Gareth Bloomfield

Superoxide signalling required for multicellular development of Dictyostelium.

Reactive oxygen species are known to have a signalling role in many organisms. In bacteria and yeast various response systems have evolved to combat oxidative stress which are triggered by reactive oxygen species. Mammals and plants are known to actively generate reactive oxygen species such as superoxide during signalling responses to a variety of extracellular factors. We report here the generation of superoxide as a signalling molecule in early development of Dictyostelium discoideum. Dictyostelium grows as single amoebae but, on starvation, the single cells aggregate to form a multicellular organism. Superoxide is generated in response to a secreted factor during the transition to the multicellular phase of development. Scavenging superoxide, either pharmacologically or by overexpressing the enzyme superoxide dismutase, inhibits the formation of the aggregate. This report of the use of superoxide as a signalling molecule in a lower eukaryote as it switches to a multicellular phase suggests that this signalling mechanism arose early in the evolution of multicellular organisms, perhaps as a necessary consequence of the need to diversify the number and type of signalling pathways available to facilitate intercellular communication.

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.

Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium

Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures. DOI: http://dx.doi.org/10.7554/eLife.04940.001

Dictyostelium transcriptional responses to Pseudomonas aeruginosa: common and specific effects from PAO1 and PA14 strains

BackgroundPseudomonas aeruginosa is one of the most relevant human opportunistic bacterial pathogens. Two strains (PAO1 and PA14) have been mainly used as models for studying virulence of P. aeruginosa. The strain PA14 is more virulent than PAO1 in a wide range of hosts including insects, nematodes and plants. Whereas some of the differences might be attributable to concerted action of determinants encoded in pathogenicity islands present in the genome of PA14, a global analysis of the differential host responses to these P. aeruginosa strains has not been addressed. Little is known about the host response to infection with P. aeruginosa and whether or not the global host transcription is being affected as a defense mechanism or altered in the benefit of the pathogen. Since the social amoeba Dictyostelium discoideum is a suitable host to study virulence of P. aeruginosa and other pathogens, we used available genomic tools in this model system to study the transcriptional host response to P. aeruginosa infection.ResultsWe have compared the virulence of the P. aeruginosa PAO1 and PA14 using D. discoideum and studied the transcriptional response of the amoeba upon infection. Our results showed that PA14 is more virulent in Dictyostelium than PA01using different plating assays. For studying the differential response of the host to infection by these model strains, D. discoideum cells were exposed to either P. aeruginosa PAO1 or P. aeruginosa PA14 (mixed with an excess of the non-pathogenic bacterium Klebsiella aerogenes as food supply) and after 4 hours, cellular RNA extracted. A three-way comparison was made using whole-genome D. discoideum microarrays between RNA samples from cells treated with the two different strains and control cells exposed only to K. aerogenes. The transcriptomic analyses have shown the existence of common and specific responses to infection. The expression of 364 genes changed in a similar way upon infection with one or another strain, whereas 169 genes were differentially regulated depending on whether the infecting strain was either P. aeruginosa PAO1 or PA14. Effects on metabolism, signalling, stress response and cell cycle can be inferred from the genes affected.ConclusionOur results show that pathogenic Pseudomonas strains invoke both a common transcriptional response from Dictyostelium and a strain specific one, indicating that the infective process of bacterial pathogens can be strain-specific and is more complex than previously thought.

The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA.

The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type amoebae induced dupA expression and resulted in transiently increased ERK1 phosphorylation, suggesting that dupA and ERK1 are part of a response to bacteria. Indeed, over 500 of the genes misregulated in the dupA(-) mutant were regulated in response to L. pneumophila infection, including some thought to have immune-like functions. MAP kinase phosphatases are known to be highly upregulated in macrophages challenged with L. pneumophila. Thus, DupA may regulate a MAP kinase response to bacteria that is conserved from amoebae to mammals.

Uses and abuses of macropinocytosis

ABSTRACT Macropinocytosis is a means by which eukaryotic cells ingest extracellular liquid and dissolved molecules. It is widely conserved amongst cells that can take on amoeboid form and, therefore, appears to be an ancient feature that can be traced back to an early stage of evolution. Recent advances have highlighted how this endocytic process can be subverted during pathology – certain cancer cells use macropinocytosis to feed on extracellular protein, and many viruses and bacteria use it to enter host cells. Prion and prion-like proteins can also spread and propagate from cell to cell through macropinocytosis. Progress is being made towards using macropinocytosis therapeutically, either to deliver drugs to or cause cell death by inducing catastrophically rapid fluid uptake. Mechanistically, the Ras signalling pathway plays a prominent and conserved activating role in amoebae and in mammals; mutant amoebae with abnormally high Ras activity resemble tumour cells in their increased capacity for growth using nutrients ingested through macropinocytosis. This Commentary takes a functional and evolutionary perspective to highlight progress in understanding and use of macropinocytosis, which is an ancient feeding process used by single-celled phagotrophs that has now been put to varied uses by metazoan cells and is abused in disease states, including infection and cancer. Summary: This Commentary provides an evolutionary view of macropinocytosis, while discussing recent breakthroughs in understanding of underlying mechanisms and their subversion by pathogens and tumour cells.

