Abstract
The type III capsular polysaccharide of group B streptococci (GBS) consists of a linear backbone with short side chains ending in residues of N-acetylneuraminic acid, or sialic acid. The presence of sialic acid on the surface of the organism inhibits activation of the alternative pathway of complement and is thought to be an important element in the virulence function of the capsule. We showed previously that a mutant strain of GBS that expressed a sialic acid-deficient, or asialo, form of the type III polysaccharide was avirulent, supporting a virulence function for capsular sialic acid. We now report the derivation of an asialo capsule mutant from a highly encapsulated wild-type strain of type III GBS, strain COH1, by insertional mutagenesis with transposon Tn916 delta E. In contrast to the wild-type strain, the asialo mutant strain COH1-11 was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat model of GBS infection. The asialo mutant accumulated free intracellular sialic acid, suggesting a defect subsequent to sialic acid synthesis in the biosynthetic pathway leading to capsule sialylation. The specific biosynthetic defect in mutant strain COH1-11 was found to be in the activation of free sialic acid to CMP-sialic acid: CMP-sialic acid synthetase activity was present in the wild-type strain COH1 but was not detected in the asialo mutant strain COH1-11. One of the two transposon insertions in the asialo mutant COH1-11 mapped to the same chromosomal location as one of the two Tn916 insertions in the previously reported asialo mutant COH31-21, identifying this site as a genetic locus necessary for expression of CMP-sialic acid synthetase activity. These studies demonstrate that the enzymatic synthesis of CMP-sialic acid by GBS is an essential step in sialylation of the type III capsular polysaccharide.
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Selected References
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- Baker C. J., Barrett F. F. Group B streptococcal infections in infants. The importance of the various serotypes. JAMA. 1974 Nov 25;230(8):1158–1160. [PubMed] [Google Scholar]
- Baltimore R. S., Kasper D. L., Baker C. J., Goroff D. K. Antigenic specificity of opsonophagocytic antibodies in rabbit anti-sera to group B streptococci. J Immunol. 1977 Feb;118(2):673–678. [PubMed] [Google Scholar]
- Boulnois G. J., Roberts I. S., Hodge R., Hardy K. R., Jann K. B., Timmis K. N. Analysis of the K1 capsule biosynthesis genes of Escherichia coli: definition of three functional regions for capsule production. Mol Gen Genet. 1987 Jun;208(1-2):242–246. doi: 10.1007/BF00330449. [DOI] [PubMed] [Google Scholar]
- Boyer K. M., Gadzala C. A., Burd L. I., Fisher D. E., Paton J. B., Gotoff S. P. Selective intrapartum chemoprophylaxis of neonatal group B streptococcal early-onset disease. I. Epidemiologic rationale. J Infect Dis. 1983 Nov;148(5):795–801. doi: 10.1093/infdis/148.5.795. [DOI] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Dillon H. C., Jr, Khare S., Gray B. M. Group B streptococcal carriage and disease: a 6-year prospective study. J Pediatr. 1987 Jan;110(1):31–36. doi: 10.1016/s0022-3476(87)80283-4. [DOI] [PubMed] [Google Scholar]
- Edwards M. S., Kasper D. L., Jennings H. J., Baker C. J., Nicholson-Weller A. Capsular sialic acid prevents activation of the alternative complement pathway by type III, group B streptococci. J Immunol. 1982 Mar;128(3):1278–1283. [PubMed] [Google Scholar]
