A bacterial factor induces changes in cysteine proteinase forms in the cellular slime mould Dictyostelium discoideum.

The electrophoretic pattern of cysteine proteinases in axenically grown myxamoebae of Dictyostelium discoideum can be altered by the addition of either Gram-negative (Klebsiella aerogenes, Escherichia coli) or Gram-positive (Micrococcus lysodeikticus, Bacillus subtilis) bacteria to the culture. No changes occurred, however, if either yeast or latex beads were used in place of bacteria. The changes involved the simultaneous loss of proteinases characteristic of the axenic cells (the A-forms) and the acquisition of those found in cells which have been grown on bacteria (the B-forms). Using K. aerogenes the conversion was complete within 4 h. Extracellular proteinase activity was unaffected during this period. After the D. discoideum cells had been lysed, no equivalent change in proteinase band pattern could be produced either by prolonged incubation of cell extracts or by treatment with proteinases. An identical conversion could be induced in cultures of myxamoebae by a factor, cysteine proteinase converting factor (CPCF), present in the 15,000 g supernatant of a sonicated suspension of K. aerogenes. CPCF was macromolecular, as demonstrated by both ultrafiltration and gel filtration, acid-precipitable, but was soluble in ethanol or alkali. Its activity was unaffected by treatment with trypsin. The results suggested that CPCF might be a component of the bacterial cell wall, and since its activity was affected by lysozyme treatment, peptidoglycan is implicated. The results can be interpreted in terms of a novel nutrient-dependent post-translational change which affected most of the cysteine proteinases present in D. discoideum myxamoebae.     

Cystein proteinase changes during macrocyst formation in Dictyostelium mucoroides

Cysteine proteinases were detected in vegetative myxamoebae of Dictyostelium mucoroides DM7 using chromogenic substrates and by electrophoretic analysis (gelatin-SDS-PAGE) which revealed three enzymes, dmCP30, dmCP35 and dmCP46 (a minor form). During the initial stages of macrocyst formation the cysteine proteinaes were secreted and disappeared almost completely from the cells. High extracellular levels of activity towards N-benzoyl-L-prolyl-L-phenylalanyl-L-arginine 4-nitroanilide and of dmCP30 persisted throughout macrocyst development. Three new intracellular proteinases, dmCP31, dmCP36 and dmCP40, were produced as macrocysts formed but their activity was only detected by gelatin-SDS-PAGE. Their appearance was specific to the developmental pathway leading to macrocyst formation. This is the first direct evidence for the accumulation of cysteine proteinases during a developmental process in a cellular slime mould.     

Increase in proteinase activity in the cellular slime mould Polysphondylium pallidum induced by bacteria

Abstract Polysphondylium pallidum strain PPHU8 grown in association with bacteria contains aspartic and cysteine proteinases. When myxamoebae were grown in axenic medium the contribution of cysteine proteinases was much lower. The proteinase activity could be altered by addition of heat-killed bacteria to axenically growing cells. This was detected as an increase in the specific activity towards N -benzoyl-L-prolyl-L-phenylalanyl-L-arginine- p -nitroanilide, a cysteine proteinase substrate, and by the appearance of cysteine proteinase bands after electrophoretic analysis. The changes were inhibited by cycloheximide, azide and dinitrophenol. All the available evidence suggests that they are due to the de novo synthesis of cysteine proteinases.     

Multiple cysteine proteinase forms during the life cycle of Dictyostelium discoideum revealed by electrophoretic analysis.

