PNEUMO-CELL

Regulation of pneumolysin in the human pathogen Streptococcus pneumoniae: A single cell approach

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 Obiettivo del progetto (Objective)

'Streptococcus pneumoniae is a major pathogen causing invasive (pneumonia, meningitis, bacteraemia) and non-invasive (acute otitis media, sinusitis) disease in young children and in elderly and/or immuno-compromised adults. The molecular mechanisms underlying S. pneumoniae virulence are not fully understood. Single-cell methodologies, including the sub-cellular localization of proteins by fluorescence microscopy, have been instrumental in studying fundamental processes in bacteria and these methodologies are potentially of great use to investigate S. pneumoniae virulence. Recently, we have developed a cell biology toolkit for S. pneumoniae, allowing us to perform gene expression and protein localization studies at the single cell level in live cells. A major S. pneumoniae virulence factor is pneumolysin which is encoded by the ply gene. Pneumolysin is highly immunogenic and binds to cholesterol in the membrane of host cells and its haemolytic activity induces cell lysis. Pneumolysin is one of the best characterized virulence factors of S. pneumoniae, but surprisingly little is known about its transcriptional regulation. Interestingly, pneumolysin is produced in the cytoplasm of S. pneumoniae and can only be released by lysis of the producer. This represents a fascinating paradox: if all cells lyse at the same time, the clonal lineage becomes extinct. Thus, mechanisms must exist to ensure that not all cells release or produce pneumolysin at the same time. This proposal is aimed at unraveling the molecular mechanisms underlying heterogeneous pneumolysin gene regulation and release using single cell analytical techniques. The host institute has excellent knowledge and facilities to perform S. pneumococcus molecular genetics and cell biology, and together with the single cell biology expertise of the researcher, this partnership should provide important insights into S. pneumoniae pathogenesis.'

Introduzione (Teaser)

Bacterial pathogenesis often depends on the production of specific toxins. Using single cell analytical techniques, European researchers unveiled the mechanism by which Streptococcus pneumoniae regulates toxin production.

Descrizione progetto (Article)

Streptococcus pneumoniae is a major human pathogen that can be present in nearly 60% of the population with no apparent symptoms. The bacterium can switch to a pathogenic state, gain access to the blood and cause meningitis and pneumonia. To achieve this, S. pneumoniae turns on the expression of certain virulence factors such as pneumolysin, a well-characterised toxin.

The ply gene encodes pneumolysin and it has a well-conserved sequence among different clinical isolates. At high concentrations, pneumolysin makes holes in the cell membrane and ruptures infected cells while at low concentrations it triggers inflammation.

Paradoxically, pneumolysin is produced within the bacterium and can only reach the host cell after lysis. This means that if all bacteria lyse, then the population would fail to propagate suggesting the existence of mechanisms for the controlled release of pneumolysin. Recent evidence suggests that only part of a bacterial population undergoes lysis as a form of self-destructive cooperation.

The EU-funded 'Regulation of pneumolysin in the human pathogen Streptococcus pneumoniae: A single cell approach' (PNEUMO-CELL) project worked on devising analytical tools for studying the mechanism of self-destructive cooperation in S. pneumoniae. In this context, researchers developed fluorescent proteins, flow cytometry assays and time-lapse microscopy protocols for visualising S. pneumoniae at the single cell level.

They discovered that pneumolysin is produced by all the cells within a bacterial population. Using time-lapse microscopy, they observed that pneumolysin gets attached to the cell wall and production increases just before lysis. This indicated that the phenomenon of self-destructive cooperation could partly involve transcriptional regulation of the ply gene. Besides locating the promoter of the five-gene operon including the ply gene, scientists also identified five genes with suspected involvement in the transcriptional regulation of ply gene.

Taken together, the results of the PNEUMO-CELL project provide important insight into the molecular mechanisms that regulate the production of the toxin, pneumolysin. Understanding the mechanism of microbial pathogenesis will undoubtedly provide solutions for their effective eradication.