Sentinel cells, symbiotic bacteria and toxin resistance in the social amoeba Dictyostelium discoideum

Abstract

The social amoeba Dictyostelium discoideum is unusual among eukaryotes in having both unicellular and multicellular stages. In the multicellular stage, some cells, called sentinels, ingest toxins, waste and bacteria. The sentinel cells ultimately fall away from the back of the migrating slug, thus removing these substances from the slug. However, some D. discoideum clones (called farmers) carry commensal bacteria through the multicellular stage, while others (called non-farmers) do not. Farmers profit from their beneficial bacteria. To prevent the loss of these bacteria, we hypothesize that sentinel cell numbers may be reduced in farmers, and thus farmers may have a diminished capacity to respond to pathogenic bacteria or toxins. In support, we found that farmers have fewer sentinel cells compared with non-farmers. However, farmers produced no fewer viable spores when challenged with a toxin. These results are consistent with the beneficial bacteria Burkholderia providing protection against toxins. The farmers did not vary in spore production with and without a toxin challenge the way the non-farmers did, which suggests the costs of Burkholderia may be fixed while sentinel cells may be inducible. Therefore, the costs for non-farmers are only paid in the presence of the toxin. When the farmers were cured of their symbiotic bacteria with antibiotics, they behaved just like non-farmers in response to a toxin challenge. Thus, the advantages farmers gain from carrying bacteria include not just food and protection against competitors, but also protection against toxins.

Keywords: Dictyostelium; bacteria; farmers; innate immunity; symbiosis; toxin resistance.

Figure 1.

Cartoon of D. discoideum slug migration. For each clone, we deposited spore and bacteria suspensions in a line on one side of each non-nutrient agar plate containing EtBr, a toxin that is taken up by sentinel cells. We allowed phototropic slugs to form and migrate towards a light source across embedded slides to facilitate imaging of slug trails. (Online version in colour.)

Figure 2.

Sentinel cell number varies with farmer/non-farmer status. (a) Fluorescent images of sentinel cells in D. discoideum slug trails. Sentinel cells are visible as bright red to orange round spots when stained with ethidium bromide (EtBr) and illuminated for imaging. Representative examples of EtBr-stained slug trails from one random farmer and one random non-farmer. Scale bar is 250 µm. (b) Farmers have fewer sentinel cells compared with non-farmers. We counted and averaged the number of sentinel cells found in five slug trails for each of 10 farmers and 10 non-farmers. We found farmers have significantly fewer sentinel cells than non-farmers (F_1,18 = 13.04, p = 0.002), as displayed in this box plot.

Figure 3.

Farmers are unharmed under toxic conditions and spores produced remain equally viable as under control conditions. (a) Non-farmer spore production is significantly reduced in toxic conditions, but farmer spore production is unchanged. (b) Importantly, farmer and non-farmer spores, whether untreated or treated with a toxin during development, remain equally viable. Significant differences in spore production or spore viability are indicated by different letters, which reflect results of a post hoc Tukey's HSD test. Error bars show s.e.m.

Figure 4.

Protection against the toxic effect of EtBr is lost when farmer-associated bacteria are removed. We compared spore production of five farmer clones either with (uncured) or without (cured) their farmer-associated bacteria to five non-farmers treated under the same conditions as a control. These clones were grown with or without EtBr (toxin). Spore production for cured farmers is reduced to the same level of harm as seen for non-farmers (either cured or uncured). The curing process had no significant effect on non-farmer spore production in either condition. Significant differences in spore production between conditions are indicated by different letters, which reflect results of a post hoc Tukey's HSD test. Error bars show s.e.m.