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Sequence variation in the Streptococcus pneumoniae pneumolysin gene affecting haemolytic activity and electrophoretic
R A Lock1, Q Y Zhang, A M Berry
1Molecular Microbiology Unit, Women's and Children's Hospital, North Adelaide, Australia.
Abstract:
When 30 clinical isolates of Streptococcus pneumoniae, representing 16 capsular serotypes, were analysed by Western blot for production of the haemolytic toxin pneumolysin (Ply), all strains produced an immunoreactive band of similar intensity. However, six isolates of serotype 8 and two of type 7F expressed Ply whose mobility on SDS-PAGE was anomalously slow. Culture lysates from these strains also had low haemolytic activities compared with those for clinical isolates of other serotypes, suggesting the possibility of mutations affecting specific activity. Genes encoding Ply from one type 8 isolate and one type 7F isolate were cloned into Escherichia coli and sequenced. Compared with the published sequence for Ply, the deduced amino acid sequence for the type 8 Ply variant contained three amino acid substitutions, and the type 7F variant four amino acid substitutions. Both variants also had Val270 and Lys271 deleted. The variant Ply proteins were purified from recombinant E. coli expressing the cloned genes, and shown to have substantially reduced specific haemolytic activities [6.8 x 10(4) haemolytic units (HU)/mg and 2.3 x 10(4) HU/mg for type 8 Ply and type 7F Ply respectively] compared with Ply itself (1.2 x 10(6) HU/mg). Studies with chimeric toxin gene constructs indicated that both the reduced haemolytic specific activity and the anomalous electrophoretic mobility of the variant Plys were attributable to a single amino acid substitution (Thr172-->Ile).
Insights
Certain Streptococcus pneumoniae strains produce a less active pneumolysin (Ply) toxin due to specific mutations. This reduced toxin activity and altered electrophoretic mobility stem from a single amino acid substitution.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Streptococcus pneumoniae causes various infections.
- Pneumolysin (Ply) is a key hemolytic toxin produced by S. pneumoniae.
- Variations in Ply activity can impact bacterial virulence.
Purpose of the Study:
- To investigate the cause of reduced hemolytic activity and altered electrophoretic mobility of Ply in specific S. pneumoniae serotypes.
- To identify the genetic basis for these observed Ply variants.
Main Methods:
- Western blot analysis of Ply production in clinical isolates.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to assess Ply mobility.
- Gene cloning into Escherichia coli, DNA sequencing, and protein purification.
- Determination of specific hemolytic activities of wild-type and variant Ply proteins.
- Construction and analysis of chimeric toxin genes.
Main Results:
- Six serotype 8 and two serotype 7F S. pneumoniae isolates showed anomalously slow Ply mobility on SDS-PAGE and reduced hemolytic activity.
- Sequencing revealed amino acid substitutions and deletions in the Ply genes of these variants.
- Purified variant Ply proteins exhibited significantly lower specific hemolytic activities compared to wild-type Ply.
- Chimeric toxin studies identified a single amino acid substitution (Thr172-->Ile) as responsible for both reduced activity and altered mobility.
Conclusions:
- Specific mutations in Streptococcus pneumoniae can lead to variants of pneumolysin with reduced hemolytic activity and altered electrophoretic properties.
- The Thr172-->Ile substitution is a key determinant of these altered Ply characteristics.
- Understanding these toxin variants is crucial for comprehending S. pneumoniae pathogenesis and developing targeted interventions.