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J. Biol. Chem., Vol. 282, Issue 38, 27562-27571, September 21, 2007

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NeuA Sialic Acid O-Acetylesterase Activity Modulates O-Acetylation of Capsular Polysaccharide in Group B Streptococcus^*

Amanda L. Lewis, Hongzhi Cao^¶, Silpa K. Patel, Sandra Diaz^||**, Wesley Ryan^¶, Aaron F. Carlin, Vireak Thon^¶, Warren G. Lewis, Ajit Varki^||**1, Xi Chen^¶, and Victor Nizet^¶¶

From the Glycobiology Research and Training Center, Departments of Pediatrics, ^||Medicine, and ^**Cellular and Molecular Medicine, School of Medicine, and the ^¶¶Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California, the Scripps Research Institute, Biochemistry Department, La Jolla, California 92037, the Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, and the ^¶Department of Chemistry, University of California, Davis, California 95616

Group B Streptococcus (GBS) is a common cause of neonatal sepsis and meningitis. A major GBS virulence determinant is its sialic acid (Sia)-capped capsular polysaccharide. Recently, we discovered the presence and genetic basis of capsular Sia O-acetylation in GBS. We now characterize a GBS Sia O-acetylesterase that modulates the degree of GBS surface O-acetylation. The GBS Sia O-acetylesterase operates cooperatively with the GBS CMP-Sia synthetase, both part of a single polypeptide encoded by the neuA gene. NeuA de-O-acetylation of free 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac₂) was enhanced by CTP and Mg²⁺, the substrate and co-factor, respectively, of the N-terminal GBS CMP-Sia synthetase domain. In contrast, the homologous bifunctional NeuA esterase from Escherichia coli K1 did not display cofactor dependence. Further analyses showed that in vitro, GBS NeuA can operate via two alternate enzymatic pathways: de-O-acetylation of Neu5,9Ac₂ followed by CMP activation of Neu5Ac or activation of Neu5,9Ac₂ followed by de-O-acetylation of CMP-Neu5,9Ac₂. Consistent with in vitro esterase assays, genetic deletion of GBS neuA led to accumulation of intracellular O-acetylated Sias, and overexpression of GBS NeuA reduced O-acetylation of Sias on the bacterial surface. Site-directed mutagenesis of conserved asparagine residue 301 abolished esterase activity but preserved CMP-Sia synthetase activity, as evidenced by hyper-O-acetylation of capsular polysaccharide Sias on GBS expressing only the N301A NeuA allele. These studies demonstrate a novel mechanism regulating the extent of capsular Sia O-acetylation in intact bacteria and provide a genetic strategy for manipulating GBS O-acetylation in order to explore the role of this modification in GBS pathogenesis and immunogenicity.

Received for publication, January 12, 2007 , and in revised form, July 5, 2007.

^* This work was supported by National Institutes of Health Grants P01-HL57345 and GM32373 (to A. V.), R01-HD051796 (to V. N.), and R01-GM076360 (to X. C.); a Gianinni Family Foundation postdoctoral fellowship award (to A. L.); and an American Heart Association established investigator award (to V. N.). 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.

¹ To whom correspondence should be addressed: University of California San Diego School of Medicine, La Jolla, CA 92093-0687. Tel.: 858-534-2214; Fax: 858-534-5611; E-mail: a1varki{at}ucsd.edu.

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