiquitination in Dictyostelium

Signal transduction through G-protein-coupled receptors (GPCRs) is central for the regulation of virtually all cellular functions, and it has been widely implicated in human diseases. These receptors activate a common molecular switch that is represented by the heterotrimeric G-protein generating a number of second messengers (cAMP, cGMP, DAG, IP3, Ca2+ etc.), leading to a plethora of diverse cellular responses. Spatiotemporal regulation of signals generated by a given GPCR is crucial for proper signalling and is accomplished by a series of biochemical modifications. Over the past few years, it has become evident that many signalling proteins also undergo ubiquitination, a posttranslational modification that typically leads to protein degradation, but also mediates processes such as protein-protein interaction and protein subcellular localization. The social amoeba Dictyostelium discoideum has proven to be an excellent model to investigate signal transduction triggered by GPCR activation, as cAMP signalling via GPCR is a major regulator of chemotaxis, cell differentiation, and multicellular morphogenesis. Ubiquitin ligases have been recently involved in these processes. In the present review, we will summarize the most significant pathways activated upon GPCRs stimulation and discuss the role played by ubiquitination in Dictyostelium cells.