Ectopical expression of bacterial collagen-like protein supports its role as adhesin in host-parasite coevolution

For a profound understanding of antagonistic coevolution, it is necessary to identify the coevolving genes. The bacterium Pasteuria and its host, the microcrustacean Daphnia, are a well-characterized paradigm for co-evolution, but the underlying genes remain largely unknown. A genome-wide association study suggested a Pasteuria collagen-like protein 7 (Pcl7) as a candidate mediating parasite attachment and driving its coevolution with the host. Since Pasteuria ramosa cannot currently be genetically manipulated, we used Bacillus thuringiensis to express a fusion protein of a Pcl7 carboxy- terminus from P. ramosa and the amino-terminal domain of a B. thuringiensis collagen-like protein (CLP). Mutant B. thuringiensis (Pcl7-Bt) spores but not wild-type B. thuringiensis (WT-Bt) spores, attached to the same site of susceptible hosts as P. ramosa. Furthermore, Pcl7-Bt spores attached readily to susceptible host genotypes, but only slightly to resistant host genotypes. These findings indicated that the fusion protein was properly expressed and folded and demonstrated that indeed the C-terminus of Pcl7 mediates attachment in a host genotype-specific manner. These results provide strong evidence for the involvement of a CLP in the coevolution of Daphnia and P. ramosa and open new avenues for genetic epidemiological studies of host–parasite interactions.

Methods

Daphnia were individually placed into a 96-well plate containing 150 μl of ADaM per well. A 10 μl of spore solution containing ~500 fluorescently labelled P. ramosa spores were added to each well and incubated in the dark for 5 min. For B. thuringiensis, 10 μl of spore solution containing ~50 000 labelled B. thuringiensis spores were added to each well and incubated in the dark for 5 min. The entire liquid volume in each well was removed and replaced with 150 μl fresh ADaM. This washing step was repeated twice, after which the entire liquid volume in each well was removed. The Daphnia were placed individually on a microscopy slide using a toothpick. A glass cover slide was applied to the Daphnia gently to avoid crushing it. Spore attachment was assessed by carefully assessment the entire depth of the field of vision in transparent animal, while the animal was alive.

To demonstrate the different phenotypes, we took pictures through the transparent body wall of the Daphnia. Using living animals is necessary to see the spores in the oesophagus, but puts limits on the quality of the pictures, as animals move and the body tissues surrounding the oesophagus blur the picture. Images were taken using Leica Application Suite (v. 4.12, using package ‘montage’) with a Leica DM6 B (Leica Microsystems, Wetzlar, Germany) microscope fitted with a Leica DFC 7000T camera and a GFP Filter cube (Excitation Filter BP 470/40).