​hgc.​jp/​~mdehoon/​software/​cluster/​software.​htm#ctv) [28]. Average linkage was used for clustering. The Java Tree View program [28] was used to show the clustering result. Results Hypercytotoxicity of complex IV isolates in vitro Cytotoxicity against a broad range of cell types is a hallmark of B. bronchiseptica GS-7977 mouse infection in vitro[11, 12, 14, 16, 23]. To measure relative levels of cytotoxicity, human epithelial cells (HeLa), murine monocyte-macrophage derived cells (J774A.1), or human pneumocyte-derived cells (A549) were infected with an array of complex I or complex IV B. bronchiseptica isolates (Figure 1A-C). These strains represent different multilocus sequence types (STs),

Fosbretabulin concentration and they were isolated from both human and non-human hosts (Table 1). Lactate dehydrogenase (LDH) release was used as a surrogate marker for cell death, and RB50, an extensively characterized complex I rabbit isolate classified as ST12, was used as a positive control for cytotoxicity [20]. An isogenic RB50 derivative with a deletion in bscN, which encodes the ATPase required for T3SS GDC 0032 cost activity [15], served as a negative control. Figure

1 Cytotoxicity of complex I and complex IV B. bronchiseptica isolates. A. HeLa, B. J774A.1, or C. A549 cells were infected with the indicated strains at a multiplicity of infection (MOI) of 50 in 24-well plates for 3 h. Following infection, release of lactate dehydrogenase (LDH) into culture medium was measured as described in Materials and Methods. Complex I and complex IV strains are designated by blue or red bars, respectively. P values were calculated by an unpaired two-tailed Student’s t Bumetanide test. For HeLa (Figure 1A) and J774A.1 cells (Figure 1B), single time point assays showed a distinct trend, in which complex IV strains displayed higher levels of cytotoxicity than complex I isolates. For A549 cells the results were more dramatic (Figure 1C). Unlike other cell types previously

examined [11, 16, 29], A549 cells are nearly resistant to cell death mediated by the RB50 T3SS (see RB50 vs. RB50ΔbscN; Figure 1C). Similarly, other complex I strains displayed little or no cytotoxicity against these cells. In striking contrast, incubation with complex IV isolates resulted in significant levels of cell death (p < 0.0001; Figure 1C). For A549 cells, strains D444 (ST15), D445 (ST17), D446 (ST3) and Bbr77 (ST18) were 10- to 15-fold more cytotoxic than RB50. Parallel assays measuring bacterial attachment to A549 cells did not detect significant differences between complex I and complex IV isolates, indicating that relative levels of adherence are not responsible for the observed differences in cytotoxicity (Additional file 1 Table S1). Kinetic studies were performed next to increase the resolution of the analysis. We examined relative levels of cytotoxicity conferred by five complex IV strains towards HeLa, J774A.