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.


Introduction
Antagonistic coevolution between hosts and parasites has been suggested to be a major driver in evolution, presumably underlying diverse biological phenomena, such as the 2. Material and methods

Preparation of electrocompetent cells and electroporation
To prepare electrocompetent cells, 100 ml of fresh LB-ls was inoculated at OD 600 nm 0.01 from an overnight culture and grown to OD 600nm 0.08 at 37°C, 180 r.p.m.The culture was distributed to two 50 ml falcon tubes and put on ice for 20 min.The tubes were spun down at 4°C, 8000 r.p.m. for 8 min, and the supernatant was removed.The pellets were then washed with 30 ml of cold sterile water containing 15% glycerol (AppliChem, A1123,1000), and the tubes were spun down at 4°C, 8000 r.p.m. for 8 min.This washing step was repeated twice.The supernatant was discarded, and the pellet was suspended in 1 ml of cold sterile water containing 15% glycerol.Finally, 100 µl aliquots were stored at −80°C or used directly for electroporation.
For electroporation of plasmid DNA, DNA (100 ng) was added to thawed, electrocompetent cells (100 µl) in a 1 mm electroporation cuvette on ice.For E. coli DC10B, a single pulse at 1.8 V (Gene Pulser Xcell Electroporation Systems, Bio-Rad Laboratories) was applied.For B. thuringiensis, a single pulse at 2.5 V was applied.Immediately after the pulse, 900 µl of pre-warmed SOC media was added, and the entire volume was transferred to a fresh 1.5 ml Eppendorf tube.The tube was incubated and shaken at 37°C, 180 r.p.m. for 1 h.Samples were spun down for 4 min at 11 000 r.p.m.We removed 900 µl of supernatant and plated the remaining volume (100 µl) on LB agar containing the respective antibiotics.Plates were incubated overnight at 37°C.

Isolation of genomic DNA
Genomic DNA was isolated using a DNeasy Blood & Tissue Kit (Qiagen GmbH, Hilden, Germany).A 2 ml overnight culture of B. thuringiensis in TSB media was spun down at 8000 r.p.m. for 4 min.The supernatant was discarded, and the pellet was resuspended in 200 µl of AL Buffer and 20 µl of proteinase K.The sample was incubated at 56°C, 500 r.p.m. for 10 min and 200 µl of >99% ethanol was added to it.It was then mixed thoroughly, transferred to a DNeasy Mini spin column and spun down at 10 000 r.p.m. for 1 min; the flow through was discarded.Then 500 µl of Buffer AW1 was added and spun down at 10 000 r.p.m. for 1 min, and the flow through was discarded.Finally, 500 µl of Buffer AW2 was added and spun down at 13 200 r.p.m. for 1 min.The column was transferred to a fresh 1.5 ml Eppendorf tube, and the DNA was eluted with 50 µl sterile water, incubated at room temperature for 1 min and centrifuged at 10 000 r.p.m. for 30 s.The concentration of DNA was measured using a Colibri Microvolume Spectrometer (Berthold Technologies, Bad Wildbad, Germany).

