Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella

The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. In this study, we infected single-cell stages of D. discoideum with different Legionella species. Intracellular growth of Legionella in this new host system was compared with their growth in the natural host Acanthamoeba castellanii . Transmission electron microscopy of infected D. discoideum cells revealed that legionellae reside within the phagosome. Using confocal microscopy, it was observed that replicating, intracellular, green fluorescent protein (GFP)-tagged legionellae rarely co-localized with fluorescent antibodies directed against the lysosomal protein DdLIMP of D. discoideum . This indicates that the bacteria inhibit the fusion of phagosomes and lysosomes in this particular host system. In addition, Legionella infection of D. discoideum inhibited the differentiation of the host into the multicellular fruiting stage. Co-culture studies with profilin-minus D. discoideum mutants and Legionella resulted in higher rates of infection when compared with infections of wild-type amoebae. Because the amoebae are amenable to genetic manipulation as a result of their haploid genome and because a number of cellular markers are available, we show for the first time that D. discoideum is a valuable model system for studying intracellular pathogenesis of microbial pathogens.     

Just the right size: cell counting in Dictyostelium

The regulation of tissue and organism size plays an essential, but poorly understood, role in multicellular development. Genes have been identified that affect body and organ size in a number of animals. Two recently identified genes, smlA and countin, are required for the proper function of a cell-counting mechanism that regulates organism size in the eukaryotic microorganism Dictyostelium discoideum. The discovery of this process now allows the study of size regulation in a simple multicellular system.     

A cell number counting factor regulates Akt/protein kinase B to regulate Dictyostelium discoideum group size

Little is known about how individual cells can organize themselves to form structures of a given size. During development, Dictyostelium discoideum aggregates in dendritic streams and forms groups of approximately 20,000 cells. D. discoideum regulates group size by secreting and simultaneously sensing a multiprotein complex called counting factor (CF). If there are too many cells in a stream, the associated high concentration of CF will decrease cell-cell adhesion and increase cell motility, causing aggregation streams to break up. The pulses of cyclic AMP (cAMP) that mediate aggregation cause a transient translocation of Akt/protein kinase B (Akt/PKB) to the leading edge of the plasma membrane and a concomitant activation of the kinase activity, which in turn stimulates motility. We found that countin- cells (which lack bioactive CF) and wild-type cells starved in the presence of anticountin antibodies (which block CF activity) showed a decreased level of cAMP-stimulated Akt/PKB membrane translocation and kinase activity compared to parental wild-type cells. Recombinant countin has the bioactivity of CF, and a 1-min treatment of cells with recombinant countin potentiated Akt/PKB translocation to membranes and Akt/PKB activity. Western blotting of total cell lysates indicated that countin does not affect the total level of Akt/PKB. Fluorescence microscopy of cells expressing an Akt/PKB pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion protein indicated that recombinant countin and anti-countin antibodies do not obviously alter the distribution of Akt/PKB PH-GFP when it translocates to the membrane. Our data indicate that CF increases motility by potentiating the cAMP-stimulated activation and translocation of Akt/PKB.     

Cells respond to and bind countin,a component of a multisubunit cell number counting factor

In Dictyostelium discoideum counting factor (CF), a secreted approximately 450-kDa complex of polypeptides, inhibits group and fruiting body size. When the gene encoding countin (a component of CF) was disrupted, cells formed large groups. We find that recombinant countin causes developing cells to form small groups, with an EC(50) of approximately 3 ng/ml, and affects cAMP signal transduction in the same manner as semipurified CF. Recombinant countin increases cell motility, decreases cell-cell adhesion, and regulates gene expression in a manner similar to the effect of CF. However, countin does not decrease adhesion or group size to the extent that semipurified CF does. A 1-min exposure of developing cells to countin causes an increase in F-actin polymerization and myosin phosphorylation and a decrease in myosin polymerization, suggesting that countin activates a rapid signal transduction pathway. (125)I-Labeled countin has countin bioactivity, and binding experiments suggest that vegetative and developing cells have approximately 53 cell-surface sites that bind countin with a K(D) of approximately 1.5 ng/ml or 60 pm. We hypothesize that countin regulates cell development through the same pathway as CF and that other proteins within the complex may modify the activity of countin and/or have independent size-regulating activities.     

