Previous Article | Next Article 
Infection and Immunity, December 2002, p. 7161-7164, Vol. 70, No. 12
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.12.7161-7164.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Neuraminidase Expressed by Streptococcus pneumoniae Desialylates the Lipopolysaccharide of Neisseria meningitidis and Haemophilus influenzae: a Paradigm for Interbacterial Competition among Pathogens of the Human Respiratory Tract
Elizabeth A. Shakhnovich, Samantha J. King, and Jeffrey N. Weiser*
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Received 21 May 2002/
Returned for modification 9 July 2002/
Accepted 28 August 2002

ABSTRACT
Both
Neisseria meningitidis and
Haemophilus influenzae are capable
of mimicking host structures by decorating their lipopolysaccharides
with sialic acid. We show that a neuraminidase expressed by
Streptococcus pneumoniae (NanA) is able to desialylate the cell
surfaces of both these species, which reside in and possibly
compete for the same host niche.

TEXT
Several bacterial species mimic host structures by sialylation
of cell surface components (
6). Among the major pathogens originating
in the human respiratory tract, both
Neisseria meningitidis and some isolates of
Haemophilus influenzae express a sialyltransferase
that adds sialic acid

-2,3 linked to galactose as a terminal
structure on their lipopolysaccharide (LPS) (
5,
7). The addition
of sialic acid promotes survival by decreasing the bactericidal
effect of complement through interaction with factor H (
15).
For
N. meningitidis, sialic acid is obtained from cytidine monophospho-
N-acetylneuraminic
acid (CMP-NANA), which only some strains are able to synthesize
(
9). For
H. influenzae, sialic acid is obtained from environmental
sources of 5-acetylneuraminic acid (Neu5Ac) (
8).
Streptococcus pneumoniae (the pneumococcus), which is also a common member of the flora of the human upper respiratory tract, has been shown to cleave sialic acid-containing substrates with
-2,3 and
-2,6 linkages to galactose as well as those with
-2,6 linkages to N-acetylgalactosamine (16). The pneumococcus expresses several distinct neuraminidases, including NanA and NanB (1, 2). In some strains, there is also a nanB homolog, nanC, the expression and activity of which have not yet been described. It has been suggested that neuraminidase activity promotes colonization by exposing host cell receptors otherwise covered by sialic acid (19). In this study, we test the hypothesis that an additional target of pneumococcal neuraminidase is sialic acid attached to the cell surface of other members of the nasopharyngeal flora.
N. meningitidis strain N3 or nontypeable H. influenzae strain H122 was grown in the presence or absence of CMP-NANA or Neu5Ac, respectively (Table 1.) Western analysis revealed that growth in the presence of a source of sialic acid corresponded with the loss of the monoclonal antibody (MAb) 3F11 epitope, recognizing lacto-N-neotetraose, a terminal LPS structure to which sialic acid is added in both species (Fig. 1 and 2) (9, 10). The loss of this epitope was associated with the presence of a higher-molecular-weight band in proteinase K-treated lysates in silver-stained Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Treatment of N. meningitidis (up to 2 x 108 CFU) or H. influenzae (2 x 106 CFU) with purified neuraminidase obtained from Clostridium perfringens (50 mU/ml) (Sigma-Aldrich Co., St. Louis, Mo.) resulted in expression of the MAb 3F11 epitope and loss of the higher-molecular-weight band, confirming that the differences in the LPS were caused by sialylation.
The effect of the pneumococcus in vitro was tested by incubation
of
N. meningitidis or
H. influenzae under conditions allowing
for LPS sialylation with culture supernatants of
S. pneumoniae grown to the mid-log phase in C+Y medium (
17). Incubation of
N3 or H122 for 30 min at 37°C with the supernatant fraction
of growth medium from pneumococcal strain P2 or P394 (10
