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A number of single amino acid substitutions throughout Streptococcus pneumoniae Cps2C were found to affect its function and confer either a mucoid or a small colony phenotype. These mutants exhibit significant changes in capsular polysaccharide (CPS) profile relative to that of wild-type pneumococci. The introduced mutations affect either polymerization or ligation of CPS to the cell wall and/or Cps2D phosphorylation. [ABSTRACT FROM AUTHOR]

The capacity of Streptococcus pneumoniae to produce capsular polysaccharide (CPS) is essential for virulence. The CPS biosynthesis proteins CpsB, CpsC, and CpsD function to regulate CPS production via tyrosine phosphorylation of CpsD. This mechanism of regulating CPS production is important for enabling S. pneumoniae to cause invasive disease. Here, we identify mutations affecting the attachment of CPS to the cell wall. These mutations were located in cpsC, such that CpsC functioned independently from CpsD tyrosine phosphorylation. These mutants produced WT levels of CPS, but were unable to cause bacteremia in mice after intranasal challenge. This finding suggests that cell-wall attachment of CPS is essential for invasive pneumococcal disease; production of WT levels of CPS alone is not sufficient. We also show that cpsB mutants, which lack the phosphotyrosine-protein phosphatase, produced less CPS than the WT strain, but attached substantially more CPS to their cell wall. Thus, the phosphorylated form of CpsD promotes attachment of CPS to the cell wall. [ABSTRACT FROM AUTHOR]

Four genes, cpsA-cpsD, at the 5' end of the capsular polysaccharide (CPS) biosynthesis locus are conserved in nearly all of the 90 known serotypes of Streptococcus pneumoniae. In the present study, the impact that mutations in cpsA, cpsB, and cpsD have on CPS production and on virulence in mice infected via systemic and intranasal routes was investigated. Strains exhibiting rough colony morphologies (in which either the cpsB or cpsD gene had been deleted) were avirulent, but a smooth, partially encapsulated strain (in which the cpsA gene had been deleted) was as virulent as the wild-type strain. Interestingly, mucoid strains containing mutations affecting the [YGX] 3-repeat domain of CpsD were unable to cause bacteremia after intranasal challenge of CD1 mice, even though such strains were capable of killing BALB/c mice after intraperitoneal challenge. In our model, the ability of S. pneumoniae to regulate, via CpsD phosphorylation, CPS production was required for its transition from the lung to the bloodstream. [ABSTRACT FROM AUTHOR]

In Streptococcus pneumoniae, CpsB, CpsC, and CpsD are essential for encapsulation, and mutants containing deletions of cpsB, cpsC, or cpsD exhibit rough colony morphologies. CpsD is an autophosphorylating proteintyrosine kinase, CpsC is required for CpsD tyrosine phosphorylation, and CpsB is a phosphotyrosine-protein phosphatase. We have previously shown that autophosphorylation of CpsD at tyrosine attenuates its activity and consequently reduces the level of encapsulation and negatively regulates CPS production. In this study, we further investigated the role of the carboxy-terminal (YGX)[sub 4] repeat domain of CpsD in encapsulation. A CpsD truncation mutant in which the entire (YGX)[sub 4] repeat domain was removed was indistinguishable from a strain in which the entire cpsD gene had been deleted, indicating that the carboxy-terminal (YGX)[sub 4] tail is required for CpsD activity in capsular polysaccharide production. Double mutants having a single tyrosine residue at position 2, 3, or 4 in the (YGX)[sub 4] repeat domain and lacking CpsB exhibited a rough colony morphology, indicating that in the absence of an active protein-tyrosine phosphatase, phosphorylation of just one of the tyrosine residues in the (YGX)[sub 4] repeat was sufficient to inactivate CpsD. When various mutants in which CpsD had either one or combinations of two or three tyrosine residues in the (YGX)[sub 4] repeat domain were examined, only those with three tyrosine residues in the (YGX)[sub 4] repeat domain were indistinguishable from the wild-type strain. The mutants with either one or two tyrosine residues exhibited mucoid colony morphologies. Further analysis of the mucoid strains indicated that the mucoid phenotype was not due to overproduction of capsular polysaccharide, as these strains actually produced less capsular polysaccharide than the wild-type strain. Thus, the tyrosine residues in the (YGX)[sub 4] repeat domain are essential for normal functioning of CpsD. [ABSTRACT FROM AUTHOR]

Examines the Streptococcus pneumoniae (S. pneumoniae) capsule biosynthesis protein, a manganese-dependent phosphotyrosine-protein phosphatase. Recognition of S. pneumoniae as the cause of invasive diseases in humans; Capacity of S. pneumoniae to produce a polysaccharide capsule; Construction of S. pneumoniae containing a defined mutation.