A phosphatidylinositol (PI) kinase gene family in Dictyostelium discoideum: biological roles of putative mammalian p110 and yeast Vps34p PI 3-kinase homologs during growth and development.

Three groups of phosphatidylinositol (PI) kinases convert PI into PI(3)phosphate, PI(4)phosphate, PI(4,5) bisphosphate, and PI(3,4,5)trisphosphate. These phosphoinositides have been shown to function in vesicle-mediated protein sorting, and they serve as second-messenger signaling molecules for regulating cell growth. To further elucidate the mechanism of regulation and function of phosphoinositides, we cloned genes encoding five putative PI kinases from Dictyostelium discoideum. Database analysis indicates that D. discoideum PIK1 (DdPIK1), -2, and -3 are most closely related to the mammalian p110 PI 3-kinase, DdPIK5 is closest to the yeast Vps34p PI 3-kinase, and DdPIK4 is most homologous to PI 4-kinases. Together with other known PI kinases, a superfamily of PI kinase genes has been defined, with all of the encoded proteins sharing a common highly conserved catalytic core domain. DdPIK1, -2, and -3 may have redundant functions because disruption of any single gene had no effect on D. discoideum growth or development. However, strains in which both of the two most highly related genes, DdPIK1 and DdPIK2, were disrupted showed both growth and developmental defects, while double knockouts of DdPIK1 and DdPIK3 and DdPIK2 and DdPIK3 appear to be lethal. The delta Ddpik1 delta Ddpik2 null cells were smaller than wild-type cells and grew slowly both in association with bacteria and in axenic medium when attached to petri plates but were unable to grow in suspension in axenic medium. When delta Ddpik1 delta Ddpik2 null cells were plated for multicellular development, they formed aggregates having multiple tips and produced abnormal fruiting bodies. Antisense expression of DdPIK5 (a putative homolog of the Saccharomyces cerevisiae VPS34) led to a defect in the growth of D. discoideum cells on bacterial lawns and abnormal development. DdPIK5 complemented the temperature-sensitive growth defect of a Schizosaccharomyces pombe delta Svps34 mutant strain, suggesting DdPIK5 encodes a functional homolog of yeast Vps34p. These observations indicate that in D. discoideum, different PI kinases regulate distinct cellular processes, including cell growth, development, and protein trafficking.     

CbfA, the C-module DNA-binding factor, plays an essential role in the initiation of Dictyostelium discoideum development

We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.     

Two components of a secreted cell number-counting factor bind to cells and have opposing effects on cAMP signal transduction in Dictyostelium

A secreted 450-kDa complex of proteins called counting factor (CF) is part of a negative feedback loop that regulates the size of the groups formed by developing Dictyostelium cells. Two components of CF are countin and CF50. Both recombinant countin and recombinant CF50 decrease group size in Dictyostelium. countin- cells have a decreased cAMP-stimulated cAMP pulse, whereas recombinant countin potentiates the cAMP pulse. We find that CF50 cells have an increased cAMP pulse, whereas recombinant CF50 decreases the cAMP pulse, suggesting that countin and CF50 have opposite effects on cAMP signal transduction. In addition, countin and CF50 have opposite effects on cAMP-stimulated Erk2 activation. However, like recombinant countin, recombinant CF50 increases cell motility. We previously found that cells bind recombinant countin with a Hill coefficient of approximately 2, a KH of 60 pm, and approximately 53 sites/cell. We find here that cells also bind 125I-recombinant CF50, with a Hill coefficient of approximately 2, a KH of approximately 15 ng/ml (490 pm), and approximately 56 sites/cell. Countin and CF50 require each other's presence to affect group size, but the presence of countin is not necessary for CF50 to bind to cells, and CF50 is not necessary for countin to bind to cells. Our working hypothesis is that a signal transduction pathway activated by countin binding to cells modulates a signal transduction pathway activated by CF50 binding to cells and vice versa and that these two pathways can be distinguished by their effects on cAMP signal transduction.     

