Wild-type Amoebae Research Articles

Cooperative predation in the social amoebae Dictyostelium discoideum.

The eukaryotic amoeba Dictyostelium discoideum is commonly used to study sociality. The amoebae cooperate during development, exhibiting altruism, cheating, and kin-discrimination, but growth while preying on bacteria has been considered asocial. Here we show that Dictyostelium are cooperative predators. Using mutants that grow poorly on Gram-negative bacteria but grow well on Gram-positive bacteria, we show that growth depends on cell-density and on prey type. We also found synergy, by showing that pairwise mixes of different mutants grow well on live Gram-negative bacteria. Moreover, wild-type amoebae produce diffusible factors that facilitate mutant growth and some mutants exploit the wild type in mixed cultures. Finding cooperative predation in D. discoideum should facilitate studies of this fascinating phenomenon, which has not been amenable to genetic analysis before.

Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1+) or repressed by antisense small RNA-mediated gene silencing (ICP1−). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1+ strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1− strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1+ amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1− amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.

A new human 3D-liver model unravels the role of galectins in liver infection by the parasite Entamoeba histolytica.

Investigations of human parasitic diseases depend on the availability of appropriate in vivo animal models and ex vivo experimental systems, and are particularly difficult for pathogens whose exclusive natural hosts are humans, such as Entamoeba histolytica, the protozoan parasite responsible for amoebiasis. This common infectious human disease affects the intestine and liver. In the liver sinusoids E. histolytica crosses the endothelium and penetrates into the parenchyma, with the concomitant initiation of inflammatory foci and subsequent abscess formation. Studying factors responsible for human liver infection is hampered by the complexity of the hepatic environment and by the restrictions inherent to the use of human samples. Therefore, we built a human 3D-liver in vitro model composed of cultured liver sinusoidal endothelial cells and hepatocytes in a 3D collagen-I matrix sandwich. We determined the presence of important hepatic markers and demonstrated that the cell layers function as a biological barrier. E. histolytica invasion was assessed using wild-type strains and amoebae with altered virulence or different adhesive properties. We showed for the first time the dependence of endothelium crossing upon amoebic Gal/GalNAc lectin. The 3D-liver model enabled the molecular analysis of human cell responses, suggesting for the first time a crucial role of human galectins in parasite adhesion to the endothelial cells, which was confirmed by siRNA knockdown of galectin-1. Levels of several pro-inflammatory cytokines, including galectin-1 and -3, were highly increased upon contact of E. histolytica with the 3D-liver model. The presence of galectin-1 and -3 in the extracellular medium stimulated pro-inflammatory cytokine release, suggesting a further role for human galectins in the onset of the hepatic inflammatory response. These new findings are relevant for a better understanding of human liver infection by E. histolytica.

Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism

Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis.

Bacterial Discrimination by Dictyostelid Amoebae Reveals the Complexity of Ancient Interspecies Interactions

Amoebae and bacteria interact within predator-prey and host-pathogen relationships, but the general response of amoeba to bacteria is not well understood. The amoeba Dictyostelium discoideum feeds on, and is colonized by, diverse bacterial species, including Gram-positive [Gram(+)] and Gram-negative [Gram(-)] bacteria, two major groups of bacteria that differ in structure and macromolecular composition. Transcriptional profiling of D. discoideum revealed sets of genes whose expression is enriched in amoebae interacting with different species of bacteria, including sets that appear specific to amoebae interacting with Gram(+) or with Gram(-) bacteria. In a genetic screen utilizing the growth of mutant amoebae on a variety of bacteria as a phenotypic readout, we identified amoebal genes that are only required for growth on Gram(+) bacteria, including one that encodes the cell-surface protein gp130, as well as several genes that are only required for growth on Gram(-) bacteria, including one that encodes a putative lysozyme, AlyL. These genes are required for parts of the transcriptional response of wild-type amoebae, and this allowed their classification into potential response pathways. We have defined genes that are critical for amoebal survival during feeding on Gram(+), or Gram(-), bacteria that we propose form part of a regulatory network that allows D. discoideum to elicit specific cellular responses to different species of bacteria in order to optimize survival.

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.

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.

