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Microbes & Immunity Brachyspira pilosicoli novel outer membrane proteins

with distinct specificity. Foldseek identified the closest the protein as uncharacterized, structure-based evidence
structural match with an uncharacterized protein from strongly supports its function as a transporter of
a Spirochaete bacterium (Tables 2 and S3). PANNZER hydrophobic substrates. Sequence comparison across nine
annotated BP951000_RS08285 as Trep, while eggNOG- B. pilosicoli strains revealed an insertion at position 255
mapper linked it to TonB-independent uptake pathways. and six variations (L12, S20, N22, A117, R187, and S253)
BLASTp analysis showed no sequence homologs in other (Figure 3H, Tables 3 and S4). Mapping these onto the
spirochetes (e.g., Treponema, Borrelia, and Leptospira), model placed N22 and A117 in the ECL region, S253 in
indicating species-specific uniqueness. MSA across nine B. the ICL region, R187 in the β-barrel TM region, and the
pilosicoli strains revealed 43 variations (Table S5). remaining variations in the N-terminal domain (Table S4).
3.2.2.7. BP951000_RS04505 3.2.2.9. BP951000_RS01090
BP951000_RS04505 is annotated as VspH in B. pilosicoli. BP951000_RS01090 is annotated as a hypothetical protein in
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As described in Section 3.2.3, Vsps mediate bacterial NCBI but as a VspH in UniProt. SignalP predicted a secretory
adherence to host cells. SignalP predicted no signal signal peptide. The AlphaFold 3 model revealed a 14-stranded
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peptide, whereas LipoP predicted the presence of β-barrel architecture (Figure 3I). Sequence-based annotation
N-terminal TMHs. The AlphaFold 3 model revealed a using PANNZER supported the VspH designation. DALI
16-stranded β-barrel (Figure 3G). analysis identified K. oxytoca CymA (PDB ID: 4V3G) as the
closest structural homolog (Tables 2 and S3). As discussed
Structural alignment identified the closest match with
E. coli K-12 PgaA (PDB ID: 4Y25) (Tables 2 and S3). As in Section 3.2.13, CymA functions as a diffusion channel
discussed in Section 3.2.2, PgaA facilitates polysaccharide for bulky substrates. Foldseek further indicated similarity
translocation across the OM. Additionally, Foldseek to an uncharacterized protein from a Chitinophagaceae
identified its best alignment with a bacterial polysaccharide bacterium. Sequence comparison across nine B. pilosicoli
OM secretin from E. coli K-12, supporting this hypothesis. strains revealed a single variation, E258, located in the ECL
PANNZER annotated the protein as a cell surface region (Figure 3I; Tables 3 and S4). Structural alignment
protein. Based on both structural and sequence analyses, of BP951000_RS04760, BP951000_RS02055, BP951000_
BP951000_RS04505 is likely involved in polysaccharide RS04505, and BP951000_RS01090 using the US-align server
secretion across the OM. Notably, its homolog in B. resulted in an RMSD of 4.00 Å (Figure S6).
hyodysenteriae (VspH) has been evaluated as a potential 3.2.2.10. BP951000_RS06935
vaccine candidate. Sequence comparison across nine B.
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pilosicoli strains revealed 247 amino acid variations and BP951000_RS06935 is annotated as a hypothetical
multiple deletions, indicating high variability throughout protein in UniProt and NCBI. It is predicted to contain a
the protein (Figure 3G and Table S5). secretory signal peptide. The AlphaFold 3 model revealed
a 12-stranded β-barrel structure with six ECLs (Figure 3J).
3.2.2.8. BP951000_RS08455 Structural homology search identified the closest match
BP951000_RS08455 is annotated as a PorV/PorQ family as the β-barrel domain of the E. coli hemoglobin-
protein in UniProt and as a hypothetical protein in binding protease (Hbp) autotransporter (PDB ID: 3AEH)
NCBI. PorV and PorQ are integral components of the (Tables 2 and S3). Hbp, a member of the serine protease
type IX secretion system, involved in protein export in autotransporters of Enterobacteriaceae family, consists
Gram-negative members of the Fibrobacteres–Chlorobi– of an N-terminal serine protease passenger domain and
Bacteroidetes superphylum. The AlphaFold 3 model a C-terminal β-barrel that facilitates its extracellular
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revealed a 14-stranded β-barrel structure with seven ECLs secretion. While BP951000_RS06935 lacks a
and an interior blocked by an N-terminal hatch domain periplasmic or passenger domain, its β-barrel homology
(Figure 3H). Foldseek identified structural similarity to a suggests a role in substrate translocation across the OM.
PorV/PorQ family protein, and the best DALI match was Foldseek identified the best match with a 12-stranded
E. coli long-chain fatty acid transporter FadL (PDB ID: β-barrel OMP from Cluster of Orthologous Groups (COG)
2R88) (Tables 2 and S3). Like FadL, BP951000_RS08455 4313 of Pseudomonas putida F1, which has a dynamic
adopts a monomeric 14-stranded β-barrel, though it lateral opening that permits the passage of hydrophobic
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lacks the lateral opening formed by the inward bend in a molecules. Sequence-based annotation by PANNZER
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β-strand that is essential for fatty acid transport in FadL. linked BP951000_RS06935 to Toxin A. Collectively,
Despite this structural difference, the remaining structural structure- and sequence-based analyses suggest that
resemblance suggests a potential role in transporting BP951000_RS06935 may act as an autotransporter or
hydrophobic molecules. Although PANZZER annotated facilitate hydrophobic molecule transport. Sequence
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Volume 2 Issue 4 (2025) 96 doi: 10.36922/MI025230050