Ectopical expression of bacterial collagen-like protein supports its role as adhesin in host-parasite coevolution
Data files
Mar 13, 2024 version files 23.80 KB
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Huessy-Bumann-Ebert_Data-Manuscript.xlsx
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README.md
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Abstract
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.
https://doi.org/10.5061/dryad.bk3j9kdjk
Description of the data and file structure
The data associated with this work are organized in an excel file with three sheets.
Each sheet refers to part of the entire dataset and are linked to the figures in the manuscript.
Each sheet also includes explanations to the names of the variables and abbreviations.
Description of variables:
Fig. 3:
- Attachment: Indicates whether spores attached (1) or no attachment (0) was observed
- Isolate: Indicates which spores were used for the attachment test
C1 C1 P. ramosa clone
WT-Bt B. thuringiensis wild-type spores
Pcl7-Bt Mutant B. thuringiensis wild-type spores expressing Pcl7
- Resistotype: Indicates the resistance phenotype to the C1 P. ramosa clone
R Resistant, no attachment of C1
S Suscpetible, attachment of C1
- Daphnia_clone: Indicates the Daphnia magna clone used for the test.
Fig. 4: Competitive attachment assay for C1 P. ramosa
- Count: Indicates the number of attached C1 P. ramosa spores
- Isolate: Indicates which spores were used for the attachment test
C1 Individuals were exposed to 50 labelled C1 P. ramosa spores
WT-Bt Individuals were exposed to ~20’000 B. thuringiensis WT-Bt spores prior to the addition of 50 C1 P. ramosa spores
Pcl7-Bt Individuals were exposed to ~20’000 Pcl7-Bt spores prior to the addition of 50 C1 P. ramosa spores
- Clone: Indicates the D. magna clone used for the assay
Fig. 5: Competitive attachment assay for C19 P. ramosa
- Count Indicates the number of attached C19 P. ramosa spores
- Isolate Indicates which spores were used for the attachment test
C19 Individuals were exposed to 50 labelled C19 P. ramosa spores
WT-Bt Individuals were exposed to ~20’000 B. thuringiensis WT-Bt spores prior to the addition of 50 C19 P. ramosa spores
Pcl7-Bt Individuals were exposed to ~20’000 Pcl7-Bt spores prior to the addition of 50 C19 P. ramosa spores
- Clone Indicates the D. magna clone used for the assay
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).