Actin cytoskeleton staining revealed that cells rounded up buy Talazoparib at MOI 500:1 (Fig. 1A), followed by detachment from the substrate. At MOI 1000:1 a cytotoxic effect on the cell monolayer was observed (white arrows and detail). To determine the minimal infection requirements for cell rounding, cells were infected with different MOIs for 2 to 5 h. Cell rounding was observed when cells were infected at MOI 500:1 for 4 h (Fig. 1B, top). To investigate whether the cytotoxic effect was strain dependent two additional K. pneumoniae strains were tested. Strains 43816 (serotype K2) and 1850 (serotype K35) also induced cell rounding (Fig. 1B, middle and bottom, respectively).

CPS amounts expressed by these strains, 238 and 35 μg per 105 c.f.u., respectively, are lower than that expressed by strain 52145 (339 μg per 105 c.f.u.), indicating that Klebsiella-induced cytotoxicity is not absolutely dependent on the amount of CPS expressed. Figure 1 K. pneumoniae triggers a cytotoxic effect during infection of

A549 carcinoma lung epithelial cells. A. Infection of A549 lung epithelial cells with K. pneumoniae 52145. MOIs used were 200:1 (top), 500:1 (middle) and 1000:1 (bottom panel and detail). Infections were carried out for 5 h in all cases. Non infected cells are shown for comparison (top left). Cells were fixed and stained for immunofluorescence. Actin cytoskeleton was labelled with phalloidin-RRX (red). White arrows show cell rounding and cytotoxicity. B. A549 epithelial cells were infected with K. pneumoniae strains 52145, 43816 and 1850 at MOI 500:1 for 4 h. Infected cells were fixed and learn more stained with phalloidin-RRX for immunofluorescence as indicated above. C. UV killed K. pneumoniae 52145 was used to infect cells at MOI 500:1 during 4 h (top). K. pneumoniae 52145 was used for a mock infection (MOI 500:1). After 4 h the bacterial suspension Mannose-binding protein-associated serine protease was

UV irradiated and used to infect a confluent cell monolayer for 4 h (middle). To assess the need of presence of live bacteria to induce cell rounding, infection was carried out at MOI 500:1 during 4 h, after which the supernatant was collected, centrifuged and filtered (0.2 Tm, nitrocellulose) to obtain a primed bacteria-free medium, which was then added to a new epithelium monolayer for 4 h (bottom). Infected cells were fixed and stained for immunofluorescence as described above. Next, we asked whether live bacteria are necessary to induce cell rounding. The bacterial inoculum was killed by UV radiation and used to infect cells (MOI 500:1, 4 h). Under these conditions, strain 52145 did not induce cell rounding (Fig. 1C, upper). In order to corroborate this observation, a mock infection was carried out, i.e. same infection conditions as before, but in a tissue culture well without cells. After 4 h, the bacterial suspension was UV irradiated and used to infect a confluent cell monolayer for 4 h. Cell rounding was not observed (Fig. 1C, middle).