Characterization of the β-barrel assembly machinery in fusobacterium.

Abstract

Fusobacterium nucleatum is a Gram-negative, anaerobic human oral microbiome constituent. Though a steady state commensal, this pathobiont plays a role in periodontal disease, endodontic infections, pre-term births, and colorectal cancer. F. nucleatum achieves its pathogenicity through a class of outer membrane proteins (OMPs) called adhesins. Several key adhesins present as β-barrel OMPs in the form of Type VA secretion system autotransporters or porins. The β-Barrel Assembly Machinery (BAM) complex plays a vital role in the biogenesis of β-barrel OMPs in Gram-negative bacteria. The E. coli BAM complex is composed of five proteins, OMP BamA and accessory lipoproteins BamB-E. Though there is diversity in the number of BAM complex components among Gram-negative bacteria, BamA and BamD are consistently essential for viability. However, preliminary searches of the F. nucleatum genome have revealed only the presence of BamA, the integral membrane protein of the complex, to the exclusion of any apparent periplasmic auxiliary proteins, and the structure of F. nucleatum BamA (FnBamA) remains unresolved. Thus, the objective of this study is to determine both the structure of FnBamA and to identify the accessory proteins with which it binds. We employ techniques including X-ray crystallography and cryo-electron microscopy (EM) to accomplish the structural aims of this proposal and have obtained the first-ever structure of FnBamA using cryo-EM. Pull-down assays coupled with proteomics elucidate the identity and nature of proteins accessory to BamA. This research is innovative in its exploration of membrane protein biology in F. nucleatum through examining BAM, a difficult system unstudied in this challenging anaerobic organism. We expect to advance the mitigation of F. nucleatum pathogenesis in the oral microbiome and beyond by detecting the binding partners and solving the structure of FnBamA.