8 CFU/ml)
resulted in loss of sialylation (Fig.
1 and
2). No effect was
seen in control supernatants containing the C+Y growth medium
alone. The addition of CMP-NANA (50 µg/ml) during incubation
of N3 with the pneumococcal supernatants had no effect on desialylation,
suggesting that under these conditions, the activity of the
neuraminidase was more efficient than that of the meningococcal
sialyltransferase (data not shown). The ability to desialylate
the LPS of N3 and H122 was noted in culture supernatant from
strain P1252, but not that from P1247 or P1253, indicating that
nanA is required for this activity (Fig.
1 and
2). The lack
of activity in P1247 cells or culture supernatant, even when
tested after growth to the stationary phase, when NanB expression
is optimal, suggests that
nanB does not contribute to the desialylation
of the LPS (data not shown) (
3). The neuraminidase activities
of strains P2 (cell fraction) and P394 (culture supernatant
fraction) were quantified by comparison to that of purified
C. perfringens neuraminidase in serial dilutions (Fig.
3.) The
results demonstrate that 5 mU of neuraminidase activity was
sufficient for complete desialylation of 2
x 10
8 meningococci.
The neuraminidase activity for strain P394 was estimated at
12 mU per supernatant fraction for 10
6 cells and 0.3 mU/10
6 cells for the cell fraction of strain P2. Thus, we estimate
that the supernatant derived from one P394 cell is sufficient
to desialylate about 1,000 meningococci under these conditions.
In contrast, the activity of one P2 cell was sufficient to desialylate
only about 25 meningococci. P394 contains a frameshift upstream
of the C-terminal cell wall LPXTGX-anchoring motif in
nanA,
explaining why >95% of the enzyme activity was found in the
culture supernatant rather than the cell fraction. Since the
assay conditions would predict more efficient access of the
enzyme to its target in the secreted form, the secretion of
NanA in P394 could account for its higher level of activity.
For P2, the neuraminidase activity was divided between the culture
supernatant (28% of activity) and cell fractions (72% of activity),
indicating partial release of the enzyme (data not shown).
We have previously shown that high concentrations of hydrogen
peroxide produced by the aerobic metabolism of the pneumococcus
may be inhibitory or bactericidal in vitro to other species
that reside in the same environment, including
H. influenzae and
N. meningitidis (
14). The findings in this study describe
a second mechanism whereby
S. pneumoniae could interfere with
the biology of potential competitors. In the case of desialylation
of the cell surfaces of other members of the microflora, the
pneumococcus appears to be specifically targeting a mechanism
involving bacterial adaptation to its host. It remains to be
determined whether interspecies competition occurs in the heavily
colonized human upper respiratory tract in which each of the
three species examined here resides. In this regard, several
previous reports suggest that the pneumococcus may have inhibitory
effects on
H. influenzae in the natural host. During exacerbations
of chronic bronchitis,
H. influenzae was isolated less frequently
during periods when the pneumococcus was present compared to
periods when it was absent (
11). Recent results from a randomized
double-blind trial of the pneumococcal conjugate vaccine showed
that a decrease in the incidence of carriage and otitis media
caused by pneumococcal types in the vaccine was associated with
an 11% increase in disease due to
H. influenzae in the vaccine
group (
4). Disease caused by the meningococcus is less common,
and we are not aware of a similar inverse association with the
pneumococcus having been reported. Clinical observations about
S. pneumoniae and
H. influenzae, however, point out the need
to understand the potential interactions of microorganisms,
since manipulation of the human microflora may lead to unanticipated
problems.