In vitro Effect of Garlic Extract and Metronidazole Against Neoparamoeba pemaquidensis, Page 1987 and Isolated Amoebae from Atlantic Salmon

Neoparamoeba pemaquidensis believed to be the most prevalent parasite of Atlantic salmon industry in Australia. In the present study, the in vitro effects of crude extract of garlic and metronidazole were investigated using a primary culture toxicity assay. Garlic extract appeared to be completely effective at killing a cultured strain (NP251002) of Neoparamoeba pemaquidensis in vitro at a dilution of 1:100 with in 24 h. The number of viable Amoebae after using garlic extract in lower dilutions (1:200, 1:400, 1:800, 1:1000) for 24 h, also were significantly lower than in the control group. Garlic extract was also efficacious at killing wild type Amoebae that isolated from the diseased fish showing clinical signs of AGD. Metronidazole had no clear effect against Neoparamoeba pemaquidensis (NP251002) even in a concentration of 50 mg L(-1) for 24 h. However some morphological changes have occurred in metronidazole-treated Amoebae after 5 days of exposure.

Antisense Inhibition of Entamoeba histolytica Cysteine Proteases Inhibits Colonic Mucus Degradation

The exact role Entamoeba histolytica cysteine proteases play in overcoming the colonic mucus barrier, as a prerequisite to epithelial cell disruption, is not known. Herein, we determined whether E histolytica trophozoites expressing the antisense transcript to cysteine protease 5 (EhCP5) could degrade colonic mucin and destroy epithelial cells. Cysteine protease-deficient amoebae were generated by antisense inhibition of EhCP5, and assayed for proteolytic activity against [(35)S]cysteine-labeled mucin from LS 174T, and HT-29F Cl.16E cells. Recombinant EhCP5 mucinase activity was also assessed. Disruption of an intact mucus barrier and epithelial cell invasion by amoebae were measured using high mucin producing LS 174T and HT-29 Cl.16E monolayers or Chinese hamster ovary (CHO) cells devoid of a mucus barrier. Trophozoites with reduced cysteine protease activity were ineffective at degrading [(35)S]cysteine-labeled colonic mucin compared to wild-type amoebae by >60%. However, bioactive recombinant EhCP5 degraded >45% of purified native mucin, which was specifically inhibited by the cysteine proteinase (CP) inhibitor, E-64. Cysteine protease-deficient trophozoites could not overcome a protective intact mucus barrier and disrupt LS 174T or HT-29F Cl.16 cell monolayers; however, they readily adhere to and disrupt CHO monolayers devoid of a mucus barrier. These findings unravel a central role for E histolytica CPs as key virulence factors in disrupting an intact mucus barrier in the pathogenesis of intestinal amoebiasis.

On the effects of cycloheximide on cell motility and polarisation in Dictyostelium discoideum

BackgroundCycloheximide is a protein synthesis inhibitor that acts specifically on the 60S subunit of eukaryotic ribosomes. It has previously been shown that a short incubation of Dictyostelium discoideum amoebae in cycloheximide eliminates fluid phase endocytosis.ResultsWe found that treatment with cycloheximide also causes the amoebae to retract their pseudopodia, round up and cease movement. Furthermore, fluid phase endocytosis, phagocytosis and capping cease in the presence of 2 mM cycloheximide, although membrane uptake, as measured using FM1-43, is unaffected. In the presence of cycloheximide, aggregation-competent amoebae sensitive to cAMP, although round, can still localise CRAC, ABP120, PI3K and actin polymerisation in response to a micropipette filled with cAMP. The behaviour of wild-type amoebae in the presence of cycloheximide is surprisingly similar to that of amoebae having a temperature-sensitive version of NSF at the restrictive temperature.ConclusionOur results may suggest that, upon cycloheximide treatment, either a labile protein required for polarised membrane recycling is lost, or a control mechanism linking protein synthesis to membrane recycling is activated.

Ca2+ regulation in the absence of the iplA gene product in Dictyostelium discoideum

BackgroundStimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA- mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis.ResultsWe investigated Ca2+-fluxes and the effect of their disturbance on chemotaxis and development of iplA- cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca2+-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn2+ was necessary to observe agonist-induced Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly loaded with Ca2+, again with a similar shift in sensitivity in the mutant. In addition, basal [Ca2+]i was significantly lower in iplA- than in wild type amoebae.ConclusionThese results support the view that [Ca2+]i-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The iplA- strain retains the capacitative Ca2+-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure.

Evidence that hsp90 is involved in the altered interactions of Acanthamoeba castellanii variants with bacteria.

