Identification of cpsD, a gene essential for type III capsule expression in group B streptococci.

We showed previously that a mutant strain of group B Streptococcus (GBS) defective in capsule production was avirulent. This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild-type strain of type III GBS, COH1, by transposon mutagenesis with Tn916 delta E. The mutant, COH1-13, was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat sepsis model compared with the wild-type strain. No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain. The Tn916 delta E insertion site in COH1-13 was mapped to the same chromosomal location as the Tn916 insertion site in the unencapsulated type III mutant COH31-15 reported previously. Nucleotide sequencing of DNA flanking the insertion site in COH1-13 revealed an open reading frame, designated cpsD, with significant homology to the rfbP gene of Salmonella typhimurium. RfbP encodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step in O-polysaccharide synthesis. A particulate fraction of a lysate of wild-type strain GBS COH1 mediated the transfer of galactose from UDP-galactose to an endogenous acceptor. The galactose-acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane-associated. Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1-13. These results, together with the similarity in deduced amino acid sequence between cpsD and rfbP suggest that cpsD encodes a galactosyl transferase essential for assembly of the GBS type III capsular polysaccharide.

Identification of a genetic locus essential for capsule sialylation in type III group B streptococci.

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.

Molecular analysis of a region of the group B streptococcus chromosome involved in type III capsule expression.

Type III group B streptococci (GBS) are the most common cause of neonatal sepsis and meningitis in the United States. The important role of the type III polysaccharide capsule and of the terminal sialic acid moiety of the capsule in the virulence of GBS has been demonstrated by using Tn916 mutagenesis. Several of the transposon insertion sites that resulted in defective type III capsule synthesis were located in a 30-kilobase (kb) region of the chromosome. Hybridization analysis of two other type III strains that differed in their relative virulence and of GBS serotypes Ia, Ib, Ic, and II showed that this region of the chromosome was highly conserved. A repetitive 1.4-kb sequence was found only in the 30-kb region of the more virulent type III strain, COH 1. The Escherichia coli maxicell in vivo expression system and an in vitro coupled transcription-translation system successfully identified the proteins expressed from the 30-kb region. Comparison of the proteins expressed from the same DNA fragments in these two assays indicated that some of these proteins may contain leader sequences that would ultimately result in their secretion to the cell surface. Identification and further characterization of the genes and their products will provide the foundation for understanding the genetic and biochemical events in GBS capsular polysaccharide production.

IS861, a group B streptococcal insertion sequence related to IS150 and IS3 of Escherichia coli.

A 1,442-base-pair (bp) insertion sequence (IS861) was identified in the type III group B streptococcal (GBS) strain COH-1. It is flanked by 26-bp imperfect inverted repeats and contains two open reading frames, 1 and 2, encoding 141- and 277-amino-acid proteins, respectively. A 3-bp target sequence, ACA, is duplicated and flanks each inverted repeat. IS861 shares greater than 30% homology with IS3 and IS150 of Escherichia coli, primarily in the region of their putative transposases. Northern (RNA) analysis revealed that RNA is actively transcribed in vivo by IS861 and 17- and 36-kilodalton proteins were synthesized in E. coli maxicell assays. Multiple copies of IS861 were observed throughout the chromosome of COH-1, and one of the copies is located near genes involved in GBS capsule synthesis. IS861 is the first insertion sequence identified in GBS. Its role in GBS and the significance of its relationship to the phylogenetically similar insertion sequences typified by IS150 and IS3 of E. coli are unknown.

Tn916 delta E: a Tn916 transposon derivative expressing erythromycin resistance.

The tetracycline resistance gene encoded within the transposon Tn916 was replaced with the gene encoding erythromycin resistance from the plasmid pVA838. The derivative transposon of Tn916 was designated Tn916 delta E and was introduced into the Streptococcus faecalis chromosome by protoplast transformation. The conjugation/transposition functions of Tn916 delta E were similar to those observed for Tn916 in S. faecalis and Tn916 delta E was capable of self-conjugation at frequencies similar to those of other S. faecalis and Group B Streptococcus. This transposon will be useful for mutagenesis studies in gram-positive organisms, especially in those species where erythromycin resistance is a more desirable selectable marker.

Transposon mutagenesis of type III group B Streptococcus: correlation of capsule expression with virulence.

The capsular polysaccharide of type III group B Streptococcus (GBS) is thought to be a major factor in the virulence of this organism. Transposon mutagenesis was used to obtain isogenic strains of a GBS serotype III clinical isolate (COH 31r/s) with site-specific mutations in the gene(s) responsible for capsule production. The self-conjugative transposon Tn916 was transferred to strain COH 31r/s during incubation with Streptococcus faecalis strain CG110 on membrane filters. Eleven transconjugant clones did not bind type III GBS antiserum by immunoblot. Immunofluorescence, competitive ELISA, and electron microscopy confirmed the absence of detectable GBS type III capsular polysaccharide in one of the transconjugants, COH 31-15. Southern hybridization analysis with a Tn916 probe confirmed the presence of the transposon sequence within each mutant. A 3.0-kilobase EcoRI fragment that flanked the Tn916 sequence was subcloned from mutant COH 31-15. This fragment shared homology with DNA from the other GBS serotypes, suggesting a common sequence for capsulation shared by organisms of different capsular types. Loss of capsule expression resulted in loss of virulence in a neonatal rat model. We conclude that a gene common to all capsular types of GBS is required for surface expression of the type III capsule and that inactivation of this gene by Tn916 results in the loss of virulence.

Pharmacokinetics of ciprofloxacin in cystic fibrosis.

We studied the pharmacokinetics of ciprofloxacin in 12 adult males with and 12 adult males without cystic fibrosis (CF). In a randomized crossover sequence, the subjects received 200 mg intravenously or 750 mg orally. With intravenous dosing, subjects also received 651 mg of iothalamate, a marker of glomerular filtration, and 700 mg of antipyrine, an indicator of hepatic oxidative drug metabolism. Pharmacokinetic parameters were determined by model independent methods. In the CF subjects, the ciprofloxacin concentration in serum during the first hour after intravenous administration was higher, and the oral absorption rate was slower. Other parameters did not differ between the groups. Mean concentrations in serum 5 min postinfusion were 3.08 and 2.14 micrograms/ml, and mean peak concentrations after oral dosing were 3.24 and 3.34 micrograms/ml in subjects with and without CF, respectively. Mean values for elimination half-life in all subjects were 4.8 and 5.0 h after intravenous and oral administration, respectively. The mean renal clearances in all subjects after intravenous and oral administration were 19.4 and 14.5 liters/h and accounted for 64 and 47% of the total clearance, respectively. These values were significantly greater than renal iothalamate clearance, indicating that tubular secretion contributed to the renal clearance of ciprofloxacin. A total of 69 and 35.4% of the administered ciprofloxacin was recovered from the urine within 48 h after intravenous and oral administration, respectively. The mean bioavailability was 71.2% and did not differ between the groups. We conclude that similar dosing regimens can be used to treat patients with CF and their normal counterparts.