Proteinases of the cellular slime mould Dictyostelium discoideum have been analysed using electrophoresis on polyacrylamide gels containing gelatin (gelatin/PAGE). Multiple proteinase forms were apparent in vegetative myxamoebae, but the presence of individual enzyme forms depended on the manner in which the cells were grown. Axenic cells had a characteristic A-pattern of proteinases consisting of six bands, the most active enzymes having apparent Mr values of 51,000 and 45,000 (these have been named ddCP51 and ddCP45, respectively). Some of the proteinases were also present in the medium, the major extracellular form was ddCP42, a 42,000-Mr enzyme. Cells grown in association with bacteria had a distinct B-pattern with three main enzymes that had apparent Mr values of 48,000, 43,000 and 38,000. All of the A- and B-pattern proteinases were most active at acid pH in the presence of dithiothreitol and were inhibited by various agents such as trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E64), leupeptin and chymostatin, which inactivate cysteine proteinases. One of the enzymes, ddCP30, was identified as cysteine proteinase B which had been purified and characterized previously [North, M.J. & Whyte, A. (1984) J. Gen. Microbiol. 130, 123-134]. During starvation of axenic cells in shaken suspensions some of the vegetative proteinases disappeared, ddCP42 was released from the cells and one new enzyme with an apparent Mr of 48,000 appeared. Addition of cyclic AMP had little effect on these changes. When the axenically grown myxamoebae underwent development on filters, similar changes in band pattern were observed and the aggregation stage was characterized by the presence of three cysteine proteinase bands (apparent Mr values of 48,000, 45,000 and 43,000). Proteinases, especially ddCP42, were released from the cells and could be collected from the buffer-saturated pads which supported the filters. The results demonstrate that cysteine proteinases are present throughout growth and development of D. discoideum and that the forms present are subject to nutritional and developmental regulation.     

Differential in vitro activation and deactivation of cysteine proteinases isolated during spore germination and vegetative growth of Dictyostelium discoideum.

Acid-activatable cysteine proteinases of Dictyostelium discoideum were first identified in spore extracts of strain SG1 using gelatin/SDS/PAGE, followed by acid treatments. Here we utilized the technique of acid activation to identify cryptic cysteine proteinases throughout auto-induced and heat-induced spore germination of D. discoideum strain SG2 and SG1. The major acid-activatable cysteine proteinase identified in SG2 and SG1 spore extracts was ddCP38 (D. discoideum cysteine proteinase with a molecular mass of 38 kDa) and ddCP48, respectively. Further investigation of these enzymes revealed that they were also base deactivatable with a treatment of ammonium chloride directly following acid activation. However, the most intriguing observation was the reversibility of the effects of base deactivation on the enzymes following a second treatment with acetic acid. Thus, we hypothesize that, unlike most mammalian cysteine proteinases which generally require the cleavage of a pro-peptide region for activation, these cysteine proteinases of D. discoideum likely undergo reversible conformational changes between latent and active forms. Moreover, we were able to detect these cryptic cysteine proteinases in the vegetative cells and early aggregates of both strains SG1 and SG2. Studies using 4-[(2S, 3S)-3-carboxyoxiran-2-ylcarbonyl-L-leucylamido]buty lguanidine, a cysteine proteinase inhibitor, revealed that acid activation of a portion of these proteinases was still achievable even after incubation with the inhibitor, further supporting the concept of two stable and reversible conformational arrangements of the enzymes. Thus, we speculate that the pH shuffles that modulate proteinase conformation and activity in vitro may be a reflection of the in vivo regulation of these enzymes via H+-ATPases and ammonia.     

Spore Germination in Strains of Dictyostelium discoideum and Other Members of the Dictyosteliaceae

Spores of all strains of Dictyostelium discoideum tested in this study germinated after a heat shock of 45 C for 30 min. Whereas the strains differed in their rates of germination, the rate for each strain was constant. A correlation existed between the rate of germination and the rate of vegetative growth when spores were inoculated into bacterial streaks. Heat shock clearly increased spore germination in D. purpureum, but the response was less dramatic than in D. discoideum. Enhancement also occurred in D. rosarium, but only in media containing peptone. Strains of D. mucoroides gave varied responses, and these could be divided into those which required mutrients for spore germination and those which did not. The spores of Polysphondylium pallidum were resistant to mild heat (45 C), but were not activated; peptone was required for germination. In contrast, the microcysts of this species were heat-labile and required no added nutrients for excystment.     