Molecular biology
Plasmids were constructed by Gibson Assembly [56].Flanking regions (~750 bp) of the target locus were PCR-amplified from genomic DNA (with primers 1 and 2 as well as 5 and 6, table 2) using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China), and primers were designed with SnapGene (v.5.2, Gibson assembly tool).The 501 bp C-terminal pcl7 sequence was synthesized (LubioScience GmbH, Zurich, Switzerland), and PCR-amplified using primers 3 and 4. The pMAD-I-SceI vector was amplified with primers 7 and 8 in a long-range PCR.The resulting three fragments and the vector were fused using the Hifi DNA Assembly Master Mix (New England Biolabs, Ipswich, MA) at 50°C for 1 h.The reaction mix contained approximately 40 ng of each fragment and 150 ng of vector royalsocietypublishing.org/journal/rsos R. Soc.Open Sci.11: 231441 for a total volume of 20 µl.E. coli DC10B was transformed with the resulting product.Plasmid DNA was purified from an overnight culture using a plasmid miniprep kit (ZymoPURE, ZymoResearch).Sequence-verified plasmids were electroporated into B. thuringiensis.Cells were incubated at 28°C for 1 h and plated on TSB agar containing 5 µg ml −1 erythromycin.
The allelic exchange procedure was done as described [53] with some modifications.B. thuringiensis integrants were isolated by shifting transformants obtained by electroporation from 28 to 37°C in the presence of 5 µg ml −1 erythromycin.pMAD-I-SceI cannot replicate in B. thuringiensis at 37 °C, so only cells which integrated the plasmid into the chromosome could propagate.Colonies were screened for the orientation of their insert using PCR (with primers 9 and 10, table 2).Colonies that harboured the right-hand insert were pooled and inoculated in 4 ml fresh TSB medium containing 5 µg ml −1 erythromycin at 37°C, 180 r.p.m. overnight.The overnight culture was diluted 1:100 in 50 ml fresh TSB medium containing 5 µg ml −1 erythromycin and incubated at 37°C, 180 r.p.m. to an OD 600 nm of 0.8 to prepare electrocompetent cells.The cells were transformed with pBKJ223 and plated on TSB agar containing 8 µg ml −1 tetracycline.Colonies were pooled and inoculated in 4 ml fresh TSB medium containing 8 µg ml −1 tetracycline at 37°C, 180 r.p.m. for 6 h.Serial dilutions (10 −1 , 10 −2 , 10 −3 and 10 −4 ) were prepared and 100 µl of each dilution was plated on TSB agar plates containing 8 µg ml −1 tetracycline.Single colonies were patched on TSB agar with 8 µg ml −1 tetracycline as well as TSB agar with 5 µg ml −1 erythromycin to screen for loss of erythromycin resistance.Erythromycin-sensitive clones were picked and stored in 50 µl LB-Glycerol (15%) at −20°C.The clones were screened for pMAD-I-SceI plasmid used for making deletion mutants, containing I-SceI restriction site that can be cleaved by the homing endonuclease I-SceI, Apr, Eryr [54] Notes: Six D. magna clones with different resistance phenotypes from the C1 P. ramosa clone were maintained in an artificial culture medium (ADaM) under standard laboratory conditions (20°C, 16:8 day:night cycle, Tetradesmus obliquus as food) as previously described in [55].
Clones with confirmed allelic exchange were inoculated into 4 ml fresh TSB media and incubated at 37°C, 180 r.p.m. overnight.Genomic DNA was prepared and Sanger-sequenced (Microsynth AG, Balgach, Switzerland).Correct clones were inoculated in 4 ml fresh TSB and incubated at 37°C, 180 r.p.m. overnight.The next day freezer stocks were generated and stored at −80°C for later use.

Sporulation and purification of spores
Spores were purified as described by [57] with some modifications.An overnight culture of B. thuringiensis in 4 ml of TSB was grown at 37°C, 180 r.p.m.The overnight culture was diluted 1:100 in 50 ml fresh TSB media containing 0.1 mmol MnSO 4 (PanReac, AppliChem) and grown at 37°C, 180 r.p.m. for 10 days.Cultures were transferred to a 50 ml falcon tube and stored on ice for 20 min.The tubes were spun down at 4°C, 10 000 r.p.m. for 10 min.The pellet was suspended in 20 ml of 50 mM Tris-HCL (pH 7.2; PanReac, AppliChem) with an addition of 50 µg ml −1 lysozyme (from hen egg whites, Fluka).Samples were incubated at 37°C, 180 r.p.m. for 1 h.The sample was spun down at 4°C, 10 000 r.p.m. for 10 min, and the pellet was suspended in cold sterile water.This washing step was repeated twice.The pellet was then suspended in 5 ml 0.05% SDS solution (Sigma, D6750-10G) by vortexing and incubated for 5 min at room temperature.The sample was then spun down at 4°C, 10 000 r.p.m. for 10 min, and the pellet was suspended in cold sterile water.This washing step was repeated five times.After the final washing step, the pellet was suspended in 5 ml of cold, sterile water and stored at 4°C for later use.Spores were counted using a Neubauer-improved chamber (Paul Marienfeld GmbH, Lauda-Konigshofen, Germany) with a chamber depth of 0.1 mm.

Fluorescent labelling of spores
Pasteuria ramosa spores were isolated by homogenizing infected Daphnia in ADaM followed by centrifugation at room temperature, 8000 r.p.m. for 5 min.Bacillus thuringiensis spores were thawed and centrifuged at room temperature, 8000 r.p.m. for 5 min.Pasteuria ramosa or B. thuringiensis pellets were suspended in 0.8 ml of 0.1 M sodium bicarbonate (pH 9.1; Sigma, S5761-500G) with 2 mg ml −1 of fluorescein-5(6)-isothiocyanate (Sigma-Aldrich, St Louis, MO).The samples were then incubated in the dark at room temperature, 1600 r.p.m. shaking for 2 h, followed by centrifugation at room temperature, 8000 r.p.m. for 4 min.The pellet was suspended in 0.8 ml sterile water and centrifuged at room temperature, 8000 r.p.m. for 4 min; the supernatant was then removed.This washing step was repeated three times.Spore suspensions were stored in sterile water at 4°C in the dark for further use.