High relatedness in a social amoeba: the role of kin-discriminatory segregation

A major challenge for social theory is to explain the importance of kin discrimination for the evolution of altruism. One way to assess the importance of kin discrimination is to test its effects on increasing relatedness within groups. The social amoeba Dictyostelium discoideum aggregates to form a fruiting body composed of dead stalk and live spores. Previous studies of a natural population showed that where D. discoideum occurs in the soil, multiple clones are often found in the same small soil samples. However, actual fruiting bodies usually contain only one clone. We here performed experiments to gauge the effect of kin-discriminatory segregation on increasing relatedness. We mixed co-occurring clones from this population using a relatedness level found in small soil samples. We found a lower proportion of uniclonal fruiting bodies and a lower level of relatedness compared with natural fruiting bodies. We found that the amount of relatedness increase attributable to kin-discriminatory segregation was small. These findings suggest a relatively minor influence of kin-discriminatory segregation on relatedness in D. discoideum. We discuss our results comparing with the results of previous studies, including those of wild clones and laboratory mutants. We ask why wild clones of D. discoideum exhibit a low degree of kin-discriminatory segregation, and what alternative factors might account for high relatedness in D. discoideum.     

A protein in crude cytosol regulates glucose-6-phosphatase activity in crude microsomes to regulate group size in Dictyostelium

Dictyostelium discoideum form groups of approximately 2 x 10(4) cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multipolypeptide complex called counting factor (CF). The CF signal transduction pathway involves CF-repressing internal glucose levels by increasing the K(m) of glucose-6-phosphatase. Little is known about how this enzyme is regulated. Glucose-6-phosphatase is associated with microsomes in both Dictyostelium and mammals. We find that the activity of glucose-6-phosphatase in crude microsomes from cells with high, normal, or low CF activity had a negative correlation with the amount of CF present in these cell lines. In crude cytosols (supernatants from ultracentrifugation of cell lysates), the glucose-6-phosphatase activity had a positive correlation with CF accumulation. The crude cytosols were further fractionated into a fraction containing molecules greater than 10 kDa (S>10K) and molecules less than 10 KDa (S<10K). S>10K from wild-type cells strongly repressed the activity of glucose-6-phosphatase in wild-type microsomes, whereas S>10K from countin(-) cells (cells with low CF activity) significantly increased the activity of glucose-6-phosphatase in wild-type microsomes by decreasing K(m). The regulatory activities in the wild-type and countin(-) S>10Ks are heat-labile and protease-sensitive, suggesting that they are proteins. S<10K from both wild-type and countin(-) cells did not significantly change glucose-6-phosphatase activity. Together, the data suggest that, as a part of a pathway modulating multicellular group size, CF regulates one or more proteins greater than 10 KDa in crude cytosol that affect microsome-associated glucose-6-phosphatase activity.     

Adhesion mutants of Dictyostelium discoideum lacking the saccharide determinant recognized by two adhesion-blocking monoclonal antibodies

A mutant of Dictyostelium discoideum, strain HL260, was isolated based on its failure to bind d-41, a monoclonal antibody that blocks developmentally regulated cell-cell adhesion. The mutant fails to normally acquire cell-cell adhesion as assayed with cells shaken in 10 mM EDTA, but aggregates and and constructs fruiting bodies. Other mutant strains, HL216 and HL220, previously shown to have impaired cell-cell adhesion, also lack the determinant that binds d-41. The three strains all carry mutations in a gene designated mod B, which directs a post-translational modification of several developmentally regulated D. discoideum glycoproteins. Diploids formed between independent mod B mutant haploid strains also lack this determinant and show marked impairment of cell-cell adhesion in EDTA, indicating that mutations in mod B, rather than other mutations not shared by the haploid strains, are related to the adhesion defect. The results are consistent with other evidence that an oligosaccharide carried on several developmentally regulated glycoproteins plays an essential role in EDTA-resistant cell-cell adhesion in D. discoideum. However, this type of adhesion is not essential for morphogenesis in that the only defect detected thus far in mod B mutant strains is that they construct relatively smaller fruiting bodies that contain fewer spores.     

