It is likely that K. pneumoniae also produces outer membrane vesicles. In fact, the extracellular toxic complex described by Straus [5, 24] could be considered a preparation of outer membrane vesicles. It is then tempting to speculate that outer membrane vesicles could be associated with K. pneumoniae cytotoxicity

described in our study. Future studies will aim to address this possibility. On the other hand, our results clearly establish that CPS is necessary for the induction of cytotoxicity. CPS is a virulence factor for several pathogens, including Streptococcus PARP inhibitor review pneumoniae, Neisseria meningitidis, Haemophilus influenzae type b and E. coli K1 [32–34]. Of note, no previous reports link the presence of CPS to cytotoxicity. However, just the presence of CPS is not sufficient for K. pneumoniae-induced cytotoxicity because capsulated UV-killed bacteria or purified CPS did not induce this effect. Given the limited current knowledge about K. pneumoniae virulence factors, we can only speculate on the

nature of bacterial factor(s) that, together with CPS, could promote cytotoxicity in the host. Signature-tagged mutagenesis approaches have identified several virulence factors [35, 36], but none of them resemble those triggering the cytotoxicity by other bacterial pathogens. All K. pneumoniae STI571 clinical isolates are capsulated, inferring the importance of CPS for virulence. Likewise, CPS is necessary for virulence in an in vivo pneumonia model [15, 35] and for Klebsiella-induced cytotoxicity (this work). However, our data indicate that CPS-dependent cytoxicity is necessary but not sufficient for Klebsiella virulence because strains Docetaxel in vivo 43816 and 1850 are less virulent

than strain 52145 and the three of them trigger cytotoxicity. This could be explained by differences in the amount of CPS expressed by these strains, although strain 43816 is also considered to be heavily capsulated. The absence of complete correlation between in vitro and in vivo studies has been previously described for other K. pneumoniae isolates. Struve et al., showed that CPS expression reduced K. pneumoniae adhesion to gut and bladder epithelium, when compared to a noncapsulated mutant. However, the presence/absence of CPS had no effect on the colonisation of the gastrointestinal tract, but did play a role in colonisation of the urinary tract [37]. On the other hand, it has been recently postulated that there is an association between CPS serotype, virulence in mice and humans, and frequency of isolation in clinical settings [38]. However, the bacterial strains tested in this study express CPS belonging to serotypes considered to have high potential of causing disease [38], and strains 52145 and 43816 express the same CPS serotype. Nevertheless, Klebsiella infections should be looked at as the outcome of specific interactions between pathogen and host cells. Indeed, factors on both pathogen and host sides may be involved in the progression of the infection.