Ludwig Eichinger

The genome of the social amoeba Dictyostelium discoideum

The social amoebae are exceptional in their ability to alternate between unicellular and multicellular forms. Here we describe the genome of the best-studied member of this group, Dictyostelium discoideum. The gene-dense chromosomes of this organism encode approximately 12,500 predicted proteins, a high proportion of which have long, repetitive amino acid tracts. There are many genes for polyketide synthases and ABC transporters, suggesting an extensive secondary metabolism for producing and exporting small molecules. The genome is rich in complex repeats, one class of which is clustered and may serve as centromeres. Partial copies of the extrachromosomal ribosomal DNA (rDNA) element are found at the ends of each chromosome, suggesting a novel telomere structure and the use of a common mechanism to maintain both the rDNA and chromosomal termini. A proteome-based phylogeny shows that the amoebozoa diverged from the animal–fungal lineage after the plant–animal split, but Dictyostelium seems to have retained more of the diversity of the ancestral genome than have plants, animals or fungi.

Sequence and analysis of chromosome 2 of Dictyostelium discoideum

The genome of the lower eukaryote Dictyostelium discoideum comprises six chromosomes. Here we report the sequence of the largest, chromosome 2, which at 8 megabases (Mb) represents about 25% of the genome. Despite an A + T content of nearly 80%, the chromosome codes for 2,799 predicted protein coding genes and 73 transfer RNA genes. This gene density, about 1 gene per 2.6 kilobases (kb), is surpassed only by Saccharomyces cerevisiae (one per 2 kb) and is similar to that of Schizosaccharomyces pombe (one per 2.5 kb). If we assume that the other chromosomes have a similar gene density, we can expect around 11,000 genes in the D. discoideum genome. A significant number of the genes show higher similarities to genes of vertebrates than to those of other fully sequenced eukaryotes. This analysis strengthens the view that the evolutionary position of D. discoideum is located before the branching of metazoa and fungi but after the divergence of the plant kingdom, placing it close to the base of metazoan evolution.

Large scale multiplex PCR improves pathogen detection by DNA microarrays

BackgroundMedium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA.ResultsTo increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000.ConclusionOur data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.

Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum

BackgroundThe social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum.ResultsWe have produced a draft genome sequence of another group dictyostelid, Dictyosteliumpurpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict.ConclusionsThe findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.

Dictyostelium transcriptional host cell response upon infection with Legionella.

Differential gene expression of Dictyostelium discoideum after infection with Legionella pneumophila was investigated using DNA microarrays. Investigation of a 48 h time course of infection revealed several clusters of co‐regulated genes, an enrichment of preferentially up‐ or downregulated genes in distinct functional categories and also showed that most of the transcriptional changes occurred 24 h after infection. A detailed analysis of the 24 h time point post infection was performed in comparison to three controls, uninfected cells and co‐incubation with Legionella hackeliae and L. pneumophilaΔdotA. One hundred and thirty‐one differentially expressed D. discoideum genes were identified as common to all three experiments and are thought to be involved in the pathogenic response. Functional annotation of the differentially regulated genes revealed that apart from triggering a stress response Legionella apparently not only interferes with intracellular vesicle fusion and destination but also profoundly influences and exploits the metabolism of its host. For some of the identified genes, e.g. rtoA involvement in the host response has been demonstrated in a recent study, for others such a role appears plausible. The results provide the basis for a better understanding of the complex host‐pathogen interactions and for further studies on the Dictyostelium response to Legionella infection.

Phylogeny-wide analysis of social amoeba genomes highlights ancient origins for complex intercellular communication

Dictyostelium discoideum (DD), an extensively studied model organism for cell and developmental biology, belongs to the most derived group 4 of social amoebas, a clade of altruistic multicellular organisms. To understand genome evolution over long time periods and the genetic basis of social evolution, we sequenced the genomes of Dictyostelium fasciculatum (DF) and Polysphondylium pallidum (PP), which represent the early diverging groups 1 and 2, respectively. In contrast to DD, PP and DF have conventional telomere organization and strongly reduced numbers of transposable elements. The number of protein-coding genes is similar between species, but only half of them comprise an identifiable set of orthologous genes. In general, genes involved in primary metabolism, cytoskeletal functions and signal transduction are conserved, while genes involved in secondary metabolism, export, and signal perception underwent large differential gene family expansions. This most likely signifies involvement of the conserved set in core cell and developmental mechanisms, and of the diverged set in niche- and species-specific adaptations for defense and food, mate, and kin selection. Phylogenetic dating using a concatenated data set and extensive loss of synteny indicate that DF, PP, and DD split from their last common ancestor at least 0.6 billion years ago.

