TM7a genome mining, epitope selection and antibody generation.

For selection of candidate proteins to serve as immunogens, we used the single cell genomic data generated by Marcy et al.², the only oral TM7 genomes available at the time we initiated this work. We used the TM7a (6806 protein genes) and TM7c datasets (590 protein genes), based on the annotations available in IMG (https://img.jgi.doe.gov)³, with the expectation that some of the genes may represent organisms other than TM7⁴. We first selected genes encoding proteins associated with membrane processes, based on COG categories (transport functions, cell wall/membrane/envelope biogenesis, extracellular structures), using the IMG assignments. The corresponding proteins were analyzed for the presence of transmembrane anchoring helical domains using TMHMM v.2.0⁵. From the list of proteins with such domains, we eliminated the ones annotated as hypothetical as well as those shorter than 100 amino acids, as we aimed for final selection of relatively large proteins with known function. The proteins that passed those criteria (117 combined between TM7a and TM7c) were analyzed by blastp against the proteomes of all oral bacteria downloaded from the Human Oral Microbiome Database (http://www.homd.org/). Forty eight of the proteins had high sequence similarity (>90%) to proteins from species of Leptotrichia (Fusobacteria), indicating they are likely contaminants, as previously reported⁴. We then individually analyzed the remaining sequences for number of transmembrane domains, number and size of predicted extracellular domains, availability of three-dimensional structures for homologues from other bacteria, and experimental data related to abundance or prior use as immunogens. The top pick was penicillin-binding protein 2 (PBP2), which belongs to a class of proteins with important roles in bacterial peptidoglycan synthesis⁶. Both high resolution crystal structure data⁷ and multiple reports that anti-PBP2 antibodies can bind to the cell surface of pathogenic bacteria^{8, 9} were available. The closest homologues to the TM7a PBP2 present in genomes of human oral bacteria had 30–40% pairwise sequence identity (species of Selenomonas, Leptotrichia, Fusobacterium, Streptococcus). To test the applicability of the approach with a protein for which 3D structure or antigenic data were not available, we also selected CpsC as a target, as cellular studies indicated a cell surface-exposed domain^{10, 11}. CpsC belongs to the polysaccharide co-polymerases (PCPs) superfamily and is involved in capsular polysaccharide biosynthesis¹². We found no homologues in the oral bacterial genomes with a pairwise identity to TM7a CpsC over 30%.

For selecting peptides to serve as antigens, we analyzed the two protein sequences for antigenic regions and peptide hydrophilicity using the online servers ABCpred¹³, BepiPred¹⁴, IEBD Analysis Resource (http://tools.immuneepitope.org/bcell) and Bio-Synthesis peptide design tools (https://www.biosyn.com), using a 16–18 amino acid-peptide threshold. We used Bio-Synthesis (Lewisville, TX, USA) for peptide synthesis and antibody production. For PBP2, we identified 7 peptides that could serve as antigens and for CpsC we identified four peptides (Supplementary Fig. 1). We next analyzed the location of the 7 peptides from TM7a on the 3D structure of the protein based on the homologous regions of the E. coli PBPB structure (PDB 3vma) using PyMOL (https://pymol.org/2/). Further details on structural modeling and visualization are presented in the comparative structural analysis section. The selected antigen peptide (SGNIKYAKQRQKTVLTRMV) was one of the three peptides with the most exposed residues. In the case of CpsC, because no experimental 3D structure was available, we selected an antigenic peptide (ESAIQEFKEQSKSLYGNS) that is in a helical region of the predicted extracellular domain (Supplementary Fig.1).

The two selected peptides were commercially synthesized by Bio-Synthesis (Lewisville, TX, USA), with cysteine added at N (CpsC) or C terminus (PBP2), purified and injected in rabbits. For antisera production, Bio-Synthesis used a 70-day protocol, with 5 booster doses and 4 test bleeds followed by ELISA. We received serum batches confirmed by ELISA to be reactive against the antigenic peptides. For IgG purification we used HiTrap Protein A HP antibody purification columns (GE Bio-Sciences (Pittsburgh, PA, USA) according to manufacturer’s protocol. For fluorescent IgG labelling of 100 μg IgG aliquots we used the Alexa Fluor™ 488 Antibody Labeling Kit from ThermoFisher (A20181) according to manufacturer’s instructions.