Length regulation in the Dictyostelium discoideum slug is a late event.

Time-lapse video light microscopy was used to study the emergence and maturation of the migratory slug from a D. discoideum aggregate. The anterior part, the tip of this simple multicellular organism, establishes migration prior to the definition of the rear, and hence the length of the slug. It was found that newly formed slugs of wild-type strain WS380B can reach lengths greater than 1 cm, yet mature slugs of this strain are rarely longer than 2-3 mumm. Often the tip extended out of the aggregation mound upon an arching pillar of cells. After the tip first touched the substratum, it commenced migration with a rapid succession of movement steps. Here we show that at the initiation of migration, a differential rate of cell movement along the developing slug axis results in a series of complicated changes, before the stable and mature shape of the slug is formed. Our results lead to new conclusions about D. discoideum slug formation and shape maintenance. Evidence is presented for regulation of slug length.     

Developmental aggregation of Myxococcus xanthus requires frgA, an frz-related gene

Myxococcus xanthus is a gram-negative bacterium which has a complex life cycle that includes multicellular fruiting body formation. Frizzy mutants are characterized by the formation of tangled filaments instead of hemispherical fruiting bodies on fruiting agar. Mutations in the frz genes have been shown to cause defects in directed motility, which is essential for both vegetative swarming and fruiting body formation. In this paper, we report the discovery of a new gene, called frgA (for frz-related gene), which confers a subset of the frizzy phenotype when mutated. The frgA null mutant showed reduced swarming and the formation of frizzy aggregates on fruiting agar. However, this mutant still displayed directed motility in a spatial chemotaxis assay, whereas the majority of frz mutants fail to show directed movements in this assay. Furthermore, the frizzy phenotype of the frgA mutant could be complemented extracellularly by wild-type cells or strains carrying non-frz mutations. The phenotype of the frgA mutant is similar to that of the abcA mutant and suggests that both of these mutants could be defective in the production or export of extracellular signals required for fruiting body formation rather than in the sensing of such extracellular signals. The frgA gene encodes a large protein of 883 amino acids which lacks homologues in the databases. The frgA gene is part of an operon which includes two additional genes, frgB and frgC. The frgB gene encodes a putative histidine protein kinase, and the frgC gene encodes a putative response regulator. The frgB and frgC null mutants, however, formed wild-type fruiting bodies.     

Protein kinase B gene homologue pkbR1 performs one of its roles at first finger stage of Dictyostelium

Dictyostelium discoideum has protein kinases AKT/PKBA and PKBR1 that belong to the AGC family of kinases. The protein kinase B-related kinase (PKBR1) has been studied with emphasis on its role in chemotaxis, but its roles in late development remained obscure. The pkbR1 null mutant stays in the first finger stage for about 16 h or longer. Only a few aggregates continue to the migrating slug stage; however, the slugs immediately go back probably to the previous first finger stage and stay there for approximately 37 h. Finally, the mutant fingers diversify into various multicellular bodies. The expression of the pkbR1 finger protein probably is required for development to the slug stage and to express ecmB, which is first observed in migrating slugs. The mutant also showed no ST-lacZ expression, which is of the earliest step in differentiation to one of the stalk cell subtypes. The pkbR1 null mutant forms a small number of aberrant fruiting bodies, but in the presence of 10% of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form nonviable stalk cells. These results suggest that the mutant has defects in a system that changes the physiological dynamics in the prestalk cell region of a finger. We suggest that the arrest of its development is due to the loss of the second wave of expression of a protein kinase A catalytic subunit gene (pkaC) only in the prestalk region of the pkbR1 null mutant.     

Alternative Developmental Pathways Determined by Environmental Conditions in the Cellular Slime Mold Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum grows in the soil as a population of independent, uninucleate amoebae. Upon entrance to the stationary phase, the amoebae collect in multicellular aggregates to form organized fruiting bodies composed of spores and stalk cells. Depending upon environmental conditions, the developing aggregate either constructs the fruiting body at the site of aggregation or transforms into a structure that can migrate to a more favorable location. Environmental conditions that favor migration are (i) the accumulation of metabolite(s) produced by the aggregate and (ii) a low ionic strength in the substratum. Conditions that prevent migration or that stop a migrating slug are (i) the presence of buffer and (ii) illumination by overhead light.     

