Search

Many pathogenic bacteria express plasminogen receptors on their surface, which may play a role in the dissemination of organisms by binding plasminogen that, when converted to plasmin, can digest extracellular matrix proteins. A 45-kDa protein was purified from Streptococcus pneumoniae and confirmed as an alpha-enolase by its ability to catalyse the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate and by N-terminal sequencing. The activity of alpha-enolase was found in the cytoplasm and in whole cells. Activity was also demonstrated in cell wall fractions, which confirmed that alpha-enolase is a cytoplasmic antigen also expressed on the surface of S. pneumoniae. The plasminogen-binding activity of alpha-enolase was examined by Western blot, which showed that purified alpha-enolase was able to bind human plasminogen. Immunoblots of the purified 45-kDa alpha-enolase with 22 sera from patients with pneumococcal disease showed binding in 15 cases, indicating that pneumococcal enolase is immunogenic.

A PCR-restriction fragment length polymorphism strategy directed against the pbp2b gene was evaluated for identification of penicillin susceptibility. A total of 106 United Kingdom (U.K.), 30 Danish, and 11 Papua New Guinean strains were tested. Of the U.K. strains, all the susceptible and all but one of the resistant isolates were correctly assigned. By using conventional definitions of "not resistant" and "not susceptible," the sensitivities were 97. 5 and 94.4%, the specificities were 100 and 98.9%, the positive predictive values were 100 and 94.4%, and the negative predictive values were 93.1 and 98.9%, respectively. This technique may allow susceptible (MIC, <0.1 mg/liter) and resistant (MIC, >1 mg/liter) isolates to be distinguished in a single PCR.

The lytA gene encoding the autolysin of Streptococcus pneumoniae may be a virulence determinant. Single-strand conformational polymorphism analysis demonstrated heterogenicity throughout the gene in clinical isolates and strains from the clonal serotypes 7 and 14. Sequence analysis of part of the choline-binding domain showed that in two isolates four amino acid substitutions occurred.

In this study we have demonstrated the activity of a choline phosphate cytidylyltransferase in cell free extracts of Streptococcus pneumoniae. Southern blot analysis of restricted S. pneumoniae genomic DNA probed with the gene coding for the choline phosphate cytidylyltransferase of Saccharomyces cerevisiae demonstrated that there is homology between the S. cerevisiae cct gene and genomic DNA of S. pneumoniae. We believe that this enzyme is involved in the biosynthesis of the choline containing cell wall antigens, teichoic acid and lipoteichoic acid, catalysing the activation of choline phosphate to CDP-choline which is then incorporated into the polysaccharide moiety.

The choline-containing teichoic and lipoteichoic acids play an important part in cell wall metabolism of Streptococcus pneumoniae. We propose that a choline kinase enzyme has a role in the synthesis of these antigens. The presence of this enzyme was demonstrated in cell free extracts of S. pneumoniae by measuring the fall in ATP concentration due to phosphorylation of choline. Genomic DNA of S. pneumoniae hybridised with a probe consisting of an internal fragment of the choline kinase gene of Saccharomyces cerevisiae and one consisting of the choline binding domain of lytA.