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J. Biol. Chem., Vol. 283, Issue 10, 6359-6366, March 7, 2008

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Opacity Factor Activity and Epithelial Cell Binding by the Serum Opacity Factor Protein of Streptococcus pyogenes Are Functionally Discrete^*

Christine M. Gillen¹, Harry S. Courtney², Kai Schulze^¶, Manfred Rohde^||, Mark R. Wilson, Anjuli M. Timmer^**, Carlos A. Guzman^¶, Victor Nizet^**, G. S. Chhatwal^||, and Mark J. Walker³

From the School of Biological Sciences, University of Wollongong, New South Wales 2522, Australia, the Veterans Affairs Medical Center and Department of Medicine, University of Tennessee Health Science Center, Memphis, Tennessee 38104, the ^¶Department of Vaccinology, Helmholtz Centre for Infection Research, Braunschweig, Germany, the ^||Department of Microbial Pathogenicity, Helmholtz Centre for Infection Research, Braunschweig, Germany D-38124, and the ^**Department of Pediatrics, Division of Pharmacology and Drug Discovery, University of California, San Diego, La Jolla, California 92093-0687

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 (SOFFn) 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 SOFFn 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, SOFFn peptide, or a peptide encompassing the C-terminal repeats (FnBD), we demonstrate that adhesion to HEp-2 cells is mediated by both SOFFn and FnBD. The HEp-2 cell binding displayed by the N-terminal SOFFn 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.

Received for publication, August 14, 2007 , and in revised form, November 26, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ Recipient of an Australian Research Council Postgraduate Award.

² Supported by research funds from the Dept. of Veterans Affairs.

³ To whom correspondence should be addressed: School of Biological Sciences, University of Wollongong, Wollongong, NSW, 2522, Australia. Tel.: 0061-2-4221-3439; Fax: 0061-2-4221-4135; E-mail: mwalker{at}uow.edu.au.

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