There are many similarities between the interactions of environmental protozoa with pathogenic bacterial species and those observed in mammalian macrophages. Since single-celled protozoa predate mammalian hosts, it is likely that interactions in environmental biofilms have selected for many of the bacterial virulence mechanisms responsible for human disease. In order to better understand bacterial-phagocyte interactions, we developed a selection for Acanthamoeba castellanii variants that are more resistant to killing by bacterial pathogens. We identified four amoebal clones that display decreased phagocytosis of bacteria but no difference in uptake of latex beads compared to wild-type amoebae. These amoebal variants display differences in cellular morphology, partial resistance to killing by bacteria, more bactericidal activity, and higher frequencies of lysosome fusion with the bacterial vacuole. Three proteins are present at lower levels in these variants than in wild-type amoebae, and matrix-assisted laser desorption ionization-time of flight mass spectrometry allowed identification of two of them as actin and hsp90. We found that specific inhibitors of hsp90 produce a similar phenotypic effect in macrophages. These data suggest that hsp90 plays a role in phagocytic and, possibly, bactericidal pathways that affect interactions of phagocytic cells with bacteria.

Mutation of the Dictyostelium fbxA Gene Affects Cell-Fate Decisions and Spatial Patterning

Cell-fate decisions and spatial patterning in Dictyostelium are regulated by a number of genes. Our studies have implicated a gene called fbxA, which codes for an F-box protein, in these pathways. The FbxA protein is one of the controls on a cAMP phosphodiesterase called RegA, mediating its degradation via ubiquitin-linked proteolysis. Using marked strains, we showed that the fbxA- mutant has defective cell-type proportioning, with a dearth of prestalk cells compared to prespore cells. In this work, we show that this effect occurs earlier during the 24 hour developmental cycle than previously thought. The normal sorting of the prestalk and prespore cells in aggregates and mounds is not affected by the mutation. The mutant cells sort abnormally at the tipped mound stage, when prespore and prestalk cells normally distribute into their proper compartments. The fbxA- mutant forms pre-stalk cells in low numbers when not in chimeras, but in the presence of wild-type amoebae the mutant preferentially forms viable spores, driving the wild type to form non-viable stalk cells. In an attempt to identify the signal transduction pathway that mediates proportionality in prestalk and prespore cells, we asked whether certain signal transduction mutants were immune to the effects of the fbxA- cells and formed spores in chimeras.

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.

Ammonia hypersensitivity of slugger mutants of D. discoideum.

The weak base ammonia inhibits aggregation and culmination of wild-type amoebae of Dictyostelium discoideum. Here we have examined its effect on a series of 'slugger' mutants previously assigned to 10 complementation groups, and so-called because they remain as slugs for extended periods. We show that the mutants accumulate normal levels of ammonia and hence may be abnormally susceptible to the ammonia they produce. In agreement with this we find that representatives of the slugger complementation groups are hypersensitive to ammonia inhibition at three clearly recognisable morphological stages of development: aggregation, tip formation and culmination. This finding suggests that a common ammonia-sensitive process underlies each of these developmental events.

Growth, Development and Genetic Characteristics of Physarum polycephalum Amoebae Able to Grow in Liquid, Axenic Medium

Amoebae from natural isolates of Physarum polycephalum, unlike the plasmodial phase, are unable to grow in axenic medium. A mutant strain of amoebae, CLd-AXE, can be cultured in the liquid, semi-defined medium used for plasmodial culture but lacks some of the properties required for studies of development and gene expression. From crosses of CLd-AXE with wild-type amoebae, new amoebal strains able to grow in axenic medium have been isolated; some of these can also undergo the reversible amoeba-flagellate transformation and apogamic plasmodium development in axenic conditions. Amoebae maintained in active growth in liquid culture for several months showed little change in their properties. Subcultures made with diluted inocula indicated that the same growth rate was achieved even when single amoebae were inoculated in liquid medium. All strains produced colonies with high efficiency when replated on bacterial lawns. Measurements of DNA content by flow cytometry indicated that the majority of amoebae in liquid cultures were haploid. Homozygous diploid amoebae constructed from one strain grew less well than the haploid cells. Genetic analysis of crosses suggested that amoebae able to grow in liquid axenic medium fell into one major phenotypic class with respect to growth rate, and that mutation at only one or two loci was necessary to allow amoebae to grow in axenic medium. Diploid, heterozygous amoebae constructed by mating a mutant with a wild-type strain were unable to grow in axenic medium, indicating that at least one of the putative axe alleles was recessive.

