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Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43
Published on: September 11, 2020
Export requirements of pneumolysin in Streptococcus pneumoniae
Katherine E Price1, Neil G Greene, Andrew Camilli
1Tufts University School of Medicine and Howard Hughes Medical Institute, Department of Molecular Biology and Microbiology, Boston, Massachusetts, USA.
Abstract:
Streptococcus pneumoniae is a major causative agent of otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolysins (CDCs), is produced by virtually all clinical isolates of S. pneumoniae, and ply mutant strains are severely attenuated in mouse models of colonization and infection. In contrast to all other known members of the CDC family, Ply lacks a signal peptide for export outside the cell. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains undefined. We show that an exogenously added signal sequence is not sufficient for Sec-dependent Ply secretion in S. pneumoniae but is sufficient in the surrogate host Bacillus subtilis. Previously, we showed that Ply is localized primarily to the cell wall compartment in the absence of detectable cell lysis. Here we show that Ply released by autolysis cannot reassociate with intact cells, suggesting that there is a Ply export mechanism that is coupled to cell wall localization of the protein. This putative export mechanism is capable of secreting a related CDC without its signal sequence. We show that B. subtilis can export Ply, suggesting that the export pathway is conserved. Finally, through truncation and domain swapping analyses, we show that export is dependent on domain 2 of Ply.
Insights
Streptococcus pneumoniae releases pneumolysin (Ply) via a novel export mechanism, not dependent on autolysis or signal peptides. This conserved pathway involves Ply
Area of Science:
- Microbiology
- Molecular Biology
- Protein Secretion
Background:
- Streptococcus pneumoniae causes severe infections like pneumonia and meningitis.
- Pneumolysin (Ply), a key virulence factor, is secreted by S. pneumoniae but lacks a signal peptide.
- Existing hypotheses suggest Ply release via autolysis or an undefined nonautolytic mechanism.
Purpose of the Study:
- To elucidate the mechanism of pneumolysin (Ply) export in Streptococcus pneumoniae.
- To investigate the role of signal peptides and autolysis in Ply secretion.
- To identify the specific protein domains involved in Ply export.
Main Methods:
- Investigated Ply secretion in S. pneumoniae and Bacillus subtilis using signal sequence addition.
- Assessed Ply localization and reassociation with cells after autolysis.
- Performed truncation and domain swapping analyses of Ply.
- Utilized mouse models for S. pneumoniae colonization and infection studies.
Main Results:
- Sec-dependent secretion of Ply in S. pneumoniae was not achieved by adding an exogenous signal sequence, but was successful in B. subtilis.
- Ply released by autolysis did not reassociate with intact cells, indicating a specific export pathway.
- The export pathway is conserved and capable of secreting Ply without a signal sequence.
- Export of Ply was found to be dependent on domain 2 of the protein.
Conclusions:
- S. pneumoniae utilizes a unique, conserved export mechanism for Ply, independent of autolysis and signal peptides.
- This novel export pathway is linked to Ply's cell wall localization and relies on domain 2.
- Understanding this mechanism offers new targets for combating S. pneumoniae infections.
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