Sty, Salmonella enterica serovar typhimurium. Ype, Yersinia pestis. Pfl, Pseudomonas fluorescens. Lpn, Legionella pneumophila. Hpy, Helicobacter pylori. Ara, Agrobacterium radiobacter. Mtu, Mycobacterium tuberculosis. Figure 2 The growth curves #INCB018424 molecular weight randurls[1|1|,|CHEM1|]# of L. pneumophila wild-type JR32, the Lp ΔclpP mutant, both harboring the vector pBC(gfp)Pmip, and the complemented strain Lp ΔclpP -p clpP. Overnight cultures of mid-exponential bacterial cells were diluted into fresh medium and then incubated at (A) 25°C, (B) 30°C, (C) 37°C, and (D) 42°C, respectively. Growth was monitored by OD600 at various time points. Points indicate mean values

and error bars indicate standard deviations of three experiments. clpP homologue is required for stress tolerance in stationary phase L. pneumophila can respond to various environmental

stresses and cope with harsh conditions while entering eukaryotic hosts [12, 41]. To assess whether clpP homologue may be involved in stress response, the above three strains were grown to logarithmic or stationary phase and exposed to various stress conditions. When the logarithmic-phase cells were exposed respectively to low pH, hydrogen peroxide, potassium chloride, and heat shock, the survival S3I-201 rates of all three strains were similar and lower than those of the stationary-phase cells (data not shown). When treated with pH 4.0 citric acid for 30 minutes, WT JR32 cells in stationary phase exhibited approximately 70% survival rate. However, only about 10% of LpΔclpP mutant Celastrol cells survived (Figure 3A). Such a deficiency was rescued in the LpΔclpP-pclpP strain (Figure 3A). This result indicated that the deletion of clpP impairs the ability of L. pneumophila to respond to low-pH conditions. Similar results were also obtained in oxidative stress assay (Figure 3B). When the cells were treated with 1 mM hydrogen peroxide for 30 minutes, the survival rate of the LpΔclpP mutant was 10 ± 2.0%, much lower than that of WT cells (56 ± 8.6%; Figure 3B). In contrast, LpΔclpP-pclpP cells displayed

a CFU closely resembling that of WT cells (Figure 3B). Likewise, when cells were incubated in 57°C water bath for 20 minutes or treated with 0.3 M potassium chloride for 1 hour, the survival rate of LpΔclpP mutant was lower than that of WT and the complementation strain (Figure 3C and 3D), indicating that clpP is also required for responses to heat shock and osmotic stress. Collectively, these results indicate that ClpP homologue is involved in tolerance to multiple stresses in stationary-phase L. pneumophila. Figure 3 Impaired stress tolerance of the L. pneumophila Lp ΔclpP mutant during stationary phase. Overnight cultures of different strains were inoculated into fresh medium and grew to stationary phase (OD600 from 3.5 to 4.5), and the cells were then treated with (A) 1 mM H2O2 for 30 minutes. * p < 0.05, (B) pH 4.0 citric acid for 30 minutes. * p < 0.01, (C) 57°C heat shock for 20 minutes. * p < 0.05, or (D) 0.3 M KCl for 1 hour. * p < 0.05.