Mycobacterial protein HbhA binds human complement component C3

Mycobacterium tuberculosis and Mycobacterium avium are facultative intracel lular pathogens that are able to survive and replicate in mononuclear phago cytes. Human complement component C3 has previously been shown to mediate a ttachment and phagocytosis of these bacteria by mononuclear phagocytes. In this study, a C3 ligand affinity blot protocol was used to identify a 30-kD a C3-binding protein in M. tuberculosis and Mycobacterium smegmatis and a 3 1-kDa C3-binding protein in M. avium. The C3-binding proteins in M. tubercu losis and M. avium localized to the cell membrane fraction and partitioned to the detergent fraction during Triton X-114 phase partitioning. The C3-bi nding protein from M. tuberculosis was partially purified using a cation ex change column and was shown to bind concanavalin A. The N terminus and an i nternal fragment of the partially purified C3-binding protein were subjecte d to amino acid sequence analysis. The resulting amino acid sequences match ed the Al. tuberculosis heparin-binding hemagglutinin (HbhA) protein. Recom binant full-length HbhA and the C terminus of HbhA fused to maltose-binding protein, but not recombinant HbhA lacking the C-terminal region, bound hum an C3. Recombinant full-length HbhA coated on polystyrene beads, was found to enhance the adherence and/or phagocytosis of the coated beads to J774.A1 cells in both the presence and absence of human serum. The presence of com plement-sufficient serum increased the adherence of the HbhA-coated beads t o the J774.A1 cells in a C3-dependent manner. If HbhA within the bacterial cell membrane functions similarly to isolated HbhA, this protein may enhanc e the adherence and phagocytosis of Al. tuberculosis and M. avium to mononu clear phagocytes through the binding of C3 and interaction with C3 receptor s on mononuclear phagocytes.