Attachment assay
Daphnia were individually placed into a 96-well plate containing 150 µl of ADaM per well.Spore solution (10 µl) containing ~500 fluorescently labelled P. ramosa spores was 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 was 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 observing 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).

Competitive attachment assay
Daphnia were individually placed into a 96-well plate containing 150 µl of ADaM per well.We then added 10 µl of spore solution to each well according to each treatment-for C1/C19: 50 labelled P. ramosa spores; for WT-Bt + C1/C19: ~20 000 labelled B. thuringiensis spores followed by 50 labelled P. ramosa spores; and for Pcl7-Bt + C1/C19: ~20 000 labelled Pcl7-Bt spores followed by 50 labelled P. ramosa spores-and incubated them 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 to remove excess spores.The Daphnia were then placed individually on a microscopy slide and a glass cover slide was gently applied to the Daphnia to avoid crushing them.The number of P. ramosa spores attached to the D. magna oesophagus were then counted by manually scanning the z-stacks.

Selection of candidate collagen-like proteins
The N-terminal domain and collagen-like region of 3 out of 8 CLPs in B. thuringiensis (table 3) were selected based on the presence of the sequence motif 'LVGPTLPPIPP', likely required for incorporation into the exosporium, to construct fusions with the pcl7 CTD.

Results
To validate the role of the CTD of the Pcl7 of P. ramosa as an adhesin that recognizes the host Daphnia magna in a genotype-specific manner, we employed surface presentation on B. thuringiensis spores.Specifically, we fused the Pcl7-CTD to the N-terminal domain of the main collagen-like exosporium surface protein of B. thuringiensis (BTB_c12600, table 3).Compared to other potential fusion partners with collagen-like domain (BTB_c35490, BTB_c38740, table 3) BTB_c12600 is more similar in its N-terminal collagen-like domain to Pcl7 and contains a peptide that matches perfectly to the consensus motif for incorporation in the surface exosporium.We identified the CTD of BTB_c12600 in Bt 407 as the N-terminal 166 amino acids after the last collagen-like repeat unit (table 4).We replaced this CTD in the chromosome of Bt 407 with the 167 amino acid-long CTD of Pcl7 using two consecutive single cross-overs.The construct was verified by sequencing.The resulting recombinant Pcl7-Bt strain showed normal growth and sporulation.We induced spore formation and purified spores using standard procedures.
To determine adherence of P. ramosa and wild-type (WT-Bt) or pcl7-expressing (Pcl7-Bt) B. thuringiensis spores to D. magna, we covalently labelled the bacteria with fluorescein and tracked them in infection assays with different D. magna genotypes using fluorescence microscopy [13].C1 P. ramosa spores attached to the oesophagus of susceptible D. magna (figure 2e; 100% attachment, figure 3) but not to resistant hosts (figure 3) as previously observed.Vegetative WT-Bt and Pcl7-Bt cells showed no attachment to the D. magna oesophagus but can be observed in the filter setae of both susceptible and resistant Daphnia (figure 2c), possibly being trapped in the mucus that lines the filter-feeding apparatus.WT-Bt spores showed attachment to neither the susceptible nor resistant host (0% attachment, figure 3), while Pcl7-Bt spores attached to the oesophagus of susceptible D. magna (figure 2g).Pcl7-Bt attached at low frequency and density to resistant D. magna (figure 2f; 15% attachment, figure 3), possibly suggesting incorrect glycosylation of Pcl7 [19] in the heterologous B. thuringiensis system.Attachment of Pcl7-Bt spores to other tissues or known sites of P. ramosa attachment such as the hindgut or the external postabdomen [59] was not observed.Thus, a single domain of CLP from P. ramosa was sufficient to mediate Pasteuria-like adhesion properties for recombinant B. thuringiensis spores.
To determine if Pcl7 presented on B. thuringiensis spores can block P. ramosa adhesion to D. magna, we infected various D. magna genotypes (table 1) with 50 spores of two different P. ramosa clones after incubation with a 400-fold excess of the much smaller WT-Bt or Pcl7-Bt spores.After 5 min exposure, we counted the number of P. ramosa spores attached to the D. magna oesophagus.WT-Bt had no impact on subsequent P. ramosa adhesion for all tested D. magna and P. ramosa genotypes (figure 4).However, Pcl7-Bt diminished adhesion of C1 P. ramosa spores in four different susceptible D. magna genotypes, indicating that Pcl7 was sufficient to block adhesion sites in the host for P. ramosa.Notes: Eight candidate B. thuringiensis genes with similar N-terminal domains and amino acid sequences as bclA.Deviations from the specific sequence motif, likely required for incorporation into the exosporium, are marked in bold.Genes selected for the construction of fusions with pcl7 are highlighted in italic.Sequences were obtained from GenBank [58] (accession no.CP003889).
We also tested Pcl7-Bt against C19 P. ramosa, an isolate that shows a different host genotype dependence from C1 P. ramosa (the source of Pcl7 in Pcl7-Bt).Pcl7-Bt had no impact on C19 adhesion to D. magna DE-G1-106 (susceptible to P. ramosa C19; resistant to C1), but diminished C19 adhesion to D. magna HU-HO-2 (susceptible to P. ramosa C19 and susceptible to C1) (figure 5).Thus, Pcl7-Bt could prevent P. ramosa adhesion specifically in D. magna resistotypes with a Pcl7-receptor (enabling infection by C1), but not in D. magna resistotypes without such a receptor.As C19 uses a different receptor from C1, the blocking in HU-HO-2 was probably mediated by steric hindrance between adhering Pcl7-Bt and P. ramosa.

