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Infection and Immunity, July 2002, p. 3744-3751, Vol. 70, No. 7
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.7.3744-3751.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Neisseria Lipooligosaccharide-Specific {alpha}-2,3-Sialyltransferase Is a Surface-Exposed Outer Membrane Protein

Dawn M. Shell,1 Lisa Chiles,2 Ralph C. Judd,2 Samar Seal,1 and Richard F. Rest1*

Department of Microbiology and Immunology, MCP Hahnemann School of Medicine, Philadelphia, Pennsylvania 19129,1 Division of Biological Sciences, University of Montana, Missoula, Montana 59812-48242

Received 17 December 2001/ Returned for modification 24 January 2002/ Accepted 26 March 2002

Neisseria gonorrhoeae and Neisseria meningitidis express an ~43-kDa {alpha}-2,3-sialyltransferase (Lst) that sialylates the surface lipooligosaccharide (LOS) by using exogenous (in all N. gonorrhoeae strains and some N. meningitidis serogroups) or endogenous (in other N. meningitidis serogroups) sources of 5'-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA). Sialylation of LOS can protect N. gonorrhoeae and N. meningitidis from complement-mediated serum killing and from phagocytic killing by neutrophils. The precise subcellular location of Lst has not been determined. We confirm and extend previous studies by demonstrating that Lst is located in the outer membrane and is surface exposed in both N. gonorrhoeae and N. meningitidis. Western immunoblot analysis of subcellular fractions of N. gonorrhoeae strain F62 and N. meningitidis strain MC583 (an acapsulate serogroup B strain) performed with rabbit antiserum raised against recombinant Lst revealed an ~43-kDa protein exclusively in outer membrane preparations of both pathogens. Inner membrane, periplasmic, cytoplasmic, and culture supernatant fractions were devoid of Lst, as determined by Western blot analysis. Consistent with this finding, outer membrane fractions of N. gonorrhoeae were significantly enriched for sialyltransferase enzymatic activity. A trace of enzymatic activity was detected in inner membrane fractions, which may have represented Lst in transit to the outer membrane or may have represented inner membrane contamination of outer membrane preparations. Subcellular preparations of an isogenic lst insertion knockout mutant of N. gonorrhoeae F62 (strain ST01) expressed neither a 43-kDa immunoreactive protein nor sialyltransferase activity. Anti-Lst rabbit antiserum bound to whole cells of N. meningitidis MC583 and wild-type N. gonorrhoeae F62 but not to the Lst mutant ST01, indicating the surface exposure of the enzyme. Although the anti-Lst antiserum avidly bound enzymatically active, recombinant Lst, it inhibited Lst (sialyltransferase) activity by only about 50% at the highest concentration of antibody used. On the contrary, anti-Lst antiserum did not inhibit sialylation of whole N. gonorrhoeae cells in the presence of exogenous CMP-NANA, suggesting that the antibody did not bind to or could not access the enzyme active site on the surface of viable Neisseria cells. Taken together, these results indicate that Lst is an outer membrane, surface-exposed glycosyltransferase. To our knowledge, this is the first demonstration of the localization of a bacterial glycosyltransferase to the outer membrane of gram-negative bacteria.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, MCP Hahnemann School of Medicine, Philadelphia, PA 19129. Phone: (215) 991-8382. Fax: (215) 848-2271. E-mail: rickrest{at}drexel.edu.

Editor: D. L. Burns

Infection and Immunity, July 2002, p. 3744-3751, Vol. 70, No. 7
0019-9567/02/$04.00+0     DOI: 10.1128/IAI.70.7.3744-3751.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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