Investigation of Mutations in FadA Structure and Function

Objectives: Fusobacterium nucleatum, a gram-negative anaerobe associated with periodontal disease and preterm birth, binds to and invades host cells through a unique adhesin, FadA. Both the non-secreted (pre-FadA) and secreted mature (mFadA) forms are required for its structure and function. Crystallography of mFadA reveals a predominantly alpha-helical structure with the monomers linked together in a head-to-tail pattern and the only non-alpha helical loop exposed at the tip of the filament. We postulate that FadA filament is anchored in the inner-membrane through pre-FadA with mFadA monomers penetrating through the outer-membrane. The aim of this study is to examine the role of the loop region and the signal peptide in the structure and function of FadA. Methods: Residues in the loop and the signal peptide domain were replaced by site-directed mutagenesis. Mutant proteins were purified and subjected to structural and functional analyses such as signal peptide retention by western-blot, attachment inhibition assay and filament formation under electron microscopy. Results: All mutants were able to produce pre-and mFadA indicating that the mutations didn't affect the signal peptide retention. Two stable mutants in the loop region (F68G and Y69G) and two in the signal peptide (L-9A and L-12A) were further analyzed.Loop mutants were defective in filament formation and do not inhibit attachment of Fusobacterium to CHO cells. L-9A mutant shows a formation of filaments like the wild-type whereas L-12A mutant formed wider filaments. Only L-9A still had binding activity. Conclusion: Preliminary results indicate that the loop region is required for both oligomerization and attachment and that residue in the signal peptide also affect oligomerization. Both the loop and the signal peptide are required for proper oligomerization, which is required for the function of FadA. In addition, L-9A mutant highlight that a correct formation of the filament is required for complete function of FadA. Grant:RO1-DE14924