Loss of Dictyostelium ATG9 results in a pleiotropic phenotype affecting growth, development, phagocytosis and clearance and replication of Legionella pneumophila

Infection of Dictyostelium discoideum with Legionella pneumophila resulted in a large number of differentially regulated genes among them three core autophagy genes, ATG8, ATG9 and ATG16. Macroautophagy contributes to many physiological and pathological processes and might also constitute an important mechanism in cell‐autonomous immunity. For further studies we selected the highly conserved ATG9. In colocalization studies with GFP‐tagged ATG9 and different organelle marker proteins we neither observed colocalization with mitochondria, the ER nor lysosomes. However, there was partial colocalization with the Golgi apparatus and many ATG9‐GFP‐containing vesicles localized along microtubules and accumulated around the microtubule organizing centre. ATG9‐deficient cells had pleiotropic defects. In addition to growth defects they displayed severe developmental defects, consistent with the known role of autophagy in Dictyostelium development. Unexpectedly, the ATG9 mutant also had a strong phagocytosis defect that was particularly apparent when infecting the cells with L. pneumophila. However, those Legionellae that entered the host could multiply better in mutant than in wild‐type cells, because of a less efficient clearance in the early and a more efficient replication in the late phase of infection. We conclude that ATG9 and hence macroautophagy has a protective role during pathogen infection.

A novel type of protein kinase phosphorylates actin in the actin-fragmin complex.

Actin‐fragmin kinase (AFK) from Physarum polycephalum specifically phosphorylates actin in the EGTA‐resistant 1:1 actin‐fragmin complex. The cDNA deduced amino acid sequence reveals two major domains of approximately 35 kDa each that are separated by a hinge‐like proline/serine‐rich segment of 50 residues. Whereas the N‐terminal domain does not show any significant similarity to protein sequences from databases, there are six complete kelch repeats in the protein that comprise almost the entire C‐terminal half of the molecule. To prove the intrinsic phosphorylation activity of AFK, full‐length or partial cDNA fragments were expressed both in a reticulocyte lysate and in Escherichia coli. In both expression systems, we obtained specific actin phosphorylation and located the catalytic domain in the N‐terminal half. Interestingly, this region did not contain any of the known protein kinase consensus sequences. The only known sequence motif present that could have been involved in nucleotide binding was a nearly perfect phosphate binding loop (P‐loop). However, introduction of two different point mutations into this putative P‐loop sequence did not alter the catalytic activity of the kinase, which indicates an as yet unknown mechanism for phosphate transfer. Our data suggest that AFK belongs to a new class of protein kinases and that this actin phosphorylation might be the first example of a widely distributed novel type of regulation of the actin cytoskeleton in non‐muscle cells.

The professional phagocyte Dictyostelium discoideum as a model host for bacterial pathogens.

The use of simple hosts such as Dictyostelium discoideum in the study of host pathogen interactions offers a number of advantages and has steadily increased in recent years. Infection-specific genes can often only be studied in a very limited way in man and even in the mouse model their analysis is usually expensive, time consuming and technically challenging or sometimes even impossible. In contrast, their functional analysis in D. discoideum and other simple model organisms is often easier, faster and cheaper. Because host-pathogen interactions necessarily involve two organisms, it is desirable to be able to genetically manipulate both the pathogen and its host. Particularly suited are those hosts, like D. discoideum, whose genome sequence is known and annotated and for which excellent genetic and cell biological tools are available in order to dissect the complex crosstalk between host and pathogen. The review focusses on host-pathogen interactions of D. discoideum with Legionella pneumophila, mycobacteria, and Salmonella typhimurium which replicate intracellularly.

DICTYOSTELIUM AS MODEL SYSTEM FOR STUDIES OF THE ACTIN CYTOSKELETON BY MOLECULAR GENETICS

The actin cytoskeleton is an essential structure for most movements at the cellular and intracellular level. Whereas for contraction a muscle cell requires a rather static organisation of cytoskeletal proteins, cell motility of amoeboid cells relies on a tremendously dynamic turnover of filamentous networks in a matter of seconds and at distinct regions inside the cell. The best model system for studying cell motility is Dictyostelium discoideum. The cells live as single amoebae but can also start a developmental program that leads to multicellular stages and differentiation into simple types of tissues. Thus, cell motility can be studied on single cells and on cells in a tissue‐like aggregate. The ability to combine protein purification and biochemistry with fairly easy molecular genetics is a unique feature for investigation of the cytoskeleton and cell motility. The actin cytoskeleton in Dictyostelium harbours essentially all classes of actin‐binding proteins that have been found throughout eukaryotes. By conventional mutagenesis, gene disruption, antisense approaches, or gene replacements many genes that code for cytoskeletal proteins have been disrupted, and altered phenotypes in transformants that lacked one or more of those cytoskeletal proteins allowed solid conclusions about their in vivo function. In addition, tagging the proteins or selected domains with green fluorescent protein allows the monitoring of protein redistribution during cell movement. Gene tagging by restriction enzyme mediated integration of vectors and the ongoing international genome and cDNA sequencing projects offer the chance to understand the dynamics of the cytoskeleton by identification and functional characterisation of all proteins involved. Microsc. Res. Tech. 47:124–134, 1999.