The social amoeba Polysphondylium pallidum loses encystation and sporulation, but can still erect fruiting bodies in the absence of cellulose

Abstract

Amoebas and other freely moving protists differentiate into walled cysts when exposed to stress. As cysts, amoeba pathogens are resistant to biocides, preventing treatment and eradication. Lack of gene modification procedures has left the mechanisms of encystation largely unexplored. Genetically tractable Dictyostelium discoideum amoebas require cellulose synthase for formation of multicellular fructifications with cellulose-rich stalk and spore cells. Amoebas of its distant relative Polysphondylium pallidum (Ppal), can additionally encyst individually in response to stress. Ppal has two cellulose synthase genes, DcsA and DcsB, which we deleted individually and in combination. Dcsa- mutants formed fruiting bodies with normal stalks, but their spore and cyst walls lacked cellulose, which obliterated stress-resistance of spores and rendered cysts entirely non-viable. A dcsa-/dcsb- mutant made no walled spores, stalk cells or cysts, although simple fruiting structures were formed with a droplet of amoeboid cells resting on an sheathed column of decaying cells. DcsB is expressed in prestalk and stalk cells, while DcsA is additionally expressed in spores and cysts. We conclude that cellulose is essential for encystation and that cellulose synthase may be a suitable target for drugs to prevent encystation and render amoeba pathogens susceptible to conventional antibiotics.

Keywords: Acanthamoeba keratitis; Amoebozoa; Encystation; Polysphondylium pallidum.; cell wall biosynthesis; cellulose synthase.

Figure 1

Phylogeny of dictyostelid cellulose synthases. A. Dicytostelid phylogeny. Genome-based phylogeny of group-representative Dictyostelium species and Acanthamoeba castellani (Acas) (Romeralo et al. 2013) with numbers referring to the relevant group or clade. Dpur: D. purpureum, Ddis: D. discoideum, Dlac: D. lacteum, Ppal: Polysphondylium pallidum, Asub: Acytostelium subglobosum, Dfas: D. fasciculatum.B. Cellulose synthase phylogeny. Amoebozoan cellulose synthase genes and their closest homologs in other organisms were retrieved by BlastP search of Genbank and ongoing D. lacteum (http://sacgb.fli-leibniz.de and A. subglobosum (http://acytodb.biol.tsukuba.ac.jp) genome projects, using Ddis DcsA as bait. The regions containing the glycosyl transferase domain were aligned using Clustal Omega (Sievers et al. 2011) and subjected to phylogeny reconstruction by Bayesian inference (Ronquist and Huelsenbeck 2003). The phylogenetic tree is annotated with the functional domain architecture of the proteins, as analyzed with SMART (Schultz et al. 1998). The protein identifiers are color-coded according to species as in panel A, with grey further indicating the bacteria Leptolyngbya sp. (EKU97898) and Cyanobacterium stanieri, and tan the oomycete Pythium iwayamai. Bayesian posterior probabilities of tree nodes are indicated by colored dots.

Figure 2

Phenotype of a dcsa- mutant. A. DcsA knockout (KO) and control random integrant (RI) cells were plated on PB agar and incubated until fruiting bodies had formed. Bar: 200 μm. B. Fruiting bodies of DcsA KO6 (a, d) and RI5 (b, e) cells, and of dcsa-neo- cells, transformed with the 1.6p::DcsA expression cassette (c) were transferred to 0.001% Calcofluor White on a slide glass. Spores and stalks were photographed under phase contrast (left panels), and under UV, combined with faint phase contrast illumination. Bar: 10 μm. C. DcsA KO6 (a) and RI5 cells (b) and dcsa- cells transformed with the 1.6::DcsA (c) or 3.0p::DcsA (d) cassettes were incubated in encystation medium. Calcofluor White was added to 0.001% after 4 days and cells were photographed. Bar: 10 μm.

Figure 3

Phenotypes of dcsb- and dcsa-/dcsb- mutants. A. Dcsb- cells were developed to fruiting bodies on PB agar and to cysts in 400 mM sorbitol. Fruiting bodies were photographed in situ (bar: 200 μm), stalk cells, spores and cysts were stained with Calcofluor White and photographed under UV illumination. Bar: 10 μm. B. Wild-type P. pallidum and the dcsa-/dcsb- mutant were incubated on PB agar and photographed at the indicated time points. Bar: 200 μm. C. dcsa-/dcsb- and wild type sorogens and fruiting bodies were submerged in situ in 0.001% Calcofluor White, placed under a coverslip and photographed under phase contrast and UV illumination. Ca,b Bar: 100 μm; Cc,d,e,f Bar: 10 μm.

Figure 4

Expression patterns of DcsA and DcsB. A/B. Ppal wild-type cells transformed with the DcsA3.0::LacZ (A) and DcsB::LacZ (B) constructs were plated on nitrocellulose filters supported by PB agar. Emerging aggregates and early and late sorogens were fixed and stained with X-gal to visualize β-galactosidase activity. Bar: 50 μm. C. Cells transformed with DcsA1.6::LacZ, DcsA3.0::LacZ and DcsB::LacZ were incubated for two days in encystation medium. Cells were then fixed and stained with X-gal, counterstained with Calcofluor White to identify cysts, and photographed under UV and brightfield illumination. Bar: 10 μm.

Figure 5

Spore and cyst viability of cellulose synthase null mutants. Wild-type, DcsA RI, dcsa-, dcsb- and dcsa-/dcsb- cells were harvested from mature spore heads or from 400 mM sorbitol after 4 days of incubation. Cells were counted and shaken for 10 min with and without 0.1% Triton-X100 before being plated at 500 cells/plate on Klebsiella lawns. After 3 days the emerging colonies were counted. The number of colonies as percentage of plated cells are shown, and the data are present means and SD of two experiments with duplicate plates for each variable.