Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in ...immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing.
Stepwise visualization of membrane pore formation by suilysin, a bacteria cholesterol-dependent cytolysin. eLife 3:e04247. doi: http://dx.doi.org/10.7554/eLife.04247 Published 2 December 2014 In the ...published article there was an error in the schematic representation of suilysin prepore proteins in Figure 6, showing these proteins in the wrong orientation with respect to the membrane. The figure has been adjusted to show the correct orientation, consistent with the scientific representation in Figure 1.
Pneumolysin (PLY) is a proven virulence factor of Streptococcus pneumoniae, deeply involved in the development of pneumococcal invasive diseases. The antibacterial and adjunctive therapies currently ...used in the management of pneumococcal infection do not address the pneumolysin-induced cytotoxicity. Therefore, even if patients survive the infection, they may suffer from long-term disabilities as a consequence of the exposure to the toxin. This research seeks to address this problem by developing new approaches to the treatment of pneumococcal diseases, based on peptides or small molecules that target pneumolysin and inhibit its activity. The peptides and small molecules were evaluated for their capacity to inhibit the haemolytic activity of pneumolysin in vitro. In total, four peptides and six small molecules were selected for in vivo tests. In vivo experiments were performed using a pneumonia model, where each animal was administered PBS or peptide / small molecule in PBS intranasally, every 6 hours after intranasal infection. One peptide (P1) and three small molecules (SM6, SM10 and SM14) improved significantly the outcome of the treatment group over the control group, when looking at the CFU in the blood, disease sign score and survival comparison. Moreover, two small molecules (SM10 and SM14) tested ex vivo, showed the capacity to abolish pneumolysin cytotoxic effect on the ciliary function of rat ependymal cells. The pneumolysin inhibitors identified in this work were pointed as potential candidates to further testing in drug discovery.
Pneumolysin (PLY) is a proven virulence factor of Streptococcus pneumoniae, deeply involved in the development of pneumococcal invasive diseases. The antibacterial and adjunctive therapies currently ...used in the management of pneumococcal infection do not address the pneumolysin-induced cytotoxicity. Therefore, even if patients survive the infection, they may suffer from long-term disabilities as a consequence of the exposure to the toxin. This research seeks to address this problem by developing new approaches to the treatment of pneumococcal diseases, based on peptides or small molecules that target pneumolysin and inhibit its activity. The peptides and small molecules were evaluated for their capacity to inhibit the haemolytic activity of pneumolysin in vitro. In total, four peptides and six small molecules were selected for in vivo tests. In vivo experiments were performed using a pneumonia model, where each animal was administered PBS or peptide / small molecule in PBS intranasally, every 6 hours after intranasal infection. One peptide (P1) and three small molecules (SM6, SM10 and SM14) improved significantly the outcome of the treatment group over the control group, when looking at the CFU in the blood, disease sign score and survival comparison. Moreover, two small molecules (SM10 and SM14) tested ex vivo, showed the capacity to abolish pneumolysin cytotoxic effect on the ciliary function of rat ependymal cells. The pneumolysin inhibitors identified in this work were pointed as potential candidates to further testing in drug discovery.
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