Survival of mice infected with different <i>P. aeruginosa</i> T3SS mutant bacteria.

<p>A lethal dose of 8.1⁷ cfu of WT <i>P. aeruginosa</i> or ΔSTY or ΔSTY/ΔPopB mutant strains was instillated intratracheally in C57BL/6 mice. (n = 10 for each group, NI  =  non-infected). Mortality was monitored for 24 h. Results are representative of three independent experiments. ***P≤0.001.</p

Cell death and IL-1β maturation upon infection of the MF4/4 macrophage cell line with different <i>P. aeruginosa</i> T3SS mutants.

<p>MF4/4 cells were pre-stimulated with 100 ng/ml LPS for 4 h and infected with WT <i>P. aeruginosa</i> or ΔSTY or ΔSTY/ΔPopB mutant strains at a MOI of 100. Non infected cells prestimulated with 100 ng/ml LPS for 4 h were used as a control. <b>A.</b> LDH release in the culture medium was determined by spectrophotometry as described in materials and methods and is expressed as optical density (absorbance) at 490 nm. Results are the mean +/− SD of triplicates and are representative for four independent experiments. <b>B.</b> Caspase-3 activity in cell extracts was measured in a fluorometric assay with Ac-DEVD-AMC and is expressed as change in fluorescence over time (Δf/min). Results are the mean +/− SD of triplicates and are representative of three independent experiments. <b>C.</b> Culture supernatants (SN) were collected and analyzed for the presence of mature IL-1β by immunoprecipitation of IL-1β followed by SDS-PAGE and western blotting. The corresponding total cell lysates were analyzed for the presence of proIL-1β by western blotting. Results are representative of three independent experiments.</p

IL-1β maturation and secretion in lungs of mice infected with different <i>P. aeruginosa</i> T3SS mutants.

<p>C57BL/6 mice were infected intratracheally with 1.1⁶ cfu of WT <i>P. aeruginosa</i> or ΔSTY or ΔSTY/ΔPopB mutants, and after 4.5 h, BALF was isolated and analyzed by SDS-PAGE and western blotting for the presence of mature IL-1β. The corresponding total lung extracts were also analyzed by western blotting for the presence of pro IL-1β. Results are representative of six different mice per experimental condition.</p

Inflammatory cell infiltration and cell death in lungs of mice infected with different <i>P. aeruginosa</i> T3SS mutants.

<p>C57BL/6 mice were infected intratracheally with 1.1⁶ cfu of <i>P. aeruginosa</i> ΔSTY or ΔSTY/ΔPopB strain for 4.5 h. The cells were analyzed by flow cytometry as described in materials and methods. <b>A.</b> The number of macrophages and PMNs in BALF was counted. <b>B.</b> Total numbers of dead macrophages and PMNs were calculated as the sum of check Annexin-V^pos Sytox^neg, Annexin-V^neg Sytox^pos, and Annexin-V^pos Sytox^pos cells. <b>C.</b> The mode of macrophage death was determined by separating Annexin-V^pos Sytox^neg from Annexin-V^neg Sytox^pos cells (n = 6 mice per group; NI were used as a control). The data are expressed as means (+/− SD) and are representative of 3 independent experiments.</p

Bacterial strains and plasmids.

<p>Bacterial strains and plasmids.</p

Intracellular localization and stability of the T3SS injected TALENs.

<p>(<b>A</b>) HeLa-Venus cells were infected with the indicated strains at an MOI of 100 for 3 hours. Nuclear proteins were extracted and subjected to SDS PAGE and Western blot by anti-Flag antibody; (<b>B</b>) HeLa-Venus cells were infected with PAK-JΔ<i>STY</i>/pExoS54-F-TALEN1 and PAK-JΔ<i>STY</i>/pExoS54-F-TALEN2 at MOI of 100 each for 3 hours. At the indicated time after the termination of infection, cytoplasmic and nuclear protein extracts were prepared and subjected to SDS PAGE and Western blot assay using anti-Flag Ab. NI, no infection control.</p

Specific or shared mini-proteins in phyla.

<p>Note: The averages of species-specific, phylum-shared and domain-shared are 58.79%, 6.20% and 0.73%, respectively. Because Actinobacteria contains only one class, class-specific mini-proteins are equal to the phylum-specifics'; similarly, Spirochaetes contains one class and one order, so the order-specific mean the phylum-specific. The hypothetical proteins account for 82.80%, 86.51%, 85.77%, 79.91%, 84.49% and 95.37% of the species-specific in Euryarchaeota, Actinobacteria, Cyanobacteria, Firmicutes, Gammaproteobacteria and Spirochaetes, respectively.</p

Plasmid transfection mediated delivery of TALENs into HeLa-Venus cells.

<p>(<b>A</b>) Fluorescent intensity of HeLa-Venus cells transfected by the indicated TALEN encoding plasmid constructs. Cells were analyzed by flow cytometry 3 days after the transfection. (<b>B</b>) Sequence changes in TALEN target region among the Venus negative cells.</p

Improvement of TALEN targeting efficiency.

<p>(<b>A</b>) Multiple rounds of TALEN injection. HeLa-Venus cells were infected with PAK-JΔ<i>STY</i>/pExoS54-F-TALEN1 and PAK-JΔ<i>STY</i>/pExoS54-F-TALEN2 at MOI of 100 each for 3 hours. Bacteria were cleared and cells were cultured for 24 hours, then repeated injection at MOI of 50 or 100. The FACS data of infected cells were compared by paired t-test and ANOVA. *P<0.05; **P<0.001. Error bars indicate standard deviations of triplicate assays. (<b>B</b>) HeLa-Venus cells were either left unsynchronized (U) or synchronized (S) through double thymidine blocking and released to determine the duration of the cell cycle by FACS analysis. (<b>C</b>) HeLa-Venus cells were synchronized to S phase (S) or unsynchronized (U) and infected by PAK-JΔ<i>STY</i>/pExoS54-F-TALEN1 and PAK-JΔ<i>STY</i>/pExoS54-F-TALEN2 at MOI of 100 each for 3 hours. The FACS data of infected cell populations were compared by paired or two-sample t-test. *P<0.05; **P<0.001. Error bars indicated the standard deviations of triplicate assays.</p

Figure 4

<p>A: Patterns of BMC domain. Dashed lines mean the domain have been evolved to a part of other domain or protein family's conserved region. Green represents IPR011238 (polyhedral organelle shell protein PduT) and IPR009193 (polyhedral organelle shell protein, EutL/PduB type) in pattern4; blue represents IPR009193 (polyhedral organelle shell protein, EutL/PduB type) and IPR009307 (ethanolamine utilization EutS) in pattern 5. They are all polyhedral organelle shell proteins. B: Phylogeny of BMC domain in Cyanobacteria. Letter L and R represent the left and right domain in pattern 3 or 4, respectively. Letter r represents the domains in pattern 2 or 5.</p