Discussion
The mechanism underlying specific coevolution by negative frequency-dependent selection is believed to be driven by the interaction between host and parasite genes.Identifying these genes marks a major step towards understanding this mechanism.In the Daphnia-Pasteuria system coevolution is well characterized on a phenotypic level, but the underlying genes are still largely unknown.Here, we tested and confirmed the hypothesis that a CLP is crucial for the attachment of the Pasteuria parasite to the cuticle of its Daphnia host.Polymorphism in the attachment phenotype had previously been shown to be the most important step in the coevolution of the two antagonists [13,18], and a specific CLP (Pcl7) of P. ramosa was linked to the attachment polymorphism [19].Here, we used B. thuringiensis as a surrogate to express a functional fusion protein harbouring the globular domain of Pcl7.By replacing a single C-terminal sequence in B. thuringiensis with the one from Pasteuria Pcl7, we were able to create B. thuringiensis spores capable of attaching in vivo to the host's oesophagus wall.This result is in line with previous studies that have sought to understand CLP function in bacterial infections using deletion mutants for CLPs [60] or purifying recombinant CLPs [35].Our Pcl7-Bt spores not only attached well to four susceptible host genotypes (figures 3 and 4), but they also attached only sparsely to two resistant host genotypes (figures 3 and 5), revealing a very similar specificity to that of P. ramosa (figure 6).
Proteins associated with the spore coat in B. thuringiensis, including the BclA homologues that were altered in this study, are predominantly synthesized during sporulation [43].We anticipated

