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Microbes & Immunity Brachyspira pilosicoli novel outer membrane proteins

variation analysis across nine B. pilosicoli strains revealed BP951000_RS03405 simply as an OMP. Sequence
four variations (S9, I10, V13, and R298) (Figure 3J, alignment across nine strains of B. pilosicoli revealed 24
Tables 3 and S4). When mapped onto the structural model, variations, all located within the periplasmic region of the
these variations were present in the N-terminal and β-barrel structure (Figure 4, Tables 3 and S4).
C-terminal regions of the β-barrel structure (Table S4).
3.2.2.13. BP951000_RS00185
3.2.2.11. BP951000_RS11380 BP951000_RS00185, annotated as a hypothetical protein,
BP951000_RS11380, annotated as a hypothetical protein in contains a secretory signal peptide with a cleavage site
NCBI, was predicted to contain a secretory signal peptide. between residues 20 and 21. The AlphaFold 3 model
UniProt classified the protein as Toxin A. It has an N-terminal predicted a 12-stranded β-barrel (Figure 4C). DALI analysis
TMH, predicted by LipoP. The AlphaFold 3 model revealed identified E. coli K-12 OMPLA as the closest structural
a 12-stranded β-barrel (Figure 4A). Structural alignment match (PDB ID: 1QD6; Z-score = 18.3, RMSD = 3.1 Å)
using DALI showed the closest match to E. coli O157:H7 (Tables 2 and S3). As discussed in Section 3.2.21, OMPLA
serine protease EspP (PDB ID: 2QOM) (Tables 2 and S3), hydrolyzes acyl ester bonds in phospholipids and
a member of the serine protease autotransporters of lysophospholipids and is involved in colicin secretion. 146-148
Enterobacteriaceae family of autotransporters. These Although OMPLA contains a catalytic His–Ser–Asn triad,
141
specialized porins contain a C-terminal β-barrel that BP951000_RS00185 lacks this motif. However, adjacent
facilitates the secretion of an N-terminal virulence- Ser–Asn residues were identified at positions 25–26
associated passenger domain. Some autotransporters and 73–74. Foldseek identified structural homology to a
consist solely of the β-barrel autotransporter domain, with DUF1207 domain-containing protein from Ignavibacteria.
the passenger or toxin-encoding gene located upstream in Sequence-based annotation indicated similarity to
the genome. The EspP passenger domain exhibits serine Toxin A, a known virulence factor. Sequence variation
120
142
protease activity, cleaving host proteins such as pepsin A across nine strains of B. pilosicoli revealed 58 variations
and human coagulation factor V. 143-145 Given the structural predominantly present in the signal peptide region
similarity, BP951000_RS11380 might function as an (Figure 4C and Table S5).
autotransporter for a serine protease or virulence factor
encoded elsewhere in the genome. Foldseek identified the 3.2.2.14. BP951000_RS10320
closest structural homolog with an uncharacterized protein BP951000_RS10320, annotated as a hypothetical protein,
from Candidatus margulis. Sequence-based analysis is predicted to contain a secretory signal peptide. The
by PANNZER also aligned it with Toxin A. Sequence AlphaFold 3 model revealed a 10-stranded β-barrel with
comparison across nine B. pilosicoli strains revealed three several TM β-strands extending toward the periplasmic
variations (M126, M154, and I278), with M126 located in side, forming an elliptical pore (Figure 4D). Periplasmic
the ECL region and the other two (M154 and I278) in the loops from the TM barrel are also folded into α-helices and
TM β-barrel region (Figure 4A, Tables 3 and S4). β-strands. Structural homology using Foldseek identified
the best match with an uncharacterized protein from B.
3.2.2.12. BP951000_RS03405 murdochii. DALI analysis returned synthetic TM β-barrels
BP951000_RS03405, annotated as a hypothetical protein, as the top four hits; the fifth hit, N. meningitidis opacity-
contains a secretory signal peptide. The AlphaFold 3 associated protein A (OpcA) (PDB ID: 2VDF), was
model revealed a 12-stranded β-barrel (Figure 4B). considered the best structural homolog (Tables 2 and S3).
DALI analysis showed E. coli OMPLA as the closest OpcA is a 10-stranded β-barrel OMP that mediates
structural homolog (PDB ID: 1ILD) (Tables 2 and S3). adhesion by binding host cell proteoglycans. 149,150 Sequence
OMPLA is an acyl hydrolase that cleaves ester bonds in analysis using PANNZER also indicated homology with a
phospholipids and lysophospholipids, and contributes to cell surface protein, supporting a potential role in host cell
colicin secretion. 146-148 BP951000_RS03405, as a structural interaction and adhesion. Sequence analysis across nine B.
homolog of E. coli OMPLA, may serve similar functions pilosicoli strains revealed 25 variations, none of which were
in B. pilosicoli. Although OMPLA contains a catalytic located in the ECL region (Figure 4D, Tables 3 and S4).
histidine (His)–serine (Ser)–asparagine (Asn) triad,
BP951000_RS03405 lacks this exact motif; however, Ser 3.2.2.15. BP951000_RS05445
and Asn residues are present at adjacent positions 224 and BP951000_RS05445, annotated as a DUF3575 domain-
225. Foldseek indicated homology to a DUF1207 domain- containing protein in UniProt and as a hypothetical protein
containing protein from Ignavibacteria, suggesting in NCBI, carries a predicted secretory signal peptide
possible functional diversity, while PANNZER annotated (SignalP), although LipoP classified it as a cytoplasmic

Volume 2 Issue 4 (2025) 97 doi: 10.36922/MI025230050