1C, lower). Taken together, these findings demonstrate that K. pneumoniae strain 52145 induces a cytotoxic SC79 solubility dmso effect through a process requiring the presence of
live bacteria. K. pneumoniae-induced cytotoxicity is dependent on the presence of CPS We sought to pinpoint bacterial factor(s) responsible for strain 52145-triggered cytotoxicity. Taken into account that several studies have demonstrated the important role of CPS in the interplay between K. pneumoniae and eukaryotic host cells, we asked whether CPS might play a role in the Klebsiella-induced cytotoxicity. We studied whether an isogenic CPS mutant of 52145, strain 52K10 , would induce cytotoxicity. Immunofluorescence analysis of the actin cytoskeleton PF-6463922 datasheet of infected A549 cells showed that strain 52K10 did not induce cytotoxicity under all conditions tested, hence suggesting that CPS could be one of MK-4827 order the bacterial factors involved in 52145-triggered cytotoxicity (Fig. 2A). Furthemore, the lack of cytotoxicity during 52K10 infection was not due to a decrease in bacterial adhesion levels because 52K10 adhesion levels to A549 cells were actually higher than those displayed by CPS-expressing strains (Fig. 2B). Even though cytotoxicity by non-capsulated strain was at some extent promoted by addition of
purified CPS during infection, purified CPS alone did not trigger clonidine a cytotoxic effect (data not shown), suggesting that additional bacterial elements besides
CPS may contribute to cytotocixity during K. pneumoniae infection. Figure 2 Capsule polysaccharide (CPS) is required for cytotoxicity during K. pneumoniae infection of A549 lung epithelium. A. Infection of A549 lung epithelial cells with K. pneumoniae 52K10, a bacterial strain lacking CPS. MOIs used were 200:1 (upper), 500:1 (middle) and 1000:1 (lower panel and right detail). Infections were carried out for 5 h in all cases. Infection conditions of MOI 500:1 for 4 h were used in the bottom panel. Infected cells were fixed and stained for immunofluorescence. Actin cytoskeleton was labelled with phalloidin-RRX (red). White arrows and detail show cell spread morphology and absence of cytotoxicity. B. Adhesion levels of K. pneumoniae strains 52145 and 52K10 to A549 lung epithelial cells. Infections were carried out at MOI 100:1 for 2 h. Mean values from three independent experiments are shown (error bars = SD). To further characterize the cytotoxic effect induced by 52145, cell toxicity was assessed by four independent methods: (i) lactate dehydrogenase (LDH) release, (ii) production of formazan, (iii) analysis of DNA integrity, and (iv) uptake of ethidium bromide.