Diphospho-myo-inositol phosphates during the life cycle of Dictyostelium and Polysphondylium.

The intracellular amounts of diphospho-myo-inositol phosphates and InsP6 were determined in Dictyostelium discoideum AX2 throughout the life cycle, including exponential growth, starvation, differentiation, sporulation and spore germination. Similar experiments were performed with the closely related species Polysphondylium pallidum under conditions resulting in microcyst formation. A distinct accumulation of these compounds is observed during the early starvation phase of the cell population before the onset of the actual differentiation program. When exponentially growing D. discoideum cells were shifted to starvation conditions, a 25-fold accumulation of 5,6-bis-PP-InsP4 within 3 h was observed. In P. pallidum, the 5,6-bis-PP-InsP4 pool rises around 20-fold within 8 h during the formation of microcysts from vegetative cells. Finally, the diphosphoinositol phosphates are deposited in spores or microcysts and are degraded when spores or microcysts germinate at low cell density.     

Dissociation of proteinase-inhibitor complexes by trichloroacetate

It was demonstrated that the addition of high concentrations of the chaotrope, sodium trichloroacetate, to proteinase assays provided for a dissociation of proteinase-inhibitor complexes. The complexes evaluated contained a heat-stable, polypeptide inhibitor of cysteine proteinases isolated from the cellular slime mold, Dictyostelium discoideum. The proteinases that were present in separate complexes included either D. discoideum proteinases or the plant proteinase papain. The general assay procedures described may be useful in detection of endogenous proteinase-inhibitor complexes in many systems.     

Genetic exchanges in the macrocysts of Dictyostelium discoideum.

Crosses were made between strains of Dictyostelium discoideum involving two drug resistance markers and the mating-type locus. Over 6000 progeny from 263 individual germinated macrocysts from four single-factor crosses, five two-factor crosses and one three-factor cross were characterized. In most cases the progeny from a single macrocyst were of one genotype, although in the population of macrocysts from any two-factor cross all possible parental and recombinant genotypes were recovered. There was no evidence of linkage between any of the markers examined. No selection against progeny carrying the methanol or the cycloheximide resistance markers was found in two-factor crosses, but selection against progeny carrying both resistance markers was found in the three-factor cross. Germination of macrocysts in all crosses was poor, only once exceeding 2.5% of the total macrocyst population. A variety of crosses and back-crosses with different parental strains indicated that germination might be influenced by both extrinsic (environmental) and multiple genetic factors. About 10% of the macrocysts yielded progeny spores that were ambivalent in their mating reactions. After extensive recloning these populations could be resolved to the normal matA (formerly A1) and mata (formerly A2) mating-types and might therefore have represented aneuploids. The results obtained with D. discoideum macrocysts differ from those obtained with other cellular slime moulds--Dictyostelium mucoroides, Dictyostelium giganteum and Polysphondylium pallidum--and are reminiscent of the results reported for germinated zygospores of Phycomyces blakesleeanus.     

Glycogen synthetase and the control of glycogen synthesis in the cellular slime mould Dictyostelium discoideum during the growth (myxamoebal) phase

1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 that are grown in axenic medium containing 86mm-glucose have seven times the glycogen content of the same myxamoebae grown in the same medium but lacking added carbohydrate. 2. During the transition from the exponential to the stationary phase of growth in axenic medium containing glucose myxamoebae preferentially synthesize glycogen and can have as much as three times the glycogen content during the stationary phase as they have during the exponential phase of growth. 3. The rate of glycogen degradation by myxamoebae is, under all conditions of growth, small compared with the rate of glycogen accumulation and the changes in glycogen content thus reflect altered rates of glycogen synthesis. 4. There is no correlation between the rate of glycogen synthesis by myxamoebae and the glycogen synthetase content of the myxamoebae. 5. The activity of glycogen synthetase of D. discoideum is inhibited by a physiological concentration of ATP and this inhibition is overcome by glucose 6-phosphate. Both effects are especially marked at physiological concentrations of UDP-glucose. 6. The rate of glycogen accumulation by myxamoebae growing exponentially in axenic media can be satisfactorily accounted for in terms of the known intracellular concentrations of glucose 6-phosphate, UDP-glucose and glycogen synthetase. The rate-limiting factors controlling glycogen synthesis by the myxamoebae are apparently the substrate (UDP-glucose) and effector (glucose 6-phosphate and ATP) concentrations rather than the amount of the enzyme.     