ACKNOWLEDGMENTS
We thank R. Rest for guidance with using the meningococcus,
M. Apicella for supplying MAb 3F11, and T. DeMaria, C. Dowson,
and A. Whatmore for providing strains.
This work was supported by grants from the Public Heath Service (AI38436 and AI44231).

FOOTNOTES
* Corresponding author. Mailing address: 402A Johnson Pavilion, Department of Microbiology, University of Pennsylvania, Philadelphia, PA 19104-6076. Phone: (215) 573-3511. Fax: (215) 898-9557. E-mail:
weiser{at}mail.med.upenn.edu.

Editor: V. J. DiRita

REFERENCES
1 - Berry, A. M., R. A. Lock, and J. C. Paton. 1996. Cloning and characterization of nanB, a second Streptococcus pneumoniae neuraminidase gene, and purification of the NanB enzyme from recombinant Escherichia coli. J. Bacteriol. 178:4854-4860.[Abstract/Free Full Text]
2 - Cámara, M., G. J. Boulnois, P. W. Andrew, and T. J. Mitchell. 1994. A neuraminidase from Streptococcus pneumoniae has the features of a surface protein. Infect. Immun. 62:3688-3695.[Abstract/Free Full Text]
3 - de Saizieu, A., U. Certa, J. Warrington, C. Gray, W. Keck, and J. Mous. 1998. Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays. Nat. Biotechnol. 16:45-48.[Medline]
4 - Eskola, J., T. Kilpi, A. Palmu, J. Jokinen et al. 2001. Efficacy of a pneumococcal conjugate vaccine against acute otitis media. N. Engl. J. Med. 344:403-409.[Abstract/Free Full Text]
5 - Gilbert, M., D. Watson, A. Cunningham, M. Jennings, N. Young, and W. Wakarchuk. 1996. Cloning of the lipooligosaccharide alpha-2,3-sialyltransferase from the bacterial pathogens Neisseria meningitidis and Neisseria gonorrhoeae. J. Biol. Chem. 271:28271-28276.[Abstract/Free Full Text]
6 - Harvey, H., W. Swords, and M. Apicella. 2001. The mimicry of human glycolipids and glycosphingolipids by the lipooligosaccharides of pathogenic Neisseria and Haemophilus. J. Autoimmun. 16:257-262.[CrossRef][Medline]
7 - Hood, D., A. Cox, M. Gilbert, K. Makepeace, S. Walsh, M. Deadman, A. Cody, A. Martin, M. Mansson, E. Schweda, J. Brisson, J. Richards, E. Moxon, and W. Wakarchuk. 2001. Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae. Mol. Microbiol. 39:341-350.[CrossRef][Medline]
8 - Hood, D., K. Makepeace, M. Deadman, R. Rest, P. Thibault, A. Martin, J. Richards, and E. Moxon. 1999. Sialic acid in the lipopolysaccharide of Haemophilus influenzae: strain distribution, influence on serum resistance and structural characterization. Mol. Microbiol. 33:679-692.[CrossRef][Medline]
9 - Mandrell, R. E., J. J. Kim, C. M. John, B. W. Gibson, J. V. Sugai, M. A. Apicella, J. M. Griffiss, and R. Yamasaki. 1991. Endogenous sialylation of the lipooligosaccharides of Neisseria meningitidis. J. Bacteriol. 173:2823-2832.[Abstract/Free Full Text]
10 - Mandrell, R. E., R. McLaughlin, Y. A. Kwaik, A. Lesse, R. Yamasaki, B. Gibson, S. M. Spinola, and M. A. Apicella. 1992. Lipooligosaccharides (LOS) of some Haemophilus species mimic human glycosphingolipids, and some LOS are sialylated. Infect. Immun. 60:1322-1328.[Abstract/Free Full Text]
11 - May, R. J. 1954. Pathogenic bacteria in chronic bronchitis. Lancet ii:839-842.
12 - McGuinness, B., I. Clarke, P. Lambden, A. Barlow, J. Poolman, D. Jones, and J. Heckels. 1991. Point mutation in meningococcal porA gene associated with increased endemic disease. Lancet 337:514-517.[CrossRef][Medline]
13 - Morse, S., and L. Bartenstein. 1980. Purine metabolism in Neisseria gonorrhoeae: the requirement for hypoxanthine. Can. J. Microbiol. 26:13-20.[Medline]
14 - Pericone, C. D., K. Overweg, P. W. M. Herman, and J. N. Weiser. 2000. Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract. Infect. Immun. 68:3990-3997.[Abstract/Free Full Text]
15 - Ram, S., A. K. Sharma, S. D. Simpson, S. Gulati, D. P. McQuillen, M. K. Pangburn, and P. A. Rice. 1998. A novel sialic acid binding site on factor H mediates serum resistance of sialylated Neisseria gonorrhoeae. J. Exp. Med. 187:743-752.[Abstract/Free Full Text]
16 - Scanlon, K., W. Diren, and R. Glew. 1989. Purification and properties of Streptococcus pneumoniae neuraminidase. Enzyme 41:143-150.[Medline]
17 - Tomasz, A. 1964. A chemically defined medium for Streptococcus pneumoniae. Bacteriol. Proc. 64:29.