A plasma membrane template for macropinocytic cups

Macropinocytosis is a fundamental mechanism that allows cells to take up extracellular liquid into large vesicles. It critically depends on the formation of a ring of protrusive actin beneath the plasma membrane, which develops into the macropinocytic cup. We show that macropinocytic cups in Dictyostelium are organised around coincident intense patches of PIP3, active Ras and active Rac. These signalling patches are invariably associated with a ring of active SCAR/WAVE at their periphery, as are all examined structures based on PIP3 patches, including phagocytic cups and basal waves. Patch formation does not depend on the enclosing F-actin ring, and patches become enlarged when the RasGAP NF1 is mutated, showing that Ras plays an instructive role. New macropinocytic cups predominantly form by splitting from existing ones. We propose that cup-shaped plasma membrane structures form from self-organizing patches of active Ras/PIP3, which recruit a ring of actin nucleators to their periphery. DOI: http://dx.doi.org/10.7554/eLife.20085.001

A new Dictyostelium prestalk cell sub-type

The mature fruiting body of Dictyostelium consists of stalk and spore cells but its construction, and the migration of the preceding slug stage, requires a number of specialized sub-types of prestalk cell whose nature and function are not well understood. The prototypic prestalk-specific gene, ecmA, is inducible by the polyketide DIF-1 in a monolayer assay and requires the DimB and MybE transcription factors for full inducibility. We perform genome-wide microarray analyses, on parental, mybE− and dimB− cells, and identify many additional genes that depend on MybE and DimB for their DIF-1 inducibility. Surprisingly, an even larger number of genes are only DIF inducible in mybE− cells, some genes are only inducible in DimB− cells and some are inducible when either transcription factor is absent. Thus in assay conditions where MybE and DimB function as inducers of ecmA these genes fall under negative control by the same two transcription factors. We have studied in detail rtaA, one of the MybE and DimB repressed genes. One especially enigmatic group of prestalk cells is the anterior-like cells (ALCs), which exist intermingled with prespore cells in the slug. A promoter fusion reporter gene, rtaA:galu, is expressed in a subset of the ALCs that is distinct from the ALC population detected by a reporter construct containing ecmA and ecmB promoter fragments. At culmination, when the ALC sort out from the prespore cells and differentiate to form three ancillary stalk cell structures: the upper cup, the lower cup and the outer basal disk, the rtaA:galu expressing cells preferentially populate the upper cup region. This fact, and their virtual absence from the anterior and posterior regions of the slug, identifies them as a new prestalk sub-type: the pstU cells. PstU cell differentiation is, as expected, increased in a dimB− mutant during normal development but, surprisingly, they differentiate normally in a mutant lacking DIF. Thus genetic removal of MybE or DimB reveals an alternate DIF-1 activation pathway, for pstU differentiation, that functions under monolayer assay conditions but that is not essential during multicellular development.

Proteomic and Microarray Analyses of the Dictyostelium Zak1-GSK-3 Signaling Pathway Reveal a Role in Early Development

ABSTRACT GskA, the Dictyostelium GSK-3 orthologue, is modified and activated by the dual-specificity tyrosine kinase Zak1, and the two kinases form part of a signaling pathway that responds to extracellular cyclic AMP. We identify potential cellular effectors for the two kinases by analyzing the corresponding null mutants. There are proteins and mRNAs that are altered in abundance in only one or the other of the two mutants, indicating that each kinase has some unique functions. However, proteomic and microarray analyses identified a number of proteins and genes, respectively, that are similarly misregulated in both mutant strains. The positive correlation between the array data and the proteomic data is consistent with the Zak1-GskA signaling pathways functioning by directly or indirectly regulating gene expression. The discoidin 1 genes are positively regulated by the pathway, while the abundance of the H5 protein is negatively regulated. Two of the targets, H5 and discoidin 1, are well-characterized markers for early development, indicating that the Zak1-GskA pathway plays a role in development earlier than previously observed.

Genetics of sex determination in the social amoebae.

The social amoebae possess a sexual cycle that involves transient mutlicellularity: first a zygote attracts surrounding haploid amoebae to form a walled aggregate around it, and then cannibalizes these peripheral cells, eventually forming a dormant single‐celled macrocyst. Self‐fertile homothallic isolates occur as well as breeding groups of self‐infertile heterothallic cells, which commonly have more than two mating types. The mating‐type locus of the widely studied model organism Dictyostelium discoideum, which has three mating types, has recently been identified. Two of the three mating types are determined by single putative regulatory genes bearing no mutual similarity, while the third is specified by homologues of both of these genes. This is the first sex‐determining locus of an Amoebozoan to be described and, since none of the key regulators show homology to known proteins, may be a first glimpse of a novel mode of regulation used in these organisms. The sexual cycle of dictyostelids has been relatively neglected, but continues to yield much interesting biology as well as having the potential to add to the genetic tools available for the study of these organisms.