Search

The haploid amoeba Dictyostelium discoideum is a versatile host system for studying cellular aspects of Legionella pathogenicity. Previous studies have shown that the internalization of L. pneumophila leads to an endoplasmic reticulum (ER)-derived organelle that supports intracellular replication of the bacteria. In this study a roadmap of host-cell factors involved in this process was developed. Phagocytosis assays with specific cellular inhibitors and the effects of well defined host-cell mutants revealed that cytoplasmic calcium levels, cytoskeleton-associated proteins and the calcium-binding proteins of the ER, calreticulin and calnexin, specifically influence the uptake and intracellular growth of L. pneumophila. Confocal microscopic time series with green fluorescent protein (GFP)-tagged calnexin and calreticulin demonstrated the accumulation of both proteins in the phagocytic cup of L. pneumophila-infected host cells. In contrast to the control experiment with Escherichia coli-containing phagosomes, both proteins decorated the replicative vacuole of L. pneumophila during the entire growth phase of the bacteria. The cumulative effects of cytosolic calcium levels, the spatial distribution of calnexin and calreticulin, and the defective invasion and replication of L. pneumophila in calnexin- and calreticulin-minus cells suggest that these factors are part of a regulatory system that leads to the specific vacuole of L. pneumophila.

The Dictyostelium mutant HSB1 is temperature-sensitive for development, undergoing aggregation and fruiting body formation at temperatures below 18[degrees]C but not above. In vivo G protein-linked adenylyl cyclase activation is defective in HSB1, and the enzyme is not stimulated in vitro by GTP[gamma]S; stimulation is restored upon addition of wild-type cytosol. Transfection with the gene encoding the cytosolic regulator PIA rescued the mutant. We excluded the possibility that HSB1 cells fail to express PIA and show that the HSB1 piaA gene harbors a point mutation, resulting in the amino acid exchange [G.sup.917]D. Both wild-type and HSB1 cells were also transfected with the HSB1 piaA gene. The pia[A.sup.HSB1] gene product displayed a partial inhibitory effect on wild-type cell development. We hypothesize that PIA couples the heterotrimeric G protein to adenylyl cyclase via two binding sites, one of which is altered in a temperature-sensitive way by the HSB1 mutation. When overexpressed in the wild-type background, PI[A.sup.HSB1] competes with wild-type PIA via the nonmutated binding site, resulting in dominant-negative inhibition of development. Expression of GFP-fused PIA shows that PIA is homogeneously distributed in the cytoplasm of chemotactically moving cells. Key Words: adenylyl cyclase; chemotaxis; Dictyostelium; G protein; signal transduction; slime molds.

G protein beta subunit-null mutants have impaired chemotaxis and phagocytosis because of regulation of the actin cytoskeleton that is not appropriate. Slow growth seen in the mutants reflects a severe phagocytosis defect mainly because of a failure in the actin meshwork's being organized into a phagocytic cup.

Abstract Background Phagocytosis 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. Results The 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. Conclusion The 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.

