The hemolytic effect of MFN1032 cells was much higher

than the other strains tested, at both growth temperatures (Figure 4). At 28°C, MFY162 was the only other strain showing high levels of hemolytic activity (40% lysis); MFY161 and MFY163 displayed only weak hemolytic activity (5-10% lysis). All clinical isolates showed some hemolytic activity (15% lysis) at 37°C, but at a lower level than that observed for MFN1032 one’s. The environmental strains tested were not hemolytic at 28°C and did not grow at 37°C. Figure 4 Cell-associated hemolytic activity of different fluorescent Pseudomonas strains. Cell-associated hemolytic activity (cHA %) was measured as described in the materials and methods. Results are means of at least three independent experiments. Standard deviation is shown. A: Hemolysis of RBCs selleck chemical incubated with MFN1032, MF37, C7R12, MFY161, MFY162, MFY163 at 28°C and MOI of 1. CHIR-99021 in vivo Contact was enhanced by centrifugation at 400 g for 10 min. B: Hemolysis of RBCs incubated with MFN1032, MF37, C7R12, MFY161, MFY162,

MFY163 at 37°C and MOI of 1. Contact was enhanced by centrifugation at 400 g for 10 min. ND: not determined. MF37 and C7R12 were unable to grow at 37°C. The hemolytic activities of MFN1032, MFY162 and MFY161, were maximal at their optimal growth temperature (28°C for MFN1032 and MFY162, 37°C for MFY 161). The hemolytic activity of the strain MFY163 was the same at 28°C and 37°C. Involvement of the Gac two-component system on cell-associated hemolytic activity We investigated the possible involvement of the Gac two-component system in the regulation of this cell-associated hemolytic activity using a group1 variant of MFN1032, V1. This variant strain is a gacA mutant and has impaired secreted hemolytic activity [12]. V1 was tested with or without transformation by electroporation with plasmid carrying the gacA gene

(pMP5565) or the parental plasmid pME6010, as a control [26] (Figure 5). Figure 5 Effect of GacA on MFN1032 cell-associated hemolytic activity. Palmatine Cell-associated hemolytic activity (cHA) for MFN1032 cells, V1 (gacA mutant) and V1 cells carrying the gacA gene-containing plasmid (pMP5565), or the parental plasmid pME6010 used as a control. The cHA of MFN1032 was taken as the reference value (100%); results are expressed as percent of this value (% relative cHA). The strains were grown at 28°C. Results are means of at least three independent experiments. Standard deviation is shown. Contact was enhanced by centrifugation at 400 g for 10 min. The non-transformed V1 strain displayed enhanced hemolytic activity (160% lysis), using MFN1032 as a reference value (100%). Introduction of a gacA gene in V1 cells by electroporation with pMP5565 restored wild-type hemolytic levels.