Ostreolysin A/pleurotolysin B (OlyA/PlyB) is a binary pore-forming protein complex that produces a rapid cardiorespiratory arrest. Increased tonus of the coronary vascular wall produced by OlyA/PlyB ...may lead to ischemia, arrhythmias, the hypoxic injury of cardiomyocytes and cardiotoxicity. We evaluated the effects of OlyA/PlyB in cultured vascular smooth muscle A10 cells. Fluorometric measurements using the Ca2+ indicator Fluo-4 AM and Fura-2 AM revealed that nanomolar concentrations of OlyA/PlyB increased the intracellular Ca2+ activity Ca2+i in A10 cells. This effect was absent in a Ca2+-free medium, indicating that OlyA/PlyB-induced Ca2+i increase was dependent on Ca2+ influx into cells. The increase in Ca2+i by OlyA/PlyB was partially prevented by: i) the calcium channel blockers verapamil and La3+, ii) the inhibitor of the sodium–calcium exchanger (NCX) benzamil, and iii) the iso-osmotic replacement of NaCl by sucrose. The pre-treatment of cells with the Ca2+-ATPase inhibitor thapsigargin reduced the Ca2+i increase evoked by OlyA/PlyB, whereas the plasma membrane depolarization with high K+ in the medium did not prevent OlyA/PlyB-induced Ca2+i. In summary, our data could suggest that the OlyA/PlyB-induced increase in Ca2+i is due to an influx of Ca2+ through a variety of co-existing plasma membrane Ca2+-permeable channels, Ca2+ entry through non-selective ion permeable pores formed de novo by OlyA/PlyB in the plasma membrane and calcium-induced intracellular Ca2+ release, altogether leading to disturbed Ca2+ homeostasis in A10 cells.
•Ostreolysin A/pleurotolysin B induces plasma membrane blebs formation.•Ostreolysin A/pleurotolysin B complex increases Ca2+i in A10 cells.•NCX, VDCC and internal Ca2+ release are involved in OlyA/PlyB induced Ca2+i rise.
The prsDE genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from Rhizobium leguminosarum bv. viciae. At least ...one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes (plyA and plyB) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in a prsD (protein secretion) mutant. The plyA gene is upstream of prsD but appears to be expressed at a very low level (if at all) in cultured bacteria. A plyB::Tn5 mutant has a very large reduction in degradation of EPS and CMC. Cultures of plyB mutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a plyA plyB double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca(2+)-binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS
PDF