Contribution of the Klebsiella pneumoniae capsule to bacterial aggregate and biofilm microstructures

We studied the interaction between capsule production and hydrodynamic growth conditions on the internal and macroscopic structure of biofilms and spontaneously formed aggregates of Klebsiella pneumoniae. Wild-type and capsule-deficient strains were studied as biofilms and under strong and mild hydrodynamic conditions. Internal organization of multicellular structures was determined with a novel image-processing algorithm for feature extraction from high-resolution confocal microscopy. Measures included interbacterial spacing and local angular alignment of individual bacteria. Macroscopic organization was measured via the size distribution of aggregate populations forming under various conditions. Compared with wild-type organisms, unencapsulated mutant organisms formed more organized aggregates with less variability in interbacterial spacing and greater interbacterial angular alignment. Internal aggregate structure was not detectably affected by the severity of hydrodynamic growth conditions. However, hydrodynamic conditions affected both wild-type and mutant aggregate size distributions. Bacteria grown under high-speed shaking conditions (i.e., at Reynolds' numbers beyond the laminar-turbulent transition) formed few multicellular aggregates while clumpy growth was common in bacteria grown under milder conditions. Our results indicate that both capsule and environment contribute to the structure of communities of K. pneumoniae, with capsule exerting influence at an interbacterial length scale and fluid dynamic forces affecting overall particle size.     

Co-occurrence in nature of different clones of the social amoeba,Dictyostelium discoideum

The social amoeba, Dictyostelium discoideum, produces a multicellular fruiting body and has become a model system for cell-cell interactions such as signalling, adhesion and development. However, unlike most multicellular organisms, it forms by aggregation of cells and, in the laboratory, forms genetic chimeras where there may be competition among clones. Here we show that chimera formation is also likely in nature, because different clones commonly co-occur on a very small scale. This suggests that D. discoideum will likely have evolved strategies for competing in chimeras, and that the function of some developmental genes will be competitive. Natural chimerism also makes D. discoideum a good model organism for the investigation of issues relating to coexistence and conflict between cells.     

Loss of FrmB results in increased size of developmental structures during the multicellular development of <Emphasis Type="Italic">Dictyostelium</Emphasis> cells

FERM domain-containing proteins are involved in diverse biological and pathological processes, including cell-substrate adhesion, cell-cell adhesion, multicellular development, and cancer metastasis. In this study, we determined the functions of FrmB, a FERM domain-containing protein, in the cell morphology, cell adhesion, and multicellular development of Dictyostelium cells. Our results show that FrmB appears to play an important role in regulating the size of developmental structures. frmB null cells showed prolonged aggregation during development, resulting in increased size of developmental structures, such as mounds and fruiting bodies, compared to those of wild-type cells, whereas FrmB overexpressing cells exhibited decreased size of developmental structures. These results suggest that FrmB may be necessary for limiting the sizes of developmental structures. Loss of FrmB also resulted in decreased cell-substrate adhesion and slightly increased cell area, suggesting that FrmB had important roles in the regulation of cell adhesion and cell morphology. These studies would contribute to our understanding of the intertwined and overlapped functions of FERM domain-containing proteins.     

Cell growth and morphology of Dictyostelium discoideum in space environment.

Two strains of cellular slime mold Dictyostelium discoideum, a radiation-sensitive mutant and the parental wild-type strain, were used to investigate the effects of microgravity and/or cosmic radiation on their morphology through the whole life span from spores to fruiting bodies for about 7 days in space shuttle of NASA. We found almost no effect of space environment on amoeba cell growth in both strains. It was also observed that almost the same number and shape of fruiting bodies in space compared to the control experiments on earth. These results suggest that there is little effect of microgravity and space radiation on germination, cell aggregation, cell differentiation and cell morphology in the cellular slime mold.