dictyNews Electronic Edition Volume 30, number 19 June 20, 2008 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= The Actinome of Dictyostelium discoideum in Comparison to Actins and Actin-related Proteins from Other Organisms Jayabalan M. Joseph 1, Petra Fey 2, Nagendran Ramalingam 1, XI Liu 3, Meino Rohlfs 1, Angelika A. Noegel 4, Annette Mueller-Taubenberger 1, Gernot Gloeckner 5 and Michael Schleicher 1 1 ABI/Cell Biology and Center for Integrated Protein Science (CIPSM), LMU, Muenchen, Germany 2 dictyBase, Center f. Genetic Medicine, Northwestern Univ., Chicago, IL, USA 3 Dept. Biol. II, LMU, Muenchen, Germany 4 Inst. f. Biochemistry I, Center f. Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Univ. of Cologne, Koeln, Germany 5 Leibniz-Inst. for Age Research - Fritz Lipmann Inst., Jena, Germany PLoS ONE, in press Actin belongs to the most abundant proteins in eukaryotic cells which harbor usually many conventional actin isoforms as well as actin-related proteins (Arps). To get an overview over the sometimes confusing multitude of actins and Arps, we analyzed the Dictyostelium discoideum actinome in detail and compared it with the genomes from other model organisms. The D. discoideum actinome comprises 41 actins and actin-related proteins. The genome contains 17 actin genes which most likely arose from consecutive gene duplications, are all active, in some cases develomentally regulated and coding for identical proteins (Act8-group). According to published data, the actin fraction in a D. discoideum cell consists of more than 95% of these Act8-type proteins. The other 16 actin isoforms contain a conventional actin motif profile as well but differ in their protein sequences. Seven actin genes are potential pseudogenes. A homology search of the human genome using the most typical D. discoideum actin (Act8) as query sequence finds the major actin isoforms such as cytoplasmic beta-actin as best hit. This suggests that the Act8-group represents a nearly perfect actin throughout evolution. Interestingly, limited data from D. fasciculatum, a more ancient member among the social amoebae, show different relationships between conventional actins. The Act8-type isoform is most conserved throughout evolution. Modeling of the putative structures suggests that the majority of the actin-related proteins is functionally unrelated to canonical actin. The data suggest that the other actin variants are not necessary for the cytoskeleton itself but rather regulators of its dynamical features or subunits in larger protein complexes. Submitted by: Michael Schleicher [schleicher@lrz.uni-muenchen.de] -------------------------------------------------------------------------------- Genome-wide transcriptional changes induced by phagocytosis or growth on bacteria in Dictyostelium. Alessio Sillo*1, Gareth Bloomfield#°1, Alessandra Balest*, Alessandra Balbo*, Barbara Pergolizzi*, Barbara Peracino*, Jason Skelton#, Alasdair Ivens#, and Salvatore Bozzaro*§ BMC GENOMICS, in press 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. Conclusions 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. Submitted by: Salvo Bozzaro [salvatore.bozzaro@unito.it] -------------------------------------------------------------------------------- Anna Maria Rosaria Colucci, Barbara Peracino, Salvatore Bozzaro, Pietro Alifano and Cecilia Bucci Dictyostelium discoideum as a model host for meningococcal pathogenesis Medical Science Monitor, in Press Background: The aim of the present study was to evaluate the possibility of studying meningococcal virulence in a new model organism, Dictyostelium discoideum, a haploid social soil amoeba that has been established as a host model for several human pathogens leading to discovery of novel virulence mechanisms. Material and Methods: A number of virulent and hyper-virulent N. meningitidis strains including a pair of isogenic encapsulated and un-encapsulated derivatives were used to test the ability of D. discoideum to internalize and grow in the presence of dead or living bacteria. The intracellular survival of internalized bacteria was also monitored. Results: Dead meningococci supported Dictyostelium growth and development although with some difference between strains, which was partially related to internalization rates. As well as in human cells, the capsule positively affected survival of internalized bacteria in Dictyostelium cells. Interestingly, at variance with dead meningococci, living meningococci progressively killed Dictyostelium cells. Conclusions: Our results suggest that several meningococcal virulence determinants such as the capsular polysaccharide may be remarkably effective also in Dictyostelium cells stimulating the use of this model host to search for novel meningococcal virulence determinants. Submitted by: Salvo Bozzaro [salvatore.bozzaro@unito.it] -------------------------------------------------------------------------------- A novel bioassay for evaluating soil bio-hazards using Dictyostelium as biosensor: Validation and application to the Bio-Bio Project Alessandra Balbo and Salvatore Bozzaro Fresenius Environ. Bull., in press An easy and cheap biosensor has been developed for assessing soil biohazards. The toxicity assay is based on inhibition of Dictyostelium development, a soil amoeba undergoing multicellular development and cell differentiation under starving conditions. The sensitivity of the assay was assessed in soil samples with increasing concentrations of heavy metals and by comparison with standard bio-assays on a battery of soils collected from contaminated industrial sites. Dictyostelium appears to be highly sensitive to polycyclic aromatic hydrocarbons, mineral oil and, to a lesser extent, to heavy metals. The assay has been applied in a concerted action with other bioassays within the frame of the BIO-BIO project. Submitted by: Salvo Bozzaro [salvatore.bozzaro@unito.it] -------------------------------------------------------------------------------- A new protein carrying an NmrA-like domain is required for cell differentiation and development in Dictyostelium discoideum Beatriz Núñez-Corcuera, Ioannis Serafimidis, Ernesto Arias-Palomo, Angel Rivera-Calzada and Teresa Suarez Dev. Biol., in press We have isolated a Dictyostelium mutant unable to induce expression of the prestalk-specific marker ecmB in monolayer assays. The disrupted gene, padA, leads to a range of phenotypic defects in growth and development. We show that padA is essential for growth, and we have generated a thermosensitive mutant allele, padA-. At the permissive temperature, mutant cells grow poorly; they remain longer at the slug stage during development and are defective in terminal differentiation. At the restrictive temperature, growth is completely blocked, while development is permanently arrested prior to culmination. padA- slugs are deficient in prestalkA cell differentiation and present an abnormal ecmB expression pattern. Sequence comparisons and predicted three-dimensional structure analyses show that PadA carries an NmrA-like domain. NmrA is a negative transcriptional regulator involved in nitrogen metabolite repression in Aspergillus nidulans. PadA predicted structure shows a NAD(P)+-binding domain, which we demonstrate that is essential for function. We show that padA- development is more sensitive to ammonia than wild-type cells and two ammonium transporters, amtA and amtC, appear derepressed during padA- development. Our data suggest that PadA belongs to a new family of NAD(P)+-binding proteins that link metabolic changes to gene expression and is required for growth and normal development. Submitted by: Teresa Suarez [teresa@cib.csic.es] ============================================================== [End dictyNews, volume 30, number 19]