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J. Biol. Chem., Vol. 283, Issue 2, 855-865, January 11, 2008

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Characterization of the N-Acetyl-5-neuraminic Acid-binding Site of the Extracytoplasmic Solute Receptor (SiaP) of Nontypeable Haemophilus influenzae Strain 2019^*

Jason W. Johnston¹, Nathan P. Coussens¹², Simon Allen^¶1, Jon C. D. Houtman, Keith H. Turner, Anthony Zaleski, S. Ramaswamy, Bradford W. Gibson^¶, and Michael A. Apicella³

From the Department of Microbiology and Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 and ^¶The Buck Institute for Age Research, Novato, California 94945

Nontypeable Haemophilus influenzae is an opportunistic human pathogen causing otitis media in children and chronic bronchitis and pneumonia in patients with chronic obstructive pulmonary disease. The outer membrane of nontypeable H. influenzae is dominated by lipooligosaccharides (LOS), many of which incorporate sialic acid as a terminal nonreducing sugar. Sialic acid has been demonstrated to be an important factor in the survival of the bacteria within the host environment. H. influenzae is incapable of synthesizing sialic acid and is dependent on scavenging free sialic acid from the host environment. To achieve this, H. influenzae utilizes a tripartite ATP-independent periplasmic transporter. In this study, we characterize the binding site of the extracytoplasmic solute receptor (SiaP) from nontypeable H. influenzae strain 2019. A crystal structure of N-acetyl-5-neuraminic acid (Neu5Ac)-bound SiaP was determined to 1.4Å resolution. Thermodynamic characterization of Neu5Ac binding shows this interaction is enthalpically driven with a substantial unfavorable contribution from entropy. This is expected because the binding of SiaP to Neu5Ac is mediated by numerous hydrogen bonds and has several buried water molecules. Point mutations targeting specific amino acids were introduced in the putative binding site. Complementation with the mutated siaP constructs resulted either in full, partial, or no complementation, depending on the role of specific residues. Mass spectrometry analysis of the O-deacylated LOS of the R127K point mutation confirmed the observation of reduced incorporation of Neu5Ac into the LOS. The decreased ability of H. influenzae to import sialic acid had negative effects on resistance to complement-mediated killing and viability of biofilms in vitro, confirming the importance of sialic acid transport to the bacterium.

Received for publication, August 9, 2007 , and in revised form, October 15, 2007.

^* This work was supported in part by NIAID Grants AI024616 and AI30040 from the National Institutes of Health. 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 atomic coordinates and structure factors (code 3b50) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1 and S2.

¹ These authors contributed equally to this work.

² Recipeint of support from the National Institutes of Health Interdisciplinary Immunology Postdoctoral Training Program Grant NRSA AI07260-21.

³ To whom correspondence should be addressed: 51 Newton Rd., IA City, IA 52242. Tel.: 319-335-7807; Fax: 319-335-9006; E-mail: michael-apicella{at}uiowa.edu.

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