18 - Tong, H. H., L. E. Blue, M. A. James, and T. F. DeMaria. 2000. Evaluation of the virulence of a Streptococcus pneumoniae neuraminidase-deficient mutant in nasopharyngeal colonization and development of otitis media in the chinchilla model. Infect. Immun. 68:921-924.[Abstract/Free Full Text]
19 - Tong, H. H., M. James, I. Grants, X. Liu, G. Shi, and T. F. DeMaria. 2001. Comparison of structural changes of cell surface carbohydrates in the eustachian tube epithelium of chinchillas infected with a Streptococcus pneumoniae neuraminidase-deficient mutant or its isogenic parent strain. Microb. Pathog. 31:309-317.[CrossRef][Medline]
20 - Weiser, J. N., Z. Markiewicz, E. I. Tuomanen, and J. H. Wani. 1996. Relationship between phase variation in colony morphology, intrastrain variation in cell wall physiology, and nasopharyngeal colonization by Streptococcus pneumoniae. Infect. Immun. 64:2240-2245.[Abstract]
Infection and Immunity, December 2002, p. 7161-7164, Vol. 70, No. 12
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.12.7161-7164.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
This article has been cited by other articles:
-
Uchiyama, S., Carlin, A. F., Khosravi, A., Weiman, S., Banerjee, A., Quach, D., Hightower, G., Mitchell, T. J., Doran, K. S., Nizet, V.
(2009). The surface-anchored NanA protein promotes pneumococcal brain endothelial cell invasion. JEM
206: 1845-1852
[Abstract]
[Full Text]
-
Burnaugh, A. M., Frantz, L. J., King, S. J.
(2008). Growth of Streptococcus pneumoniae on Human Glycoconjugates Is Dependent upon the Sequential Activity of Bacterial Exoglycosidases. J. Bacteriol.
190: 221-230
[Abstract]
[Full Text]
-
Severi, E., Hood, D. W., Thomas, G. H.
(2007). Sialic acid utilization by bacterial pathogens. Microbiology
153: 2817-2822
[Abstract]
[Full Text]
-
Figueira, M. A., Ram, S., Goldstein, R., Hood, D. W., Moxon, E. R., Pelton, S. I.
(2007). Role of Complement in Defense of the Middle Ear Revealed by Restoring the Virulence of Nontypeable Haemophilus influenzae siaB Mutants. Infect. Immun.
75: 325-333
[Abstract]
[Full Text]
-
Marri, P. R., Hao, W., Golding, G. B.
(2006). Gene Gain and Gene Loss in Streptococcus: Is It Driven by Habitat?. Mol Biol Evol
23: 2379-2391
[Abstract]
[Full Text]
-
Simell, B., Jaakkola, T., Lahdenkari, M., Briles, D., Hollingshead, S., Kilpi, T. M., Kayhty, H.
(2006). Serum Antibodies to Pneumococcal Neuraminidase NanA in Relation to Pneumococcal Carriage and Acute Otitis Media. CVI
13: 1177-1179
[Abstract]
[Full Text]
-
Pettigrew, M. M., Fennie, K. P., York, M. P., Daniels, J., Ghaffar, F.
(2006). Variation in the Presence of Neuraminidase Genes among Streptococcus pneumoniae Isolates with Identical Sequence Types.. Infect. Immun.
74: 3360-3365
[Abstract]
[Full Text]
-
Paterson, G. K., Mitchell, T. J.
(2006). Innate immunity and the pneumococcus. Microbiology
152: 285-293
[Abstract]
[Full Text]
-
Bergmann, S., Hammerschmidt, S.
(2006). Versatility of pneumococcal surface proteins. Microbiology
152: 295-303
[Abstract]
[Full Text]
-
LeMessurier, K. S., Ogunniyi, A. D., Paton, J. C.
(2006). Differential expression of key pneumococcal virulence genes in vivo. Microbiology
152: 305-311
[Abstract]
[Full Text]
-
King, S. J., Whatmore, A. M., Dowson, C. G.
(2005). NanA, a Neuraminidase from Streptococcus pneumoniae, Shows High Levels of Sequence Diversity, at Least in Part through Recombination with Streptococcus oralis. J. Bacteriol.
187: 5376-5386
[Abstract]
[Full Text]
-
Shah, D. S. H., Russell, R. R. B.
(2004). A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans. Microbiology
150: 1947-1956
[Abstract]
[Full Text]
-
Vimr, E. R., Kalivoda, K. A., Deszo, E. L., Steenbergen, S. M.
(2004). Diversity of Microbial Sialic Acid Metabolism. Microbiol. Mol. Biol. Rev.
68: 132-153
[Abstract]
[Full Text]
-
Swords, W. E., Moore, M. L., Godzicki, L., Bukofzer, G., Mitten, M. J., VonCannon, J.
(2004). Sialylation of Lipooligosaccharides Promotes Biofilm Formation by Nontypeable Haemophilus influenzae. Infect. Immun.
72: 106-113
[Abstract]
[Full Text]
-
Swords, W. E., Jones, P. A., Apicella, M. A.
(2003). Review: The lipo-oligosaccharides of Haemophilus influenzae: an interesting array of characters. Innate Immunity
9: 131-144
[Abstract]