AA
Notes: Amino acid sequences of the native Bt collagen-like exosporium surface protein BTB_c12600, the Pcl7 protein and the resulting Pcl7-Bt fusion protein.The Bt NTD used in the fusion protein is highlighted in italic and the Pcl7 CTD in bold.Asterisk represents the stop codon.
that the Pcl7 fusion protein would be present upon completed spore formation.Consistent with this, only spores but not vegetative Pcl7-Bt cells adhered to the host's oesophagus.The specificity of the B. thuringiensis spores to a single host tissue thus indicates the functional ectopical expression of the Pcl7 fusion protein, generating the same attachment phenotype as C1 P. ramosa spores.This attachment specificity was only seen in hosts susceptible to C1 P. ramosa, while in two resistant host genotypes, attachment was either weak or entirely absent.Thus, by replacing part of a single protein, we were able to transform B. thuringiensis from non-attaching to attaching, recreating the first step of the infection process.To test if Pcl7-Bt spores adhere to the same molecular structure in the oesophagus wall as C1 P. ramosa spores we conducted competitive attachment assays.We found that the moieties or potential receptors on the D. magna oesophagus cuticle surface are blocked by the Pcl7-Bt spores, supporting our hypothesis that the Pcl7-Bt interferes with the attachment sites of C1 P. ramosa.
The specificity of the Pcl7-Bt's attachment was less pronounced than that of the original P. ramosa pathogen, with the difference in attachment between a resistant and a susceptible host genotype being 0% and 100% for C1 P. ramosa, while it was 15% and 80% for the Pcl7-Bt spores (figure 3).Furthermore, the competitive attachment assays showed that C1 spores were not totally blocked from attachment.Some Pasteuria spores were still able to attach after the host had been treated with the Pcl7-Bt spores.Finally, to see a visible picture of attachment, we needed much higher spore concentrations of the Pcl7-Bts than the bigger P. ramosa pathogen.However, the weak signals observed are certainly caused in part by the rapid germination of B. thuringiensis spores once exposed to the Daphnia, as germinating spores do not express BclA [43].Furthermore, because P. ramosa spores are large (about 5.5 µm in diameter) and thus much more visible compared to the B. thuringiensis spores (about 1.6 × 0.8 µm) [61], more spores are required to see attachment with the fluorescent microscope.The difference in the attachment of Pcl7-Bt spores as compared to spores from C1 Pasteuria indicates the presence of factors that contribute to the attachment.The specific coevolution of P. ramosa with D. magna might have streamlined the system to a very high degree, with Pcl7 certainly playing a vital role, but for a stronger attachment, other factors are needed.One such factor may be glycosylation.BclA, and CLPs in general, are known to be highly glycosylated [62,63].The sequence polymorphism  of Pcl7 results in two predicted N-linked glycosylation sites, one of which correlates with the infection phenotype of Pasteuria [19] and thus may partly explain the attachment polymorphism.The Pcl7 fusion protein expressed in B. thuringiensis may contain different glycans from Pcl7 expressed in P. ramosa or might not be glycosylated at all.This might result in altered adhesion properties.Other fusion proteins containing other sequence polymorphisms or glycosylation sites might be used in the future to work out the details of these differences.An analysis of the specific carbohydrate moieties of the Pcl7 glycoprotein could also be done to support this hypothesis [64].However, our data show that such a potential mis-glycosylation had only a minor impact on adhesion and competition.Other factors that may contribute to attachment could be related to the spore morphology.P. ramosa spores have an unusual shape, which may have evolved to maximize adhesion.
Our study verified that the globular part of the Pcl7 protein contributes decisively to the parasite's ability to attach to the host cuticle, although the molecular mechanisms for the attachment polymorphism are still unclear, as is the composition of the host cuticle surface in the oesophagus.Previous research has shown that CLPs can bind to a variety of molecules and cell surface components such as integrins [28], glycoproteins [34], polysaccharides [65], lipoproteins [35] and mammalian collagen [66].Future studies could focus on identifying the D. magna receptor for Pcl7.

Figure 1 .
Figure 1.Graphic overview of the construction of the Pcl7-Bt fusion.(a) Pcl7 consists of three domains: an amino-terminal domain (NTD), a central collagen-like region (CLR) and a carboxy-terminal globular domain (CTD).(b) Homologous recombination can occur either through homologous regions 'X' , resulting in the regeneration of the wild-type gene and protein (c) or through 'Y' , resulting in the generation of the Pcl7-Bt fusion.Figure created with Biorender.com.

Figure 2 .
Figure 2. Attachment phenotypes using labelled spores.Microscopy images demonstrating the various attachment phenotypes using labelled spores.The red box in the schematic to the right indicates the approximate area (head region) of images (d-g).(a) Bright-field image of a D. magna (genotype (=clone) HU-HO-2) showing the entire animal with an arrow indicating the site of the oesophagus.(b) Overview of a D. magna (genotype HU-HO-2) using a GFP Filter cube showing the entire animal with an arrow indicating the location of the oesophagus.Note the weak autofluorescence of the D. magna tissue.(c) Labelled B. thuringiensis vegetative cells accumulated in the filter setae of a D. magna (genotype HU-HO-2).(d) Closeup of the D. magna foregut with an arrow indicating the position of the oesophagus, situated perpendicular to the arrow.The two bright objects left of the arrow are the autofluorescent mandibles.(e) Upper body of a susceptible D. magna (genotype HU-HO-2) with labelled C1 P. ramosa spores attached to the oesophagus (arrow).(f) Upper body of a resistant D. magna (genotype FI-Xinb3) where labelled Pcl7-Bt spores do not aggregate in the oesophagus (arrow).The faint visible light fluorescent band is attributed to autofluorescence.The beginning of the midgut, visible in the upper right corner, shows fluorescence because labelled spores have been ingested by the Daphnia.(g) Upper body of a susceptible D. magna (genotype HU-HO-2) with labelled Pcl7-Bt spores attached to the oesophagus (arrow).The midgut with its appendix (=caecum) is visible on the right.

Table 1 .
Material used in the study.

Table 2 .
Primers used in this study.

Table 3 .
Candidate collagen-like proteins in B. thuringiensis.

Table 4 .
Amino acid sequences used for the Pcl7-Bt fusion.