The one-year PROZE project consisted of four different aims, which are described below.
1. Waddlia chondrophila culture and protein techniques.
At the start of the project the protocols and techniques to culture W. chondrophila were not present at TubaScan. During the PROZE project, we successfully set-up the W. chondrophila culture, this includes the culturing of Acanthamoeba castellanii in which W. chondrophila grows. Stocks of amoeba and W. chondrophila are stored for future usage and protocols are available within TubaScan.
2. Peptide selection
The selection of the peptides started with the usage of a database from our collaborator in Lausanne (Switzerland). This database contains the genomes of 77 Chlamydiales and it is possible to compare these genes and find similarities and differences. In addition, the results from a paper by dr. Julia Lienard were used as well, as she determined among others which outer membrane protein were able to react with human sera (including W. chondrophila positive serum). The combination of these two sources led to the selection of six promising proteins.
Those six proteins were ran through BLASTp. BLAST is an online program that compares nucleotide or protein sequences to sequence databases and calculates the statistical significance of matches. After this step, two proteins were excluded because they showed more homology with related Chlamydiales. At TubaScan, we do not have the expertise to identify the peptides which are most immunogenic, therefore we send the sequence of the four proteins of interest to GenScript who provided us with the immunogenic regions of each protein. Again, we used Blastp to select the most promising peptide regions, which led to the final selection of 12 peptides. These 12 peptides were produced by GenScript with a purity of >95%.
3. Sensitivity and specificity of the outer membrane ELISA versus the peptide ELISA
After the selection and production of the peptides, we used them to develop the peptide ELISA. The peptides were used in different combinations to coat the ELISA plates. Based on the results obtained from several experiments we concluded that the peptide combination of these four peptides do not influence the results. We did found out that a sample dilution of 1:50 and detection antibody concentration of 1:5000 were optimal.
If we make combinations (max 8 peptides per coating) of all peptides, we can see differences in OD value (the final outcome of the ELISA) between Waddlia negative/ doubtful samples and Waddlia positive serum samples. However, we only observe these differences when we preselect samples with a well-defined status (negative, doubtful or positive). Once we measure a larger batch of samples we do not observe these differences anymore, and we cannot clearly distinguish between the three different groups.
We came across several challenges for the WaddliaSCAN:
a. Better distinguish between negative, doubtful and positive samples
b. Determine cross-reaction with relevant Chlamydiales
c. Validation of peptide ELISA with a second sample cohort
d. Need for a positive control
4. Zebrafish model and CRISPR/Cas9
The associate was trained by the zebrafish expert from the MMI at the VUmc in a 2-week zebrafish course. In addition, there is a postdoc in TubaScan with extensive zebrafish experience. She has learned both the Innovative Associate and a junior researcher how to perform infections. All protocols needed to perform zebrafish injections (medium preparation, W. chondrophila preparation, and injections) are prepared and stored in TubaScan’s protocol database.
We decided to use the CRISPR/Cas9 system to make a Toll-like-receptor 2 (TLR2) knock-out. TLR2 is one of the important immune receptors which recognize bacteria. All needed primers, guide RNA and Cas9 protein are ordered and tested using a housekeeping gene. All materials to make the TLR2 knock out zebrafish are present and injections has started in September.