Enzyme synthesis in the cellular slime mould Dictyostelium discoideum during the differentiation of myxamoebae grown axenically

1. Myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 were grown on different media, and were harvested either in the stationary or exponential phases of the growth cycle to yield samples of myxamoebae differing in enzymic composition. 2. Morphogenesis and cell differentiation phenomena in D. discoideum appear to be similar in myxamoebae grown and harvested under different conditions. 3. The specific activity of the enzymes beta-N-acetylglucosaminidase, acid phosphatase, alpha-mannosidase, beta-glucosidase and alkaline phosphatase have been determined during cell differentiation of myxamoebae grown and harvested under different conditions. 4. The pattern of synthesis of these enzymes, all of which have been claimed to be part of the ;developmental programme', either remains unaffected despite the origin of the myxamoebae (alkaline phosphatase) or is qualitatively similar but quantitatively affected (acid phosphatase, beta-glucosidase) or is both qualitatively and quantitatively affected by changes in the myxamoebae (alpha-mannosidase, beta-N-acetylglucosaminidase). 5. The implications of these results for the concept of a ;developmental programme' are discussed.     

Multiple acid proteinases in the cellular slime mould Dictyostelium discoideum.

Proteinase activity in the cellular slime mould Dictyostelium discoideum has been analyzed by electrophoresis on polyacrylamide gels containing denatured hemoglobin. At least eight bands due to acid proteinases have been defined using extracts of myxamoebae, four bands A-D which move faster than the fifth and major band E, a minor band E' which moves just behind E and two slow bands G and H. Fruiting body formation was accompanied by the appearance of one new proteinase band F. The proteinases were present in extracts of both axenically-grown and bacterially-grown cells. Differences between the pH dependence and stability of the individual proteinases were detected. Inhibitor studies suggested that the faster proteinases A-D may be cathepsin B-like, whilst the slower enzymes E, E' and F do not fit readily into any known group of proteinases since they were sensitive to HgCl2 but not to other inhibitors of cathepsin B and not to inhibitors of cathepsin D-like proteinases under standard conditions. None of the proteinases was apparently formed during or after preparation of extracts and the proteinases could be re-run on polyacrylamide gels to give only the band expected from the first run. The bands are believed to reflect multiple proteinase activities within the cell.     

Inhibition of early but not late proteolytic processing events leads to the missorting and oversecretion of precursor forms of lysosomal enzymes in Dictyostelium discoideum