- Edwards M. S., Nicholson-Weller A., Baker C. J., Kasper D. L. The role of specific antibody in alternative complement pathway-mediated opsonophagocytosis of type III, group B Streptococcus. J Exp Med. 1980 May 1;151(5):1275–1287. doi: 10.1084/jem.151.5.1275. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fearon D. T. Regulation by membrane sialic acid of beta1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1971–1975. doi: 10.1073/pnas.75.4.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gray B. M. ELISA methodology for polysaccharide antigens: protein coupling of polysaccharides for adsorption to plastic tubes. J Immunol Methods. 1979;28(1-2):187–192. doi: 10.1016/0022-1759(79)90340-5. [DOI] [PubMed] [Google Scholar]
- Hoiseth S. K., Moxon E. R., Silver R. P. Genes involved in Haemophilus influenzae type b capsule expression are part of an 18-kilobase tandem duplication. Proc Natl Acad Sci U S A. 1986 Feb;83(4):1106–1110. doi: 10.1073/pnas.83.4.1106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jennings H. J., Lugowski C., Kasper D. L. Conformational aspects critical to the immunospecificity of the type III group B streptococcal polysaccharide. Biochemistry. 1981 Aug 4;20(16):4511–4518. doi: 10.1021/bi00519a001. [DOI] [PubMed] [Google Scholar]
- Kasper D. L., Baker C. J., Baltimore R. S., Crabb J. H., Schiffman G., Jennings H. J. Immunodeterminant specificity of human immunity to type III group B streptococcus. J Exp Med. 1979 Feb 1;149(2):327–339. doi: 10.1084/jem.149.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Klegerman M. E., Boyer K. M., Papierniak C. K., Levine L., Gotoff S. P. Type-specific capsular antigen is associated with virulence in late-onset group B Streptococcal type III disease. Infect Immun. 1984 Apr;44(1):124–129. doi: 10.1128/iai.44.1.124-129.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kroll J. S., Zamze S., Loynds B., Moxon E. R. Common organization of chromosomal loci for production of different capsular polysaccharides in Haemophilus influenzae. J Bacteriol. 1989 Jun;171(6):3343–3347. doi: 10.1128/jb.171.6.3343-3347.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kuypers J. M., Heggen L. M., Rubens C. E. Molecular analysis of a region of the group B streptococcus chromosome involved in type III capsule expression. Infect Immun. 1989 Oct;57(10):3058–3065. doi: 10.1128/iai.57.10.3058-3065.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Martin T. R., Rubens C. E., Wilson C. B. Lung antibacterial defense mechanisms in infant and adult rats: implications for the pathogenesis of group B streptococcal infections in the neonatal lung. J Infect Dis. 1988 Jan;157(1):91–100. doi: 10.1093/infdis/157.1.91. [DOI] [PubMed] [Google Scholar]
- OUCHTERLONY O. Diffusion-in-gel methods for immunological analysis. Prog Allergy. 1958;5:1–78. [PubMed] [Google Scholar]
- Pangburn M. K., Müller-Eberhard H. J. Complement C3 convertase: cell surface restriction of beta1H control and generation of restriction on neuraminidase-treated cells. Proc Natl Acad Sci U S A. 1978 May;75(5):2416–2420. doi: 10.1073/pnas.75.5.2416. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pozsgay V., Jennings H., Kasper D. L. 4,8-anhydro-N-acetylneuraminic acid. Isolation from edible bird's nest and structure determination. Eur J Biochem. 1987 Jan 15;162(2):445–450. doi: 10.1111/j.1432-1033.1987.tb10622.x. [DOI] [PubMed] [Google Scholar]
- Rohr T. E., Troy F. A. Structure and biosynthesis of surface polymers containing polysialic acid in Escherichia coli. J Biol Chem. 1980 Mar 25;255(6):2332–2342. [PubMed] [Google Scholar]
- Rubens C. E., Heggen L. M. Tn916 delta E: a Tn916 transposon derivative expressing erythromycin resistance. Plasmid. 1988 Sep;20(2):137–142. doi: 10.1016/0147-619x(88)90016-9. [DOI] [PubMed] [Google Scholar]
