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