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Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein AI and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOFDeltaFn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOFDeltaFn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOFDeltaFn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOFDeltaFn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOFDeltaFn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone.

Mycoplasma hyopneumoniae is the causative pathogen of porcine enzootic pneumonia, an economically significant disease that disrupts the mucociliary escalator in the swine respiratory tract. Expression of Mhp 107, a P97 paralog encoded by the gene mhp107, was confirmed using ESI-MS/MS. To investigate the function of Mhp107, three recombinant proteins, F1MhP107, F2MhP107, and F3MhP107, spanning the N-terminal, central, and C-terminal regions of Mhp107 were constructed. Colonization of swine by M. hyopneumoniae requires adherence of the bacterium to ciliated cells of the respiratory tract. Recent studies have identified a number of M. hyopneumoniae adhesins that bind heparin, fibronectin, and plasminogen. F1MhP107 was found to bind porcine heparin (KD ∼90 nM) in a dose-dependent and saturable manner, whereas F3Mhp107 bound fibronectin (KD ∼180 nM) at physiologically relevant concentrations. F1MhP107 also bound porcine plasminogen (KD = 24 nM) in a dose-dependent and physiologically relevant manner. Microspheres coated with F3MhP107 mediate adherence to porcine kidney epithelial-like (PK15) cells, and all three recombinant proteins (F1MhP107-F3MhP107) bound swine respiratory cilia. Together, these findings indicate that Mhp107 is a member of the multifunctional M. hyopneumoniaeadhesin family of surface proteins and contributes to both adherence to the host and pathogenesis. [ABSTRACT FROM AUTHOR]

Serum opacity factor (SOF) is a unique multifunctional virulence determinant expressed at the surface of Streptococcus pyogenes and has been shown to elicit protective immunity against GAS infection in a murine challenge model. SOF consists of two distinct domains with different binding capacities: an N-terminal domain that binds apolipoprotein At and a C-terminal repeat domain that binds fibronectin and fibrinogen. The capacity of SOF to opacify serum by disrupting the structure of high density lipoproteins may preclude its use as a vaccine antigen in humans. This study generated mutant forms of recombinant SOF with reduced (100-fold) or abrogated opacity factor (OF) activity, for use as vaccine antigens. However, alterations introduced into the N-terminal SOF peptide (SOFΔFn) by mutagenesis to abrogate OF activity, abolish the capacity of SOF to protect against lethal systemic S. pyogenes challenge in a murine model. Mutant forms of purified SOFΔFn peptide were also used to assess the contribution of OF activity to the pathogenic processes of cell adhesion and cell invasion. Using latex beads coated with full-length SOF, SOFΔFn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOFΔFn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOFΔFn peptide is independent of OF activity. We demonstrate that while the N terminus of SOF does not directly mediate intracellular uptake by epithelial cells, this domain enhances epithelial cell uptake mediated by full-length SOF, in comparison to the FnBD alone. [ABSTRACT FROM AUTHOR]