- Rubens C. E., Wessels M. R., Heggen L. M., Kasper D. L. Transposon mutagenesis of type III group B Streptococcus: correlation of capsule expression with virulence. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7208–7212. doi: 10.1073/pnas.84.20.7208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shigeoka A. O., Pincus S. H., Rote N. S., Hill H. R. Protective efficacy of hybridoma type-specific antibody against experimental infection with group-B Streptococcus. J Infect Dis. 1984 Mar;149(3):363–372. doi: 10.1093/infdis/149.3.363. [DOI] [PubMed] [Google Scholar]
- Silver R. P., Finn C. W., Vann W. F., Aaronson W., Schneerson R., Kretschmer P. J., Garon C. F. Molecular cloning of the K1 capsular polysaccharide genes of E. coli. Nature. 1981 Feb 19;289(5799):696–698. doi: 10.1038/289696b0. [DOI] [PubMed] [Google Scholar]
- Silver R. P., Vann W. F., Aaronson W. Genetic and molecular analyses of Escherichia coli K1 antigen genes. J Bacteriol. 1984 Feb;157(2):568–575. doi: 10.1128/jb.157.2.568-575.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Troy F. A., 2nd The chemistry and biosynthesis of selected bacterial capsular polymers. Annu Rev Microbiol. 1979;33:519–560. doi: 10.1146/annurev.mi.33.100179.002511. [DOI] [PubMed] [Google Scholar]
- Van Dijk W. C., Verbrugh H. A., van der Tol M. E., Peters R., Verhoef J. Role of Escherichia coli K capsular antigens during complement activation, C3 fixation, and opsonization. Infect Immun. 1979 Aug;25(2):603–609. doi: 10.1128/iai.25.2.603-609.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vann W. F., Silver R. P., Abeijon C., Chang K., Aaronson W., Sutton A., Finn C. W., Lindner W., Kotsatos M. Purification, properties, and genetic location of Escherichia coli cytidine 5'-monophosphate N-acetylneuraminic acid synthetase. J Biol Chem. 1987 Dec 25;262(36):17556–17562. [PubMed] [Google Scholar]
- Vimr E. R., Aaronson W., Silver R. P. Genetic analysis of chromosomal mutations in the polysialic acid gene cluster of Escherichia coli K1. J Bacteriol. 1989 Feb;171(2):1106–1117. doi: 10.1128/jb.171.2.1106-1117.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- WARREN L. The thiobarbituric acid assay of sialic acids. J Biol Chem. 1959 Aug;234(8):1971–1975. [PubMed] [Google Scholar]
- Wessels M. R., Benedí W. J., Jennings H. J., Michon F., DiFabio J. L., Kasper D. L. Isolation and characterization of type IV group B Streptococcus capsular polysaccharide. Infect Immun. 1989 Apr;57(4):1089–1094. doi: 10.1128/iai.57.4.1089-1094.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wessels M. R., Kasper D. L. Antibody recognition of the type 14 pneumococcal capsule. Evidence for a conformational epitope in a neutral polysaccharide. J Exp Med. 1989 Jun 1;169(6):2121–2131. doi: 10.1084/jem.169.6.2121. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wessels M. R., Paoletti L. C., Kasper D. L., DiFabio J. L., Michon F., Holme K., Jennings H. J. Immunogenicity in animals of a polysaccharide-protein conjugate vaccine against type III group B Streptococcus. J Clin Invest. 1990 Nov;86(5):1428–1433. doi: 10.1172/JCI114858. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wessels M. R., Pozsgay V., Kasper D. L., Jennings H. J. Structure and immunochemistry of an oligosaccharide repeating unit of the capsular polysaccharide of type III group B Streptococcus. A revised structure for the type III group B streptococcal polysaccharide antigen. J Biol Chem. 1987 Jun 15;262(17):8262–8267. [PubMed] [Google Scholar]
- Wessels M. R., Rubens C. E., Benedí V. J., Kasper D. L. Definition of a bacterial virulence factor: sialylation of the group B streptococcal capsule. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8983–8987. doi: 10.1073/pnas.86.22.8983. [DOI] [PMC free article] [PubMed] [Google Scholar]