The members of the
antigen 85
protein family (
Ag85), consisting of members Ag85A, Ag85B, and Ag85C, are the predominantly secreted
proteins of mycobacteria and possess the ability to specifically interact with
fibronectin (Fn). Because Fn-
binding proteins are likely to be important
virulence factors of Mycobacterium spp.,
Ag85 may contribute to the adherence, invasion, and dissemination of organisms in host tissue. In this study, we reported the Fn binding affinity of Ag85A, Ag85B, and Ag85C from Mycobacterium avium subsp.
paratuberculosis (MAP) (K(D) values were determined from 33.6 to 68.4 nm) and mapped the Ag85-binding motifs of Fn. Fn14, a type III module located on the
heparin-binding domain II (Hep-2) of Fn, was discovered to interact with
Ag85 from MAP. The
peptide inhibition assay subsequently demonstrated that a
peptide consisting of residues 17-26 from Fn14 ((17)SLLVSWQPPR(26), termed P17-26) could interfere with Ag85B binding to Fn (73.3% reduction). In addition, single
alanine substitutions along the sequence of P17-26 revealed that the key residues involved in Ag85-Fn binding likely contribute through hydrophobic and charge interactions. Moreover, binding of
Ag85 on Fn
siRNA-transfected Caco2 cells was dramatically reduced (44.6%), implying the physiological significance of the Ag85-Fn interaction between mycobacteria and host cells during
infection. Our results indicate that
Ag85 binds to Fn at a novel motif and plays a critical role in mycobacteria adherence to host cells by initiating
infection.
Ag85 might serve as an important colonization factor potentially contributing to mycobacterial virulence.