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Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling.

Tomás A, Lery L, Regueiro V, Pérez-Gutiérrez C, Martínez V, Moranta D, Llobet E, González-Nicolau M, Insua JL, Tomas JM, Sansonetti PJ, Tournebize R, Bengoechea JA - J. Biol. Chem. (2015)

Bottom Line: Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant.The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion.Importantly, these factors do not play a redundant role.

View Article: PubMed Central - PubMed

Affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.

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K. pneumoniae PulA T2SS is required for attenuation of NF-κB activation.A, immunoblot analysis of PulA levels in the outer membranes of Klebsiella strains. An asterisk marks a protein also recognized by the anti-PulA antibody, which served as a loading control. Data are representative of three independent experiments. B, adhesion (left panel) and internalization (right panel) of Klebsiella strains to A549 cells (n = 3). C, SEAP levels released by A549 cells left untreated (control (CON), white bar) or infected with the indicated strains. Scale bars represent mean ± S.E. (n = 3). D, immunoblot analysis of IκBα and tubulin levels in A549 cells left uninfected (control) or infected for 3 h. Data are representative of three independent experiments. E, ELISA of IL-8 released by A549 cells left untreated (control, white bar) or infected for 12 h with the indicated strains (n = 3). F, immunoblots showing phosphorylated MAPKs and tubulin levels in cell extracts of A549 cells left uninfected (control, time 0) or infected with K. pneumoniae strains for different times. The results are representative of three independent experiments. G and H, immunoblot analysis of IκBα and tubulin levels in lysates of A549 cells infected with the indicated strains. The cells were left untreated, stimulated with IL-1β (1 ng/ml, 10 min), infected for 3 h, or infected for 3 h and then stimulated with IL-1β. In H, siRNA targets are indicated in the top row of each panel (AS−, AllStars control siRNA). Data are representative of three independent experiments. *, p < 0.05 (results are significantly different from the results for Kp52145; one-tailed t test). ns (not significant), p > 0.05 (for the indicated comparisons; one-tailed t test).
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Figure 6: K. pneumoniae PulA T2SS is required for attenuation of NF-κB activation.A, immunoblot analysis of PulA levels in the outer membranes of Klebsiella strains. An asterisk marks a protein also recognized by the anti-PulA antibody, which served as a loading control. Data are representative of three independent experiments. B, adhesion (left panel) and internalization (right panel) of Klebsiella strains to A549 cells (n = 3). C, SEAP levels released by A549 cells left untreated (control (CON), white bar) or infected with the indicated strains. Scale bars represent mean ± S.E. (n = 3). D, immunoblot analysis of IκBα and tubulin levels in A549 cells left uninfected (control) or infected for 3 h. Data are representative of three independent experiments. E, ELISA of IL-8 released by A549 cells left untreated (control, white bar) or infected for 12 h with the indicated strains (n = 3). F, immunoblots showing phosphorylated MAPKs and tubulin levels in cell extracts of A549 cells left uninfected (control, time 0) or infected with K. pneumoniae strains for different times. The results are representative of three independent experiments. G and H, immunoblot analysis of IκBα and tubulin levels in lysates of A549 cells infected with the indicated strains. The cells were left untreated, stimulated with IL-1β (1 ng/ml, 10 min), infected for 3 h, or infected for 3 h and then stimulated with IL-1β. In H, siRNA targets are indicated in the top row of each panel (AS−, AllStars control siRNA). Data are representative of three independent experiments. *, p < 0.05 (results are significantly different from the results for Kp52145; one-tailed t test). ns (not significant), p > 0.05 (for the indicated comparisons; one-tailed t test).

Mentions: Two transposon insertions were found in the pulC and loci. pulC is the first locus of the Klebsiella T2SS that secrets the enzyme pullulanase encoded by pulA (53). yacC encodes for a lipoprotein containing a domain related to the pulS/outS family but for which the exact function has not yet been described. To help understand the contribution of Klebsiella T2SS to NF-κB attenuation, we constructed a pulA mutant, because PulA is the only known protein secreted by K. pneumoniae T2SS (54). Immunoblot analysis confirmed the absence of the enzyme pullulanse in the outer membranes of the pulC and yacC mutants and, as expected, also in the outer membrane of the pulA mutant (Fig. 6A). The amount of cell-bound CPS expressed by the mutants was quantified, and no differences were found between the CPS expressed by the wild type (11.49 ± 1.91 mg/107 cells) and the CPS expressed by any of the mutants (52ΔpulA, 15.21 ± 3.65 mg/107 cells; 52pulC::tn5, 9.44 ± 2.91 mg/107 cells; and 52yacC::tn5, 13.26 ± 4.97 mg/107 cells; for each comparison between wild-type CPS levels and mutant levels, p > 0.05, one-tailed Student's t test). Control experiments revealed that the adhesion levels of the pulC and yacC mutants were higher than those of the pulA and Kp52145 strains, which in turn, were not significantly different (Fig. 6B). In contrast, no significant differences were found in the internalization to cells between any of the strains (Fig. 6B).


Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling.