In Streptococcus pneumoniae, the first four genes of the capsule locus (cpsA to cpsD) are common to most serotypes. By analysis of various in-frame deletion and site-directed mutants, the function of their gene products in capsular polysaccharide (CPS) biosynthesis was investigated. We found that while CpsB, C and D are essential for encapsulation, CpsA is not. CpsC and CpsD have similarity to the amino-terminal and carboxy-terminal regions, respectively, of the autophosphorylating protein-tyrosine kinase Wzc from Escherichia coli. Alignment of CpsD with Wzc and other related proteins identified conserved Walker A and B sequence motifs and a tyrosine rich domain close to the carboxy-terminus. We have shown that CpsD is also an autophosphorylating protein-tyrosine kinase and that point mutations in cpsD affecting either the ATP-binding domain (Walker A motif) or the carboxy-terminal [YGX]4 repeat domain eliminated tyrosine phosphorylation of CpsD. We describe, for the first time, the phenotypic impact of these two mutations on polysaccharide production and show that they affect CPS production differently. Whereas a mutation in the Walker A motif resulted in loss of encapsulation, mutation of the tyrosines in the [YGX]4 repeat domain resulted in an apparent increase in encapsulation and a mucoid phenotype. These data suggest that autophosphorylation of CpsD at tyrosine attenuates its activity and reduces the level of encapsulation. Additionally, we demonstrated that CpsC is required for CpsD tyrosine phosphorylation and that CpsB influences dephosphorylation of CpsD. These results are consistent with CpsD tyrosine phosphorylation acting to negatively regulate CPS production. This has implications for the function of CpsC/CpsD homologues in both Gram-positive and Gram-negative bacteria and provides a mechanism to explain regulation of CPS production during pathogenesis. [ABSTRACT FROM AUTHOR]

Determines the DNA sequence for the type 19A- and 19C-specific serotypes of the capsular polysaccharide in Streptococcus pneumoniae. Comparison of the flanking sequences and structures of the four serogroup 19cps; Structural differences between genes; Glucosyl transferase encoding; Arrangement of genes within the cps19; Putative biosynthetic pathways.

Analyzes the sequence data obtained from the 5' portion of the Streptococcus pneumoniae (S. pneumoniae) type 19A capsular polysaccharide biosynthesis locus. Homologous genes in the type 19F locus; Souther hybridization analysis of the cps loci from S. pneumoniae serotypes; Homologue content of the serotypes tested; Presence of two distinct classes of cpsC.

Investigates the molecular and genetic character of the capsule biosynthesis locus of Streptococcus pneumoniae type 23F. Amplification and cloning of the capsule locus; Proposed functions for all the protein products; Proposed biosynthetic pathway for the type 23F capsular polysaccharide.

Presents a study in which long-range PCR was used to amplify and clone a 10.5kb section of the Streptococcus pneumoniae type 19B capsule locus (cps19b), between cps19bH and cps19bK. Construction of the S. pneumoniae strain; Isolation of the type 19B cps genes; Characterization of these genes; Conclusions of the study.

Discusses the isolation, characterization and nucleotide sequence of IS1202, an insertion sequence of Streptococcus pneumoniae. Presence in the chromosomes of pneumococci belonging to other serotype; Revelation of its close linkage to type 19F capsular polysacchride synthesis locus.

Serotype 19F variants of the major Spanish multiresistant serotype 23F clone of Streptococcus pneumoniae have been proposed to have arisen by recombinational exchanges at the capsular biosynthetic locus. Members of the Spanish multiresistant serotype 23F clone and the serotype 19F variants were confirmed to be essentially identical in overall genotype, as they were indistinguishable by REP-PCR, and had identical sequences at three polymorphic housekeeping genes. Eight serotype 19F variants were studied and all had large recombinational replacements at the capsular biosynthetic locus. In all cases, one of the recombinational cross-over points appeared to be upstream of dexBwhich flanks one end of the capsular locus, and in six of the variants the other cross-over point was downstream of aliA, which flanks the other end of the locus. In two strains a recombinational cross-over point between the introduced serotype 19F capsular region and that of the Spanish serotype 23F clone could be clearly identified, within cpsN in one strain and within cpsM in the other. The differences in the recombinational junctions and sequence polymorphisms within the introduced capsular genes, suggested that the eight serotype 19F variants emerged on at least four separate occasions. Changes in capsular type by recombination may therefore be relatively frequent in pneumococci and this has implications for the long-term efficacy of conjugate pneumococcal vaccines that will protect against only a limited number of serotypes. [ABSTRACT FROM AUTHOR]