Observation

Novel Type III Effectors in Pseudomonas aeruginosa

David Burstein, Shirley Satanower, Michal Simovitch, Yana Belnik, Meital Zehavi, Gal Yerushalmi, Shay Ben-Aroya, Tal Pupko, Ehud Banin
Stephen Carlyle Winans, Editor
DOI: 10.1128/mBio.00161-15

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Figures

  • FIG 1 
    FIG 1 

    (a) Two new putative effectors are translocated into HeLa cells. (Top row) HeLa cells were infected with Δ3tox strains (active T3SS) or Δ3tox ΔpscJ strains (nonactive T3SS) expressing either unfused BlaM, ExoS, or two putative effectors (PemA and PemB) fused to BlaM. Cells stained in blue indicate successful translocation of BlaM fused to the bacterial effector. Green cells indicate no translocation (×10 magnification). (b) The two new effectors are not essential structural components of the T3SS machinery. HeLa cells were infected with a P. aeruginosa strain lacking the known effectors (Δ3tox), a strain which has a nonfunctional T3SS (Δ3tox ΔpscJ), or strains with deletions of the putative genes pemA, pemB, or both (Δ3tox ΔpemA, Δ3tox ΔpemB, or Δ3tox ΔpemA ΔpemB) expressing ExoY fused to BlaM. Cells stained in blue indicate successful translocation of BlaM fused to the bacterial effector. Green cells indicate no translocation (×20 magnification).

  • FIG 2 
    FIG 2 

    (a) The two new effectors are not cytotoxic to HeLa cells. HeLa cells were infected with wild-type P. aeruginosa or the Δ3tox or Δ3tox ΔpscJ strain and three strains lacking one (Δ3tox ΔpemA or Δ3tox ΔpemB) or both (Δ3tox ΔpemA ΔpemB) of the newly discovered effectors. Cytotoxicity was assessed by the CytoTox-ONE homogeneous membrane integrity assay after 4 h of infection. Three biological repeats were performed in duplicate. Expression of the effectors under these conditions was validated (see Fig. S5A in the supplemental material). None of the strains showed a significant change in cytotoxicity according to the one-way analysis of variance (ANOVA) test. (b) Expression of the two new effectors does not affect the growth of S. cerevisiae. pemA and pemB were expressed from the GAL10 promoter in S. cerevisiae BY4741. Growth was assessed by a yeast spotting assay. One molar sorbitol, 7 mM caffeine, 0.5 M NaCl, 0.12 µg/ml tunicamycin, and growth at 37°C were used to assess the growth effects of the effectors under various stress conditions. S. cerevisiae expressing the Legionella toxic effector lpg2975 and the empty vector pGREG523-Km were used as controls. Three biological repeats were performed. Effector expression was validated by Western blotting (Fig. S6B).

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