The Neisseria lipooligosaccharide-specific alpha-2,3-sialyltransferase is a surface-exposed outer membrane protein

Abstract

Neisseria gonorrhoeae and Neisseria meningitidis express an approximately 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 MC58 not subset 3 (an acapsulate serogroup B strain) performed with rabbit antiserum raised against recombinant Lst revealed an approximately 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 MC58 not subset 3 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.

FIG. 1.

SDS-PAGE and Western blot analyses of subcellular fractions of N. gonorrhoeae strains F62 and ST01 and N. meningitidis strain MC58⊄3. (A, left panel) Whole-cell lysates (WC), culture supernatants (Cult Sup), isolated outer membranes (OM), isolated periplasm (Peri), isolated inner membranes (IM), and isolated cytoplasm (Cyto) from N. gonorrhoeae strains F62 and ST01 were separated in an SDS-12.5% PAGE gel and stained with CBB. Lanes Cont Cult Sup contained sterile growth medium. Lanes MW contained molecular mass markers. (A, right panel) Western blot of the SDS-PAGE gel shown in the left panel probed with anti-cLst antiserum, as described in Materials and Methods. The position of the 43-kDa Lst is indicated by an arrow. (B) Whole-cell (WC), cytoplasmic, inner membrane (IM), periplasmic, and outer membrane (OM) fractions of N. meningitidis MC58⊄3 were separated in an SDS-12% PAGE gel and transferred to a polyvinylidene difluoride membrane. (Right panel) Polyvinylidene difluoride membrane probed with anti-cLst, preadsorbed with a lysate of E. coli DH5α. (Left panel) Blot reblocked with 0.02% Tween and stained with India ink. Lane MW contained prestained markers. The position of Lst at ∼43 kDa is indicated.

FIG. 2.

Inhibition of rLst activity by HA7 anti-rLst antiserum. rLst was incubated with different dilutions of HA7 antiserum and tested for sialyltransferase activity as described in Materials and Methods. The positive control was Lst mixed with assay buffer. Sialyltransferase activity was also measured after incubation with normal rabbit antiserum (NRS) isolated from rabbits prior to immunization. The data are representative of the results of two experiments.

FIG. 3.

Affinity purification of native rLst by HA7 anti-rLst antiserum. rLst was applied to a protein A column containing bound antibodies from HA7 antiserum or normal rabbit serum (NRS). The column was washed, and rLst was dissociated from the column with a three-step elution procedure as described in Materials and Methods. The wash and three elution fractions were tested for Lst sialyltransferase activity (left panel) and by Western analysis for rLst protein (right panel) as described in Materials and Methods. (Right panel) Lane 1, rLst; lane 2, NRS column, wash; lane 3, NRS column, elution 1; lane 4, NRS column, elution 2; lane 5, NRS column, elution 3; lane 6, HA7 column, wash; lane 7, HA7 column, elution 1; lane 8, HA7 column, elution 2; lane 9, HA7 column, elution 3. The positions of Lst and molecular mass markers are indicated on the left of the Western blot.

FIG. 4.

HA7 anti-Lst antiserum binding to whole cells. (A) Different concentrations of N. gonorrhoeae F62 or ST01 (lst⁻) and N. meningitidis MC58⊄3 were mixed with HA7 antiserum at a final dilution of 1:2,500 and washed, and the amount of antibody bound was detected by incubation with protein A-alkaline phosphatase as described in Materials and Methods. (B) Bacteria (5 × 10⁷ cells) were mixed with different dilutions of HA7 antiserum and examined for bound antibodies as described above. The data are representative of the results of two experiments.