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Microbes & Immunity Brachyspira pilosicoli novel outer membrane proteins

structural model revealed a β-barrel comprising 18 predicted a 16-stranded β-barrel structure with a lateral
β-strands, nine ECLs, and eight periplasmic loops gate between strands 1 and 16 (Figure 3E). DALI analysis
(Figure 3C). DALI analysis showed the highest structural revealed its closest structural match with the translocation
similarity to P. aeruginosa PAO1 porin OM channel protein and assembly module protein A (TamA) from E. coli
K (OccK)-7 (PDB ID: 4FRT) (Tables 2 and S3). The OccK (PDB ID: 4C00) (Tables 2 and S3), an Omp85 superfamily
protein family contains a ladder of basic residues (arginine protein featuring three N-terminal POTRA domains and
+ lysine = 11%) that form a positively charged channel for a C-terminal 16-stranded β-barrel. TamA facilitates
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the uptake of small, carboxyl-containing substrates. 116-119 autotransporter β-barrel membrane insertion and passenger
BP951000_RS04405 exhibited a lower arginine + lysine domain translocation into the extracellular space. 123-125
content (7.6%), suggesting that while structurally similar Foldseek also identified a structural match with an Omp85
to OccK, it may facilitate the transport of alternative domain-containing protein from Dracunculus medinensis.
substrates. Foldseek identified its closest structural match While BP951000_RS04440 closely resembles the TamA
with an uncharacterized protein from Treponema vincentii. β-barrel domain, it lacks the POTRA domains, suggesting
PANNZER annotated it as Toxin A, a known virulence functional divergence. BP951000_RS04440 is thus predicted
factor (Tables 2 and S3). 120 to be a structural homolog of TamA, but its specific role in
These findings suggest that BP951000_RS04405 is a B. pilosicoli remains to be clarified. Notably, PANNZER
porin-like protein with potential functional divergence, annotated the protein as Toxin A (Tables 2 and S3).
warranting further experimental annotation. A total of 23 Sequence comparison across nine B. pilosicoli strains
amino acid sequence variations were identified across nine revealed 10 variations (K104, K113, S117, Y124, I132, T134,
B. pilosicoli strains (Tables 3 and S4). Structural mapping N151, G153, L243, and V252) (Tables 3 and S4). Structural
showed six variations (N41, F84, K90, N98, S175, and Q183) mapping showed K104 and L243 in the ECL region, with
in the ECL region, eight (T24, N32, S34, I101, S102, N104, the remaining variations located within the TM β-barrel
I264, and T303) in the TM β-barrel region, and the remainder domain (Figure 3E and Table S4).
in the periplasmic region (Figure 3C and Table S4). 3.2.2.6. BP951000_RS08285

3.2.2.4. BP951000_RS09655 BP951000_RS08285 is annotated as a transcriptional
BP951000_RS09655 is annotated as a hypothetical protein regulating protein (Trep) in B. pilosicoli. In Pseudomonas
in NCBI and as a domain of unknown function (DUF) fluorescens, Trep catalyzes trehalose phosphorylation
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5723 domain-containing protein in UniProt. It carries a and its translocation across the OM. LipoP predicted
secretory signal peptide. The AlphaFold model predicted BP951000_RS08285 as cytoplasmic, whereas SignalP
a 16-stranded β-barrel, with strands 3–12 longer than the predicted no signal peptide. The AlphaFold 3 predicted a
rest, giving the barrel an asymmetric extracellular profile 16-stranded C-terminal β-barrel with a large N-terminal
(Figure 3D). periplasmic domain and a lateral opening between strands
1 and 16 (Figure 3F). Structure-based annotation showed
DALI analysis identified the closest structural match the closest match with TolB proteins from E. coli K-12
with the tetraheme c-type cytochrome CymA from (PDB ID: 3IAX) and Citrobacter freundii (PDB ID: 2IVZ)
Klebsiella oxytoca (PDB ID: 4V3G) (Tables 2 and S3). (Tables 2 and S3). E. coli TolB is a periplasmic protein with
CymA, a 14-stranded OMP, facilitates passive diffusion a two-domain structure: an α/β N-terminal domain and
of large molecules such as cyclodextrins and linear a six-bladed β-propeller C-terminal domain (Figure S5).
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maltooligosaccharides. 121,122 It aligned with 14 of the 16 The periplasmic domain of BP951000_RS08285 closely
β-strands in BP951000_RS09655. The comparable pore resembles the TolB C-terminal domain, suggesting
diameter suggests a similar function in passive diffusion a similar role in porin assembly and cell envelope
channels in B. pilosicoli. Structural alignment using the integrity. 128-130 However, unlike TolB, BP951000_RS08285
US-Align server yielded an RMSD of 4.28 Å, supporting high includes an additional TM β-barrel domain, indicating
structural homology. PANNZER annotated the protein as a potential functions unique to B. pilosicoli.
cell surface protein (Tables 2 and S3). Sequence comparison
across nine strains of B. pilosicoli revealed 315 variations and When the β-barrel domain alone was queried, the
multiple deletions, indicating high variability across the full top DALI hit was filamentous hemagglutinin (FHA)
length of the protein (Figure 3D and Table S5). transporter FhaC (PDB ID: 4QL0) from Bordetella
pertussis, a 16-stranded β-barrel protein that transports
3.2.2.5. BP951000_RS04440 FHA (Figure S5). While FhaC includes POTRA domains
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BP951000_RS04440, annotated as a hypothetical protein, for substrate recognition, BP951000_RS08285 lacks
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contains a secretory signal peptide. The AlphaFold 3 model them, suggesting it may function as a translocation pore

Volume 2 Issue 4 (2025) 95 doi: 10.36922/MI025230050