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Microbes & Immunity Brachyspira pilosicoli novel outer membrane proteins

BP951000_RS01280, BP951000_RS10445, BP951000_ OMBB proteome of B. pilosicoli using a consensus-based
RS00365, BP951000_RS04620, and BP951000_RS04220— computational approach.
are annotated in UniProt as SR domain-containing proteins, We predicted 42 OMBB proteins and validated their
but listed as hypothetical in NCBI. All are predicted to β-barrel architectures using AlphaFold 3. These proteins
contain a signal peptide. Five similarly annotated proteins exhibited diverse topologies, ranging from 8 to 26 strands.
(Group A) were previously discussed in Section 3.2.9. Structural homology-based functional annotation revealed
The AlphaFold 3 models revealed a conserved eight- putative homologs of BamA, LptD, TolC, TBDRs, NspA,
stranded β-barrel architecture (Figure 5B-I). Structural OmpA, FadL, and others, suggesting roles in membrane
alignment analysis using DALI showed that BP951000_ biogenesis, LPT, efflux activity, substrate uptake, host
RS01280, BP951000_RS00365, and BP951000_RS06930 colonization, and immune modulation.
closely matched with N. meningitidis NspA (PDB ID: Among the 42 OMBB proteins, seven lacked annotation
1P4T), whereas BP951000_RS04620, BP951000_RS00765, in both UniProt and NCBI. Combined structure- and
BP951000_RS03290, BP951000_RS04220, and BP951000_ sequence-based analyses enabled putative functional
RS10445 aligned best with the N-terminal β-barrel domain assignment for these hypothetical proteins. Comparative
of E. coli K-12 OmpA (PDB ID: 9FZC) (Tables 2 and S3). sequence analysis across nine B. pilosicoli strains revealed

As described in Sections 3.2.5 and 3.2.9, NspA extensive polymorphisms in 12 proteins, each containing
contributes to host colonization and immune evasion 40 or more variations, suggesting potential roles in immune
and is a potential vaccine candidate, 86-89 whereas OmpA evasion and host adaptation.
plays roles in phage recognition, conjugation, membrane This study expands current knowledge of the
integrity, diffusion, and virulence. 101-109 Foldseek analysis B. pilosicoli OMP repertoire and provides a framework for
further revealed that seven of the eight proteins showed identifying potential targets for diagnostic, prophylactic,
the closest structural match with uncharacterized proteins and therapeutic development. The functional predictions
from the Brachyspira genus. Notably, BP951000_RS01280 and structural insights reported here lay the foundation for
aligned with an OMBB protein from B. hyodysenteriae, future experimental work aimed at elucidating the precise
supporting its role as a conserved OMP component. roles of these OMPs in IS pathogenesis.

Consistent with structural data, PANNZER annotated
BP951000_RS06930, BP951000_RS00765, BP951000_ Acknowledgements
RS01280, BP951000_RS10445, and BP951000_RS04620 None.
as OMBB proteins, whereas BP951000_RS03290,
BP951000_RS00365, and BP951000_RS04220 remained Funding
uncharacterized. The convergence of structure- and Amisha Panda is a recipient of a junior research fellowship
sequence-based annotations supports their classification from the Department of Biotechnology, Government of
as OMBB proteins, although experimental validation India (grant number: DBT/2023-24/UOD/2326). Jahnvi
is needed to confirm their functional roles. Sequence Kapoor is a recipient of a junior research fellowship from

comparison across nine B. pilosicoli strains revealed the University Grants Commission, Government of India
variations at several positions (Tables 3, S4, and S5). (grant number: 231620067077).
This study did not include experimental validation
of OMBBs, which is a limitation. Future research should Conflict of interest
focus on validating these predictions through experimental The authors declare that they have no competing interests.
characterization and functional analysis.
Author contributions
4. Conclusion
Conceptualization: Sanjiv Kumar
Brachyspira pilosicoli is a globally prevalent enteric Data curation: Amisha Panda
spirochete associated with IS in both animals and Formal analysis: Amisha Panda, Jahnvi Kapoor, Ravindresh
humans. Despite its clinical significance, the molecular Chhabra, Anannya Bandyopadhyay
mechanisms underlying its pathogenesis remain poorly Investigation: Amisha Panda
understood, particularly the role of OMPs, which are Methodology: Amisha Panda, Sanjiv Kumar, Anannya
critical for nutrient uptake, adhesion, immune evasion, Bandyopadhyay
and virulence. In this study, we addressed this knowledge Software: Amisha Panda, Sanjiv Kumar
gap by systematically identifying and characterizing the Supervision: Sanjiv Kumar

Volume 2 Issue 4 (2025) 101 doi: 10.36922/MI025230050