Purification of a mycobacterial adhesin for fibronectin.
Previous studies have demonstrated that mycobacteria attach to fibronectin (FN). The attachment of mycobacteria to FN is considered to be biologically important in Mycobacterium bovis BCG therapy for superficial bladder cancer, initiation of delayed hypersensitivity to mycobacterial antigens, and the phagocytosis of mycobacteria by epithelial cells. Therefore, we purified the mycobacterial receptor for FN. Culture supernatants from 3-week cultures of Mycobacterium vaccae, which contained proteins that bound FN and inhibited the attachment of both M. vaccae and BCG to FN, were used as a source of receptor. Lyophilized M. vaccae supernatants were reconstituted in 0.02 M bis-Tris (pH 6.0) and applied sequentially to an ACA 54 gel filtration column and a DEAE-Sephacel anion-exchange column. A purified inhibitory protein of 55 kDa (p55) was obtained. The purified p55 protein was observed to bind to FN and to inhibit 125I-FN binding to viable BCG in a dose-dependent manner. Polyclonal and monoclonal antibodies to the protein were generated. The resulting polyclonal antiserum blotted a single protein band at 55 kDa in crude M. vaccae supernatants, cross-reacted with a 55-kDa BCG protein by Western blot (immunoblot), and recognized a 55-kDa band that was associated with the BCG cell wall, which is consistent with its function as a FN receptor. A monoclonal immunoglobulin M(lambda) was isolated from mice immunized with purified M. vaccae p55 protein that was not functional in Western blots but inhibited the attachment of viable BCG to FN. These studies demonstrate that a protein or antigenically related proteins with M(r)s of 55,000 function as FN receptors for at least two distinct mycobacteria.
Ratliff
T L
TL
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri.
McCarthy
R
R
Telle
W B
WB
Brown
E J
EJ
D000911:Antibodies, Monoclonal / Q000276:immunology; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D015153:Blotting, Western; D005353:Fibronectins / Q000378:metabolism*; D008970:Molecular Weight; D009161:Mycobacterium / Q000737:chemistry*; D009163:Mycobacterium bovis / Q000737:chemistry; D017469:Receptors, Fibronectin / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D005353:Fibronectins; D017469:Receptors, Fibronectin