Evidence that elevated intracellular cyclic AMP triggers spore maturation in Dictyostelium

ABSTRACT Spore maturation occurs during normal development in Dictyostelium when environmental influences induce a migrating slug to transform into a fruiting body. As the amoeboid prespore cells turn into refractile spores there is a burst of enzyme accumulation, including UDP-galactose epimerase, and at a later stage the exocytosis of preformed components of the spore coat. Evidence is presented here that this process is triggered by an elevated intracellular cAMP concentration. First, a number of rapidly developing (rde) mutants, whose cAMP metabolism had been investigated previously, are shown to be able to form spores in submerged monolayers, whereas wild-type strains are not. The phenotypes of these mutants are best explained by a derepression of the signal transduction pathway utilizing intracellular cAMP. Second and more direct, it is shown that the permeant cAMP analogues 8-Br-cAMP and 8-chlorophenylthio-cAMP, but not cAMP itself, can rapidly induce spore differentiation in wild-type amoebae incubated in submerged monolayers. These analogues also stimulate accumulation of UDP-galactose epimerase in slug cells transferred to shaken suspension. The ability to induce spore differentiation with Br-cAMP in wild-type strains provides a new technique that can be exploited in various ways. For instance, spore differentiation in strain V12M2 is induced by 8-Br-cAMP at very low cell densities, suggesting that neither cell contact nor additional soluble inducers are necessary in these conditions. In contrast NC4 cells may require an additional inducer. Spore differentiation is inhibited by the stalk-specific inducer DIF-1 suggesting that DIF-1 inhibits a target downstream of intracellular cAMP in the signal transduction pathway inducing spore differentiation.

Effects of suboptimal levels of extracellular calcium on the regulation of the cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation in Dictyostelium discoideum.

When starved wild-type amoebae of Dictyostelium discoideum were washed and incubated in 1 mM-EGTA, their ability to induce soluble cyclic AMP phosphodiesterase (PD) activity in response to either millimolar cyclic AMP or a series of nanomolar cyclic AMP pulses was reduced by 55-75%. Supplementation of EGTA-treated cells with exogenous Ca2+ stimulated PD induction in a dose-dependent fashion (EC50 = 100-200 nM free extracellular Ca2+), and enzyme production was maximal at about 1 microM free Ca2+. Ca2+ depletion also strongly impaired production of the phosphodiesterase inhibitor (PDI). In contrast, other than delaying their appearance by about 1 h, EGTA had little effect on the induction by cyclic AMP pulses of cell surface markers such as contact sites A and membrane-bound PD activity. Similar changes in both the soluble and membrane activities were observed with strain NP368, a mutant that overproduces cyclic GMP when stimulated by cyclic AMP. Thus, Ca2+ depletion does not appear to inhibit PD and PDI production by reducing intracellular cyclic GMP. To determine whether Ca2+ depletion alters signal transduction, two mutants that produce the soluble PD activities constitutively were examined. Suboptimal concentrations of free extracellular Ca2+ were found to inhibit PD production in these cells to the same degree and with the same concentration dependence as low Ca2+ inhibited PD induction by cyclic AMP in wild-type cells. These results suggest that Ca2+ depletion by EGTA probably inhibits PD and PDI production indirectly by perturbing an intracellular Ca2+ pool(s) rather than by altering a surface cyclic AMP-receptor-mediated process.

Growth and differentiation of wild type amoebae of Physarum polycephalum in liquid culture

A method for growing wild type amoebal strains of Physarum polycephalum in two-membered liquid culture is presented. The medium is a simple buffered salts solution. We found that a minimal level of divalent cation was required for growth. All amoebal strains tried to date have grown under our conditions in stationary culture. Growth under gyratory conditions was only successful at 60 rpm or less and consistent growth required a period of adaptation over several transfers. Differentiation of two apogamic strains, CL and CH1, were compared. Contrary to the results seen on agar plates, the time of onset for the first committed amoebae was identical for both strains in liquid culture. Attempts to demonstrate mating between two genetically compatible amoebal strains grown together in liquid culture were not successful.

Chemotactic response of wild-type and aggregation-defective mutants of Polysphondylium violaceum

The aggregation-specific chemoattractant for Polysphondylium violaceum is N-propionyl-γ-L-glutamyl-L-ornithine-δ-lactam ethyl ester, or glorin. Wild-type amoebae allowed to develop in liquid culture acquire increased ability to respond to glorin shortly after starvation, i.e., just prior to the time they become aggregation competent. Similarly, as development proceeds, the amoebae show decreased sensitivity to folic acid, but they show almost no response to cyclic AMP at any time during their development in liquid culture. The optimum concentrations for the chemotactic response are 10 -8 M for glorin and 10 -5–10 -6 M for folic acid. A class of aggregation-defective mutants, aggA, will not aggregate in the absence of an excreted pheromone, D factor. During development in liquid culture in the presence or absence of D factor, these aggA mutants show a chemotactic response similar to that of wild-type amoebae to folic acid and glorin. However, D factor does enhance the chemotactic response of aggA mutants to glorin. In the absence of D factor, mutant amoebae will form fruiting bodies if exposed to a chemotactic gradient of either folic acid or glorin. Under these conditions, the mutant amoebae circumvent the requirement for D factor in order to develop.