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. 2014 Feb;26(2):453-9.
doi: 10.1016/j.cellsig.2013.10.008. Epub 2013 Oct 31.

The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas

Qingyou Du  1 , Christina Schilde  1 , Elin Birgersson  1 , Zhi-hui Chen  1 , Stuart McElroy  1 , Pauline Schaap  2
Affiliations

The cyclic AMP phosphodiesterase RegA critically regulates encystation in social and pathogenic amoebas

Qingyou Du et al. Cell Signal. 2014 Feb.

Abstract

Amoebas survive environmental stress by differentiating into encapsulated cysts. As cysts, pathogenic amoebas resist antibiotics, which particularly counteracts treatment of vision-destroying Acanthamoeba keratitis. Limited genetic tractability of amoeba pathogens has left their encystation mechanisms unexplored. The social amoeba Dictyostelium discoideum forms spores in multicellular fruiting bodies to survive starvation, while other dictyostelids, such as Polysphondylium pallidum can additionally encyst as single cells. Sporulation is induced by cAMP acting on PKA, with the cAMP phosphodiesterase RegA critically regulating cAMP levels. We show here that RegA is deeply conserved in social and pathogenic amoebas and that deletion of the RegA gene in P. pallidum causes precocious encystation and prevents cyst germination. We heterologously expressed and characterized Acanthamoeba RegA and performed a compound screen to identify RegA inhibitors. Two effective inhibitors increased cAMP levels and triggered Acanthamoeba encystation. Our results show that RegA critically regulates Amoebozoan encystation and that components of the cAMP signalling pathway could be effective _targets for therapeutic intervention with encystation.

Keywords: 3′5′-adenosine monophosphate; ACG; Acanthamoeba castellani; Acanthamoeba keratitis; Acas; AcrA; Ddis; Dictyostelium discoideum; Encystation; KO; MRSA; PDE; PKA; Polysphondylium pallidum; Ppal; RI; Sensor histidine kinase; Stress signalling; adenylate cyclase G; adenylate cyclase R; cAMP; cAMP dependent protein kinase; cAMP-phosphodiesterase; knock-out; methicillin resistant Streptococcus aureus; phosphodiesterase; random integrant.

