Identification and analysis of fipA, a Fusobacterium nucleatum immunosuppressive factor gene

We have previously demonstrated that sonic extracts of Fusobacterium nucleatum FDC 364 were capable of inhibiting human T-cell responses to mitogens and antigens, The purified F. nucleatum immunosuppressive protein (FIP) is composed of two subunits of 44 and 48 kDa, Furthermore, FIP inhibits T-cell activation by arresting cells in the middle of the G(1) phase of the cell cycle; the data available to date suggest that FIP impairs the expression of the proliferating-cell nuclear antigen, To initiate delineation of FIP structure-function relationships, molecular cloning of the FIP gene was carried out. A DNA library of F. nucleatum FDC 364 was constructed by partial digestion of genomic DNA with Sau3A and screened for the production of FIP with polyclonal antibody. Twelve immunoreactive clones were identified. One of these clones contained a 3.1-kbp insert and was chosen for further study. Cell lysates were found to contain an immunoreactive band that comigrated with the 44-kDa subcomponent of the native FIP, Sequencing of the 3.1-kbp insert revealed the presence of three open reading frames (ORFs). One ORF extends from nucleotides 415 to 1620, encodes 402 amino acids, and is preceded by a ribosome-binding site, Deletion analysis and antibody elution analysis showed that this ORF encodes the 44-kDa subunit (FipA) of native FIP. A second ORF is situated upstream of fipA. However, Northern (RNA) analysis suggested that fipA is not transcribed as part of an operon but is transcribed from its own promotor, Finally, the partially purified recombinant FipA protein was capable of impairing T-cell activation in a manner consistent with the native protein. These results indicate that the two components that form the native protein are most probably distinct gene products and suggest that the 44-kDa FipA polypeptide is sufficient to mediate the immunosuppressive activities of the native protein complex.