Tomás A, Lery L, Regueiro V, Pérez-Gutiérrez C, Martínez V, Moranta D, Llobet E, González-Nicolau M, Insua JL, Tomas JM, Sansonetti PJ, Tournebize R, Bengoechea JA - J. Biol. Chem. (2015)

K. pneumoniae PulA T2SS is required for attenuation of NF-κB activation.A, immunoblot analysis of PulA levels in the outer membranes of Klebsiella strains. An asterisk marks a protein also recognized by the anti-PulA antibody, which served as a loading control. Data are representative of three independent experiments. B, adhesion (left panel) and internalization (right panel) of Klebsiella strains to A549 cells (n = 3). C, SEAP levels released by A549 cells left untreated (control (CON), white bar) or infected with the indicated strains. Scale bars represent mean ± S.E. (n = 3). D, immunoblot analysis of IκBα and tubulin levels in A549 cells left uninfected (control) or infected for 3 h. Data are representative of three independent experiments. E, ELISA of IL-8 released by A549 cells left untreated (control, white bar) or infected for 12 h with the indicated strains (n = 3). F, immunoblots showing phosphorylated MAPKs and tubulin levels in cell extracts of A549 cells left uninfected (control, time 0) or infected with K. pneumoniae strains for different times. The results are representative of three independent experiments. G and H, immunoblot analysis of IκBα and tubulin levels in lysates of A549 cells infected with the indicated strains. The cells were left untreated, stimulated with IL-1β (1 ng/ml, 10 min), infected for 3 h, or infected for 3 h and then stimulated with IL-1β. In H, siRNA targets are indicated in the top row of each panel (AS−, AllStars control siRNA). Data are representative of three independent experiments. *, p < 0.05 (results are significantly different from the results for Kp52145; one-tailed t test). ns (not significant), p > 0.05 (for the indicated comparisons; one-tailed t test).
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Figure 6: K. pneumoniae PulA T2SS is required for attenuation of NF-κB activation.A, immunoblot analysis of PulA levels in the outer membranes of Klebsiella strains. An asterisk marks a protein also recognized by the anti-PulA antibody, which served as a loading control. Data are representative of three independent experiments. B, adhesion (left panel) and internalization (right panel) of Klebsiella strains to A549 cells (n = 3). C, SEAP levels released by A549 cells left untreated (control (CON), white bar) or infected with the indicated strains. Scale bars represent mean ± S.E. (n = 3). D, immunoblot analysis of IκBα and tubulin levels in A549 cells left uninfected (control) or infected for 3 h. Data are representative of three independent experiments. E, ELISA of IL-8 released by A549 cells left untreated (control, white bar) or infected for 12 h with the indicated strains (n = 3). F, immunoblots showing phosphorylated MAPKs and tubulin levels in cell extracts of A549 cells left uninfected (control, time 0) or infected with K. pneumoniae strains for different times. The results are representative of three independent experiments. G and H, immunoblot analysis of IκBα and tubulin levels in lysates of A549 cells infected with the indicated strains. The cells were left untreated, stimulated with IL-1β (1 ng/ml, 10 min), infected for 3 h, or infected for 3 h and then stimulated with IL-1β. In H, siRNA targets are indicated in the top row of each panel (AS−, AllStars control siRNA). Data are representative of three independent experiments. *, p < 0.05 (results are significantly different from the results for Kp52145; one-tailed t test). ns (not significant), p > 0.05 (for the indicated comparisons; one-tailed t test).
Mentions: Two transposon insertions were found in the pulC and loci. pulC is the first locus of the Klebsiella T2SS that secrets the enzyme pullulanase encoded by pulA (53). yacC encodes for a lipoprotein containing a domain related to the pulS/outS family but for which the exact function has not yet been described. To help understand the contribution of Klebsiella T2SS to NF-κB attenuation, we constructed a pulA mutant, because PulA is the only known protein secreted by K. pneumoniae T2SS (54). Immunoblot analysis confirmed the absence of the enzyme pullulanse in the outer membranes of the pulC and yacC mutants and, as expected, also in the outer membrane of the pulA mutant (Fig. 6A). The amount of cell-bound CPS expressed by the mutants was quantified, and no differences were found between the CPS expressed by the wild type (11.49 ± 1.91 mg/107 cells) and the CPS expressed by any of the mutants (52ΔpulA, 15.21 ± 3.65 mg/107 cells; 52pulC::tn5, 9.44 ± 2.91 mg/107 cells; and 52yacC::tn5, 13.26 ± 4.97 mg/107 cells; for each comparison between wild-type CPS levels and mutant levels, p > 0.05, one-tailed Student's t test). Control experiments revealed that the adhesion levels of the pulC and yacC mutants were higher than those of the pulA and Kp52145 strains, which in turn, were not significantly different (Fig. 6B). In contrast, no significant differences were found in the internalization to cells between any of the strains (Fig. 6B).

Bottom Line: Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant.The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion.Importantly, these factors do not play a redundant role.

View Article: PubMed Central - PubMed

Affiliation: From the Infection and Immunity Program, Fundación de Investigación Sanitaria de las Islas Baleares (FISIB), 07110 Mallorca, Spain, the Instituto de Investigación Sanitaria de Palma (IdisPa), 07120 Mallorca, Spain, the Centro de Investigación Biomédica en Red Enfermedades Respiratorias (CIBERES), 28029 Madrid, Spain.

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Related in: MedlinePlus