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Figures

Fig. 1
Fig. 1
RegA identification. RegA homologs were retrieved by BLASTp search of all eukaryote sequences in Genbank and by tBLASTn search of ongoing D.lacteum (http://sacgb.fli-leibniz.de) and A.castellanii (Acas) (http://www.hgsc.bcm.tmc.edu) genome sequencing projects using Ddis RegA as bait. The Acas RegA protein sequence was deduced from reverse transcribed mRNA. Protein sequences were aligned using M-coffee and regions that were not unambiguously aligned were deleted. Phylogenetic relationships between aligned sequences were determined by Bayesian inference run for 1 million generations, using a mixed amino acid model, with rate variation between sites estimated by a gamma distribution with a proportion of invariable sites. The posterior probabilities of tree nodes are indicated and the tree is annotated with the functional domain architecture of the proteins. Numbers between brackets denote from which of the four major taxon groups the Dictyostelid sequences were derived . Black and grey scale bars represent number of substitutions per site and protein length in amino-acids (aa), respectively.
Fig. 2
Fig. 2
P. pallidum regA1 knock-out phenotype. A/B. Growth on bacteria. 105 cells of the Ppal rega1-strain, KO38, the random integrant strain RI15 and wild-type cells were inoculated as 1 μl droplets on K.aerogenes lawns in quadruplicate and incubated at 22 °C under ambient light. KO and RI plaques were photographed at 2 and 3 days of incubation (A) and the diameter of all plaques was measured daily with a ruler for 10 days (B). Means and SD of measurements are presented. The arrow indicates the growth front. Bar: 1 mm. C. Fruiting body morphogenesis. rega1-KO38 and wild-type cells were harvested from K.aerogenes lawns before bacteria were cleared, plated on non-nutrient agar and incubated at 22 °C under ambient light. Developing structures were photographed at 2 h intervals. Bar: 0.5 mm.
Fig. 3
Fig. 3
Proliferation, encystation and germination in rega1-mutants. A. Proliferation. Ppal wild type (WT), KO and random integrant (RI) strains were inoculated in HL5 liquid culture medium at 3 × 105 cells/ml. Aliquots were stained with Calcofluor at daily intervals to stain cyst walls, and cyst and total cell numbers were counted over a 4 day period. Wild-type cells and means and SD of two RI (RI14, RI15) and two KO (KO31, KO38) strains are presented. B. Encystation. The experiment was repeated with two RI (RI11,RI12) and two KO (KO8,KO10) from another transformation (set 1, see Fig. S2) and the data from both sets were recalculated into percentage of encysted cells. Means and SD of two strains from each set are presented. C. Cyst and spore germination. Ppal wild-type, KO and RI spores were harvested from 5-day old fruiting bodies. For cysts, wild-type, KO and RI cells were grown to stationary phase in HL5. Cultures were supplemented with 0.25 M sorbitol and incubated for 3 more days to allow mature cysts to form. Cysts (grey bars) or spores (black bars) were treated for 10 min with 0.1% Triton-X100 to lyse amoeboid cells and washed with PB. Cysts and spores were plated with K. aerogenes over 10 plates each, with 50 spores/cysts per plate, and emerging Ppal colonies were counted after 3–4 days. Means and SD of 2 experiments are presented.
Fig. 4
Fig. 4
Heterologous expression and characterization of Acas RegA. A/B. Heterologous expression. The Acas RegA cDNA was fused to a hexa-his tag in vector pET28a, expressed in E.coli, and purified by Ni+ chromatography. The column flow-through (FT) and three fractions eluted with 250 mM imidazole were size-fractionated by SDS-PAGE (A). Western blots of the size-fractionated proteins (B) were incubated with 1:2000 diluted mouse anti his-tag antibody and 1:5000 diluted peroxidase conjugated goat-anti-mouse IgG, followed by peroxidase detection. C. Acas RegA activity. 1 μl aliquots of the combined 250 mM imidazole eluate fractions of expressed Acas RegA and combined eluates obtained from the same amount of E.coli cells, transformed with empty pET28a vector, were incubated for 30 min with 10 nM 3H-cAMP and assayed for 3H-cAMP hydrolysis. D. Mg2 + dependence. Purified Acas RegA was incubated with 10 nM 3H-cAMP and increasing concentrations of MgCl2 and assayed for 3H-cAMP hydrolysis. Data are expressed as percentage of 3H-cAMP hydrolysis occurring at 0.3 mM MgCl2. E. Substrate specificity. Purified Acas RegA was incubated with 10 nM 3H-cAMP and increasing concentrations of cAMP and cGMP, and assayed for 3H-cAMP hydrolysis. Data are expressed as percentage of hydrolysis at 10 nM 3H-cAMP only. Means and SD of two experiments performed in triplicate are presented. F. The data for competition by cAMP (panel E) were converted into moles of 5′AMP produced per μg protein per min (V) at each concentration (S) and plotted as S/V against S in a Hanes plot. Intersections of the plot with the abscissa and ordinate, represent − KM and KM/Vmax values, respectively, yielding a KM of 19 μM and a Vmax of 55 nmol/min μg protein.
Fig. 5
Fig. 5
Selection of Acas RegA inhibitors and their effects on encystation. A. Inhibition. Three compounds that inhibited Acas RegA activity at concentrations < 100 μM were selected from a panel of 32 PDE inhibitors (Table S2) and tested at a concentration range of 10− 6 to 10− 2 M for inhibition of 3H-cAMP hydrolysis. Means and SD of 2 experiments performed in triplicate are presented. B–D. Encystation. Acas amoebas were resuspended in starvation buffer (SB) and incubated with 100 μM of the effective Acas RegA inhibitors dipyridamole and trequinsin, the inactive compound W-7, 0.1% DMSO (the W-7 and dipyridamole solvent) and without additives (control). At successive days, cells were harvested, stained with calcofluor, and photographed under UV and weak phase-contrast illumination to identify cysts and amoebas respectively. B/C. Images of cells incubated for 3 days with 0.1% DMSO (B) or 100 μM dipyridamole (C) and stained with calcofluor, Bar: 10 μm. D. Amoebas and cysts were counted in total sample sizes of 600 cells, and the percentage of encysted cells was determined. Means and SD of 2 experiments are presented. E. cAMP levels. Acas cells incubated with dipyridamole, W7 and 0.1% DMSO, as above, were lysed in perchloric acid at the indicated time points and assayed for total cell-associated cAMP levels. Data are standardized on the protein content of the samples and represent means and SD of 2 experiments performed in triplicate.
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