Cells treated

by Plu1962 alone displayed a dramatic decrease in density of green fluorescence (Fig. 4c). This implied that Plu1962 alone could depolymerize microtubules to a certain extent. We next investigated the possible mechanisms responsible for the rapid cell death caused by binary toxin using Apoptosis and Necrosis Assay Kit. The intact membrane of live cells excludes charged cationic dyes, such as trypan blue, propidium, or ethidium, and short incubation with these dyes results in selective Dinaciclib labeling of dead cells, while live cells show minimal dye uptake. Loss of plasma membrane integrity leading to increased permeability to PI was found to be characteristic of necrosis. Most of the CF-203 cells treated with 0.6 μmol L−1 mixture of Plu1961/Plu1962 showed strong blue fluorescence and red fluorescence. Conversely, weak blue fluorescence and no red fluorescence were detected in control cells (Supporting Information, Fig. S1). Moreover, incubation of CF-203 cells with mixture of Plu1961/Plu1962 (0.6 nM) failed to induce DNA ladder fragmentation, a hallmark of apoptosis, even after incubation for 24 h (data not shown). Taken together, we therefore assumed that Plu1961/Plu1962 exhibited necrotic cytotoxicity in CF-203 cells. Five mammalian cell lines (B16, 4T1,

HeLa, Hep 3B, HCT116) were also used to examine the cytotoxicity of binary toxin. Neither Plu1961 nor Plu1962 alone could inhibit the growth of all tested mammalian cell lines. Unexpectedly, the mixture of Plu1961/Plu1962 (1.6 μmol L−1) exhibited no CDK phosphorylation cytotoxic effect on all tested mammalian cell unless lines (data not shown). We then co-expressed Plu1961 and Plu1962 in BL21 (DE3). Lysate from BL (Bi) exhibited strong cytotoxicity against B16, 4T1, and HeLa cells (Fig. 5). Hep 3B and HCT116 cells were insensitive to BL (Bi) lysate (data not shown). In the present study, we identified a XaxAB-like binary toxin from P. luminescens, which exhibits cytotoxicity against insect midgut CF-203 cells and

some mammalian cell lines. Both Plu1961 and Plu1962 were necessary to restore full cytotoxicity against CF-203 cells. XaxAB and Plu1961/Plu1962 show no homology to any other protein with known function, indicating that they constitute a distinct family of binary toxins (Vigneux et al., 2007). Photorhabdus luminescens proliferates in the hemolymph before the insect dies and must therefore be able to escape the insect immune response. Cell-mediated immunity comes into play immediately after the insect hemocoel is penetrated by a foreign body (Ribeiro & Brehelin, 2006). It was reported that injection of wild-type E. coli into Manduca sexta resulted in rapid encapsulation of all of the bacteria by the insect hemocytes, completely clearing the infection from the hemocoel.