Lysosomal enzymes are initially synthesized as precursor polypeptides which are proteolytically cleaved to generate mature forms of the enzymatically active protein. The identification of the proteinases involved in this process and their intracellular location will be important initial steps in determining the role of proteolysis in the function and targeting of lysosomal enzymes. Toward this end, axenically growing Dictyostelium discoideum cells were pulse radiolabeled with [35S]methionine and chased in fresh growth medium containing inhibitors of aspartic, metallo, serine, or cysteine proteinases. Cells exposed to the serine/cysteine proteinase inhibitors leupeptin and antipain and the cysteine proteinase inhibitor benzyloxycarbonyl-L-phenylalanyl-L-alanine-diazomethyl ketone (Z-Phe- AlaCHN2) were unable to complete proteolytic processing of the newly synthesized lysosomal enzymes, alpha-mannosidase and beta-glucosidase. Antipain and leupeptin treatment resulted in both a dramatic decrease in the efficiency of proteolytic processing, as well as a sevenfold increase in the secretion of alpha-mannosidase and beta-glucosidase precursors. However, leupeptin and antipain did not stimulate secretion of lysosomally localized mature forms of the enzymes suggesting that these inhibitors prevent the normal sorting of lysosomal enzyme precursors to lysosomes. In contrast to the results observed for cells treated with leupeptin or antipain, Z-Phe-AlaCHN2 did not prevent the cleavage of precursor polypeptides to intermediate forms of the enzymes, but greatly inhibited the production of the mature enzymes. The accumulated intermediate forms of the enzymes, however, were localized to lysosomes. Finally, fractionation of cell extracts on Percoll gradients indicated that the processing of radiolabeled precursor forms of alpha-mannosidase and beta-glucosidase to intermediate products began in cellular compartments intermediate in density between the Golgi complex and mature lysosomes. The generation of the mature forms, in contrast, was completed immediately upon or soon after arrival in lysosomes. Together these results suggest that different proteinases residing in separate intracellular compartments may be involved in generating intermediate and mature forms of lysosomal enzymes in Dictyostelium discoideum, and that the initial cleavage of the precursors may be critical for the proper localization of lysosomal enzymes.     

Growth of myxamoebae of the cellular slime mould Dictyostelium discoideum in axenic culture

1. A simple axenic medium suitable for the growth of the myxamoebae of a strain of the cellular slime mould Dictyostelium discoideum is described. 2. Procedures suitable for the growth of this strain in liquid and on solid media are described. 3. Conditions suitable for initiating the cell differentiation of myxamoebae grown axenically are described.     

Correlation between acrasins and spore germination inhibitors in cellular slime molds.

Discadenine,3-(3-amino-3-carboxypropyl)-N6-delta 2-isopentenyladenine, which inhibits spore germination, was previously found in Dictyostelium discoideum. Studies on the distribution of discadenine in different species of cellular slime molds by high-pressure liquid chromatography showed that discadenine is present in D. discoideum, Dictyostelium purpureum, and Dictyostelium mucoroides, but not in Dictyostelium minutum, Polysphondylium violaceum, or Polysphondylium pallidum. Discadenine synthetase, which is involved in biosynthesis of discadenine with N6-delta 2-isopentenyladenine as substrate, was only detected in cells of the former three species. In addition, discadenine inhibited spore germination only in these three species. These results clearly demonstrate that discadenine is produced as an inhibitor of spore germination in the species of cellular slime molds in which the acrasin is cyclic adenosine 5'-monophosphate (AMP). This means that there is a structural and biochemical correlation between the spore germination inhibitor and the acrasin, since 5'-AMP, a direct precursor in discadenine biosynthesis, can be derived from cyclic AMP by hydrolysis with cyclic AMP phosphodiesterase.     

Adenosine 3′:5′-cyclic monophosphate-binding protein from the cellular slime mould Dictyostelium discoideum (Short Communication)

During the differentiation of myxamoebae of Dictyostelium discoideum strain Ax-2 grown in axenic medium there is a seven- to ten-fold increase in the specific activity of cyclic AMP-binding protein(s). Evidence is presented for the belief that cyclic AMP phosphodiesterase and cyclic AMP-binding protein are distinct molecular species.     

Enzyme synthesis in myxamoebae of the cellular slime mould Dictyostelium discoideum during growth in axenic culture

1. The specific activities of beta-N-acetylglucosaminidase, acid phosphatase, alpha-mannosidase, beta-glucosidase, UDP-glucose pyrophosphorylase and alkaline phosphatase have been determined in myxamoebae of the cellular slime mould Dictyostelium discoideum Ax-2 grown on different media and in different phases of the growth cycle. 2. Variations in enzymic composition occur with changes in growth medium and phase of the growth cycle. 3. The intracellular location of the enzymes studied has been determined. 4. Two enzymes, beta-N-acetylglucosaminidase and alpha-mannosidase, are not only synthesized preferentially as the myxamoebae enter the stationary phase of growth but they are also excreted. The excretion process appears to be specific, because other enzymes that occur in the same intracellular fraction are not excreted.     

