Novel mycobacteria antigen 85 complex binding motif on fibronectin

Area of Science:

• Microbiology
• Immunology
• Biochemistry

Background:

• The antigen 85 (Ag85) protein family (Ag85A, Ag85B, Ag85C) are secreted by mycobacteria and bind fibronectin (Fn).
• Fn-binding proteins are considered potential virulence factors for Mycobacterium species, possibly aiding in host tissue adherence, invasion, and dissemination.

Purpose of the Study:

• To investigate the fibronectin (Fn) binding affinity of Ag85 proteins from Mycobacterium avium subsp. paratuberculosis (MAP).
• To map the specific binding sites of Ag85 on Fn and identify key residues involved in the interaction.
• To assess the physiological significance of the Ag85-Fn interaction in mycobacterial adherence to host cells.

Main Methods:

• Determined the Fn binding affinity of MAP Ag85A, Ag85B, and Ag85C using K(D) values.
• Mapped Ag85-binding motifs on Fn, identifying Fn14 (a type III module on Hep-2) as an interaction site.
• Utilized peptide inhibition assays with specific Fn14 peptides and alanine substitutions to identify key binding residues.
• Assessed Ag85 binding to Fn siRNA-transfected Caco2 cells to evaluate physiological relevance.

Main Results:

• Ag85 proteins from MAP exhibited Fn binding with K(D) values ranging from 33.6 to 68.4 nm.
• Fn14 was identified as the specific binding module on Fn for MAP Ag85.
• A peptide (P17-26) from Fn14 significantly inhibited Ag85B binding to Fn (73.3% reduction), with key residues identified through alanine substitutions.
• Ag85 binding to Caco2 cells was reduced by 44.6% upon Fn siRNA transfection, confirming the interaction's importance.

Conclusions:

• Ag85 proteins bind fibronectin at a novel motif (Fn14), involving hydrophobic and charge interactions.
• The Ag85-Fn interaction plays a critical role in mycobacteria adherence to host cells, initiating infection.
• Ag85 may function as a significant colonization factor, contributing to mycobacterial virulence.