However, it is speculated that Gram-negative bacteria produce membrane-derived vesicles other than OMVs that originate from the inner membrane. A future study should determine whether membrane-derived vesicles from Gram-negative bacteria contain either OMVs, inner membrane vesicles or both. Klebsiella pneumoniae OMVs may interact with host cells and alter host cell biology, because these

Saracatinib vesicular components contain numerous proteins, LPS and peptidoglycans. LPS-refractory epithelial HEp-2 cells and LPS-susceptible monocyte U937 cells were treated with different amounts of K. pneumoniae OMVs to determine whether K. pneumoniae OMVs induce morphological changes and growth inhibition of the host cells. No morphological changes (Fig. 2a) or inhibited cellular growth (Fig. 2b) were observed

in either cells treated with ≤ 50 μg mL−1 (protein concentration) OMVs. Two previous studies focusing on the host cell pathology induced by K. pneumoniae showed that extracellular components released or secreted from bacteria are partly associated with host cell cytotoxicity (Straus, 1987; buy JQ1 Cano et al., 2009). Thus, we expected that K. pneumoniae OMVs would inhibit growth or induce death in either U937 cells, HEp-2 cells or both. However, OMVs from K. pneumoniae ATCC 13883 did not inhibit cell growth and were not cytotoxic to either cell type. In proteomic analysis of K. pneumoniae OMVs, we did not find any cytotoxic factors. These results suggest that OMVs from K. pneumoniae ATCC 13383 do not carry cytotoxic factors. However, whether OMVs from other K. pneumoniae strains are cytotoxic to host cells remains to be determined. To determine whether K. pneumoniae OMVs induce a proinflammatory response in vitro, HEp-2 cells were treated with 1–20 μg mL−1 (protein concentration) of K. pneumoniae OMVs for 24 h, and the expression of proinflammatory cytokine genes was analysed by RT-PCR. HEp-2 cells originating from human laryngeal

epithelial cells were used, because the respiratory tract is a common site BCKDHA for colonization of or infection by K. pneumoniae. HEp-2 cells were infected with live K. pneumoniae with a multiplicity of infection (MOI) of 1 or 10 as a positive control. Expression of IL-1β and IL-8 increased in a dose-dependent manner in respond to the K. pneumoniae OMVs (Fig. 3). MIP-1 expression was not increased. No expression of the IL-6 gene was observed (data not shown). These results indicate that K. pneumoniae OMVs elicit the expression of proinflammatory cytokine genes in epithelial cells. A proinflammatory response against OMVs has also been observed for several other Gram-negative pathogens, including Salmonella enterica serovar Typhimurium (Alaniz et al., 2007), H. pylori (Ismail et al., 2003), P. aeruginosa (Bauman & Kuehn, 2006; Ellis et al., 2010), Neisseria meningitidis (Durand et al., 2009) and Vibrio anguillarum (Hong et al., 2009).