Metabolism of the cellular slime mould Dictyostelium discoideum grown in axenic culture

1. The DNA, RNA, protein and carbohydrate contents of myxamoebae of Dictyostelium discoideum strain Ax-2 were measured after growth on bacteria or in various axenic media. 2. Myxamoebae grown in the different axenic media have similar DNA, RNA and protein contents, but there are marked differences in the contents of glycogen and free sugars. The DNA and protein contents of myxamoebae grown on bacteria are different from those in myxamoebae grown axenically. 3. Approximately half the DNA found in myxamoebae grown on bacteria is of bacterial rather than of slime-mould origin. 4. The specific activities of some enzymes (including UDP-glucose pyrophosphorylase) are higher in myxamoebae grown axenically than in myxamoebae grown on bacteria. Nevertheless the characteristic increase in the specific activity of UDP-glucose pyrophosphorylase occurring during differentiation of cells of the wild-type strain NC-4 is also found in cells grown axenically. 5. The rate of amino acid oxidation during axenic growth of the myxamoebae is decreased when the cells are supplied with glucose.     

Adenosine 3′:5′-cyclic monophosphate concentrations and phosphodiesterase activities during axenic growth and differentiation of cells of the cellular slime mould Dictyostelium discoideum

During growth of myxamoebae of Dictyostelium discoideum (strain Ax-2) in axenic medium, the myxamoebae secrete cyclic AMP. As the cells leave the exponential phase of growth and enter the stationary phase, there is an approximate doubling of the intracellular cyclic AMP content, but the amount of extracellular cyclic AMP remains proportional, at all times, to the number of myxamoebae present. During development of axenically grown myxamoebae, there is first a rise in the intracellular concentration of cyclic AMP, followed by a rise in the amount of extracellular cyclic AMP, which reaches a peak at the time of aggregation and then declines. There is a second peak in the amount of extracellular cyclic AMP found at the time of fruiting-body formation, but this second peak is not associated with a rise in the intracellular cyclic AMP concentration. Controls thus exist over the synthesis and secretion of cyclic AMP. Evidence is presented for the belief that the activity of the adenylate cyclase enzyme controls the amount of cyclic AMP synthesized rather than the activity or amount of cyclic AMP phosphodiesterase present. Similar changes occur in extracellular cyclic AMP and phosphodiesterase concentrations during incubation of myxamoebae in buffered suspensions to those occuring during the first few hours of development of such cells on solid media, but the timing of these changes is different.     

Cysteine proteinase changes during microcyst formation and germination inPolysphondylium pallidum

Microcyst formation inPolysphondylium pallidum WS320 was accompanied by a decrease in intracellular cysteine proteinase activity measured with the peptide nitroanilides Z-Arg-Arg-Nan and Bz-Pro-Phe-Arg-Nan. Some activity was released into the buffer, and secretion of that towards Z-Arg-Arg-Nan continued until encystment occurred. Cells grown in association withEscherichia coli had an electrophoretic proteinase pattern different from cells grown axenically. Microcysts formed from the two cell populations also had distinct proteinase patterns; those from bacterially grown cells retained significant quantities of proteinase ppCP22, whereas those derived from axenic cells were devoid of detectable proteinases. No significant changes in cysteine proteinase activities were observed during microcyst germination, although some changes in activity occurred subsequent to emergence. The results indicate that there is not a close correlation between particular cysteine proteinases and specific stages of microcyst formation. Intracellular proteinase loss and concomitant secretion are, however, processes typical of cellular slime molds developing in response to starvation.