Abstract Background Ras proteins are guanine-nucleotide-binding enzymes that couple cell surface receptors to intracellular signaling pathways controlling cell proliferation and differentiation, both in lower and higher eukaryotes. They act as molecular switches by cycling between active GTP and inactive GDP-bound states, through the action of two classes of regulatory proteins: a) guanine nucleotide exchange factor (GEFs) and b) GTP-ase activating proteins (GAPs). Genome wide analysis of the lower eukaryote Dictyostelium discoideum revealed a surprisingly large number of Ras Guanine Nucleotide Exchange Factors (RasGEFs). RasGEFs promote the activation of Ras proteins by catalyzing the exchange of GDP for GTP, thus conferring to RasGEFs the role of main activator of Ras proteins. Up to date only four RasGEFs, which are all non-redundant either for growth or development, have been characterized in Dictyostelium. We report here the identification and characterization of a fifth non-redundant GEF, RasGEFM. Results RasGEFM is a multi-domain protein containing six poly-proline stretches, a DEP, RasGEFN and RasGEF catalytic domain. The rasGEFM gene is differentially expressed during growth and development. Inactivation of the gene results in cells that form small, flat aggregates and fail to develop further. Expression of genes required for aggregation is delayed. Chemotaxis towards cAMP is impaired in the mutant, due to inability to inhibit lateral pseudopods. Endogenous cAMP accumulates during early development to a much lower extent than in wild type cells. Adenylyl cyclase activation in response to cAMP pulses is strongly reduced, by contrast guanylyl cyclase is stimulated to higher levels than in the wild type. The actin polymerization response to cAMP is also altered in the mutant. Cyclic AMP pulsing for several hours partially rescues the mutant. In vitro experiments suggest that RasGEFM acts downstream of the cAMP receptor but upstream of the G protein. Conclusion The data indicate that RasGEFM is involved in the establishment of the cAMP relay system. We propose that RasGEFM is a component of a Ras regulated pathway, which integrate signals acting as positive regulator for adenylyl cyclase and negative regulator for guanylyl cyclase. Altered guanylyl cyclase, combined with defective regulation of actin polymerization, results in altered chemotaxis.

Mammalian, or mechanistic, target of rapamycin complex 2 (mTORC2) regulates a variety of vital cellular processes, and its aberrant functioning is often associated with various diseases. Rictor is a peculiar and distinguishing mTORC2 component playing a pivotal role in controlling its assembly and activity. Among extant organisms, Rictor is conserved from unicellular eukaryotes to metazoans. We replaced two distinct, but conserved, glycine residues in both the Dictyostelium piaA gene and its human ortholog, RICTOR. The two conserved residues are spaced ∼50 amino acids apart, and both are embedded within a conserved region falling in between the Ras-GEFN2 and Rictor_V domains. The effects of point mutations on the mTORC2 activity and integrity were assessed by biochemical and functional assays. In both cases, these equivalent point mutations in the mammalian RICTOR and Dictyostelium piaA gene impaired mTORC2 activity and integrity. Our data indicate that the two glycine residues are essential for the maintenance of mTORC2 activity and integrity in organisms that appear to be distantly related, suggesting that they have a evolutionarily conserved role in the assembly and proper mTORC2 functioning. [ABSTRACT FROM AUTHOR]

Objectives The usefulness of university admission tests to medical schools has been discussed in recent years. In the academic year 2014-15 in Italy, several students who failed the admission test appealed to the regional administrative court ('Tribunale Amministrativo Regionale'--TAR) requesting to be included, despite their test results, and all were admitted to their respective courses. The existence of this population of students generated a control group, in order to evaluate the predictive capacity of the admission test. The aim of the present work is to discuss the ability of university admission tests to predict subsequent academic success. Setting and participants The study involved 683 students who enrolled onto the first year of the degree course in medicine in the academic year 2014-15 at the University of Turin (Molinette and San Luigi Gonzaga colleges). The students were separated into two categories: those who passed the admission test (n1=531) and those who did not pass the admission test but won their appeal in the TAR (n2=152). Outcomes The validity of the admission test was analysed using specificity, sensitivity, positive and negative likelihood ratios (LH+, LH-), receiver operating characteristic (ROC) curves, area under the ROC curve (AUC), and relative (95% CI). Results The results showed that the admission test appeared to be a good tool for predicting the academic performances in the first year of the course (AUC=0.70, 95% CI 0.64 to 0.76). Moreover, some subject areas seemed to have a greater discriminating capacity than others. In general, students who obtained a high score in scientific questions were more likely to obtain the required standards during the first year (LH+ 1.22, 95% CI 1.14 to 1.25). Conclusions Based on a consistent statistical approach, our study seems to confirm the ability of the admission test to predict academic success in the first year at the school of medicine of Turin. [ABSTRACT FROM AUTHOR]