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Microbes & Immunity Brachyspira pilosicoli novel outer membrane proteins

“OMBB protein.” Our study supports the latter annotation, roles in B. pilosicoli. Sequence comparison across nine
revealing the presence of a secretory signal peptide. strains of B. pilosicoli revealed two variations (T246 and
Structural modeling using AlphaFold 3 showed a β-barrel N499) (Tables 3 and S4), both mapped to the periplasmic
architecture comprising eight β-strands (Figure 2E). α-helical regions of the protein (Table S4).
Structural similarity assessment using the DALI server
identified the closest match with Neisseria meningitidis 3.2.1.7. BP951000_RS09000
NspA (PDB ID: 1P4T) (Tables 2 and S3). NspA, an BP951000_RS09000 is annotated as an OM efflux protein
eight-stranded β-barrel protein, is involved in bacterial in UniProt and as a TolC family protein in NCBI. It
attachment and interaction with the host immune system carries a secretory signal peptide. The AlphaFold 3 model
and has been proposed as a potential vaccine candidate. 86-89 revealed a trimeric 18-stranded β-barrel, with each
Foldseek analysis identified an uncharacterized protein of protomer contributing six β-strands (Figure 2G). Similar
Brachyspira murdochii as its closest structural homolog, to BP951000_RS05600 (Section 3.2.6), we validated this
suggesting potential species-specific divergence. structure using TrRosetta, RoseTTAFold, ESMFold, and
SWISS-MODEL. Structural alignment of monomeric
Sequence-based annotation using PANNZER predicted
the presence of an OMBB domain (Tables 2 and S3). models from all five tools yielded an RMSD value of
Together, the structure- and sequence-based data 3.85 Å (Figure S3), confirming strong agreement with
strongly support the classification of BP951000_RS03440 the AlphaFold 3 prediction. Consistent with canonical
TolC proteins, the barrel extends into the periplasm as
as an OMBB protein, likely involved in host-pathogen an α-helical tunnel (Figure 2G). Structural alignment
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interaction. MSA across B. pilosicoli strains identified identified E. coli K-12 TolC (PDB ID: 6WXI) as the closest
six variations (F24, V47, V64, N110, D169, and A197)
(Tables 3 and S4). Structural mapping localized one match (Tables 2 and S3). As described in Section 3.2.6, E. coli
colicin import,
TolC mediates hemolysin secretion,
94,95
92,93
variation (V47) to the ECL region, four variations (F24, antibiotic efflux, and bacteriophage recognition. The
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V64, N110, A197) to the TM region, and one variation Foldseek tool identified its closest structural match with
(D169) to the intracellular region of the protein (Table S4).
an uncharacterized protein from a Spirochaete bacterium.
3.2.1.6. BP951000_RS05600 PANNZER and eggNOG-mapper similarly annotated
BP951000_RS09000 as an integral OMP and efflux
BP951000_RS05600 is annotated as a putative OM transporter (Tables 2 and S3). Amino acid sequence
component of a multidrug efflux system in UniProt and comparison across nine B. pilosicoli strains revealed two
as a TolC family protein in NCBI. Both SignalP and LipoP
predicted a secretory signal peptide. The AlphaFold 3 variations: S90 (in the ECL region) (Figure 2G) and S131
(within the β-barrel domain) (Tables 3 and S4).
model revealed a trimer 18-stranded β-barrel, with each
subunit (protomer) contributing six β-strands (i.e., one- 3.2.1.8. BP951000_RS06235
third of the barrel) (Figure 2F). As no known 18-stranded BP951000_RS06235 is annotated as a TolC family protein
TolC structures have been discovered to date, we validated in UniProt. The AlphaFold 3 model predicted a trimeric
this model using TrRosetta, RoseTTAFold, ESMFold, and 12-stranded β-barrel, with each subunit contributing
SWISS-MODEL. All models showed high similarity and four β-strands (Figure 2H). As in Section 3.2.6, we
aligned closely with the AlphaFold 3 model. Alignment validated this structure using ESMFold, SWISS-MODEL,
of the five monomeric models yielded a root mean square RoseTTAFold, and TrRosetta. Structural alignment of
deviation (RMSD) of 3.96 Å, supporting model reliability monomeric models from all tools yielded an RMSD of
(Figure S2). Like canonical TolC, the TM barrel extends 3.26 Å (Figure S4), confirming model consistency. The
into the periplasm as an α-helical tunnel, connected to the barrel extends into the periplasm as an α-helical tunnel
OM (Figure 2F), with its periplasmic entry blocked, likely connecting to the OM (Figure 2H). DALI analysis showed
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90
to prevent leakage through the OM, as the β-barrel domain the highest similarity to E. coli K-12 TolC (PDB ID: 6WXI)
remains constantly open. 91 (Tables 2 and S3). Foldseek identified the closest homolog
BP951000_RS05600 showed the best structural match as TolC from a Spirochaete bacterium. PANNZER and
with E. coli K-12 TolC (PDB ID: 6WXI) (Tables 2 and S3). eggNOG-mapper consistently annotated the protein as an
TolC functions in hemolysin secretion, 92,93 colicin OM efflux protein (Tables 2 and S3), suggesting a TolC-
import, 94,95 antibiotic efflux, and serves as a bacteriophage like function. Sequence alignment across nine B. pilosicoli
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receptor. PANNZER and eggNOG-mapper respectively strains revealed variations at 18 positions: K2, N3, F5, V6,
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annotated the protein as “integral OMP TolC” and “efflux F7, I8, I10, L12, S16, S25, N33, I42, E43, L93, S105, E136,
TM transporter” (Tables 2 and S3), suggesting similar I137, and T210 (Tables 3 and S4). Mapping showed L93

Volume 2 Issue 4 (2025) 92 doi: 10.36922/MI025230050