Klebsiella pneumoniae capsule polysaccharide impedes the expression of beta-defensins by airway epithelial cells

Abstract

Human beta-defensins (hBDs) contribute to the protection of the respiratory tract against pathogens. It is reasonable to postulate that pathogens have developed countermeasures to resist them. Klebsiella pneumoniae capsule polysaccharide (CPS), but not the lipopolysaccharide O antigen, mediated resistance against hBD1 and hBD2. hBD3 was the most potent hBD against Klebsiella. We investigated the possibility that as a strategy for survival in the lung, K. pneumoniae may not activate the expression of hBDs. Infection of A549 and normal human bronchial cells with 52145-Deltawca(K2), a CPS mutant, increased the expression of hBD2 and hBD3. Neither the wild type nor the lipopolysaccharide O antigen mutant increased the expression of hBDs. In vivo, 52145-Deltawca(K2) induced higher levels of mBD4 and mBD14, possible mouse orthologues of hBD2 and hBD3, respectively, than the wild type. 52145-Deltawca(K2)-dependent upregulation of hBD2 occurred via NF-kappaB and mitogen-activated protein kinases (MAPKs) p44/42, Jun N-terminal protein kinase (JNK)-dependent pathways. The increase in hBD3 expression was dependent on the MAPK JNK. 52145-Deltawca(K2) engaged Toll-like receptors 2 and 4 (TLR2 and TLR4) to activate hBD2, whereas hBD3 expression was dependent on NOD1. K. pneumoniae induced the expression of CYLD and MKP-1, which act as negative regulators for 52145-Deltawca(K2)-induced expression of hBDs. Bacterial engagement of pattern recognition receptors induced CYLD and MKP-1, which may initiate the attenuation of proinflammatory pathways. The results of this study indicate that K. pneumoniae CPS not only protects the pathogen from the bactericidal action of defensins but also impedes their expression. These features of K. pneumoniae CPS may facilitate pathogen survival in the hostile environment of the lung.

FIG. 1.

Roles of CPS and the LPS O antigen in the susceptibility of K. pneumoniae to human β-defensins. The survival of bacteria (expressed as a percentage of the number of colonies of the same strain not exposed to agents) in the presence of 1 μg/ml of hBD1, hBD2, or hBD3 is shown. Error bars represent standard deviations from the means for three experiments, each one run in duplicate. Asterisks indicate results that are significantly different (P < 0.05 by a one-tailed t test) from those for wild-type K. pneumoniae 52145 (Kp52145).

FIG. 2.

The K. pneumoniae CPS mutant induces the expression of human β-defensins in airway epithelial cells. (A) NHBE cells were infected with either Kp52145 (filled bars), the CPS mutant 52145-Δwca_K2 (light shaded bars), or the LPS O antigen mutant 52O21 (dark shaded bars) for different times, and levels of human β-defensin (hBD1, hBD2, and hBD3) mRNA were assessed by RT-qPCR. CON, control (noninfected cells). Data are presented as means ± standard deviations (n = 3). (B) Experiments were similar to those described for panel A except that A549 cells were used instead of NHBE cells. Data are presented as means ± standard deviations (n = 4). (C) A549 cells were transfected with the hBD2 or hBD3 luciferase reporter gene and the Renilla luciferase plasmid. Cells were infected with K. pneumoniae strains, and hBD2 or hBD3 promoter activation was measured as relative luciferase activity 6 h postinfection. CON, control (noninfected cells). Data are means ± standard deviations (n = 3). Asterisks indicate results that are significantly different (P < 0.05 by a one-tailed t test) from those for noninfected cells.

FIG. 3.

β-Defensin expression in the lungs of mice after K. pneumoniae infection. Mice were either left uninfected (control [CON]) (open bars) (n = 5) or infected with wild-type K. pneumoniae 52145 (filled bars) (n = 15) or 52145-Δwca_K2 (shaded bars) (n = 15). The expression of mouse β-defensin (mBD1, mBD4, and mBD14) mRNA in whole lungs at the indicated time points postinfection was assessed by RT-qPCR (5 mice per time point). Data are means ± standard deviations. *, P < 0.05 for comparison with noninfected mice; ▵, P < 0.05 for comparison with wild-type K. pneumoniae-infected mice.

FIG. 4.

NF-κB and MAPKs are involved in the expression of hBD2 and hBD3 induced by the K. pneumoniae CPS mutant. (A) A549 cells were transfected with the hBD2 luciferase reporter gene and the Renilla luciferase plasmid. Cells were infected with 52145-Δwca_K2, and hBD2 promoter activation was measured as relative luciferase activity 6 h postinfection. Where indicated, cells were treated with chemical inhibitors 1 h prior to infection or with DMSO, which was the vehicle solution for all these compounds. The compounds used were CAPE (NF-κB inhibitor) (15 μg/ml), SB203580 (MAPK p38 inhibitor) (5 μM), SP600125 (MAPK JNK inhibitor) (10 μM), and U0126 extracellular signal-regulated kinase (ERK) (MAPK p44/p42 inhibitor) (2 μM). CON, control (noninfected cells). (B) Experiments were similar to those described for panel A except that the hBD3 luciferase reporter gene was used instead of the hBD2 luciferase reporter gene and luciferase activity was measured 16 h postinfection. Values are means ± standard deviations (n = 3) for panels A and B. *, P < 0.05 for comparison with noninfected cells; ▵, P < 0.05 for comparison with K. pneumoniae CPS mutant-infected cells treated with DMSO. (C) (Top) Immunoblot showing IκBα levels in extracts of A549 cells infected with Kp52145 or 52145-Δwca_K2 for different times. (Bottom) Immunoblot showing tubulin levels under the same conditions. The results are representative of three independent experiments. (D) Immunoblots showing phospho-p38 (P-p38), total p38 (p38), phospho-p44/42 (P-p44/42), total p44/42 (p44/42), phospho-JNK (P-JNK), and total JNK (JNK) levels in extracts of A549 cells infected with Kp52145 or 52145-Δwca_K2 for different times. The results are representative of three independent experiments. CON, noninfected cells; -, infected cells without any other treatment.

FIG. 5.

Dissection of the receptors involved in the induction of human β-defensin expression upon infection by the K. pneumoniae CPS mutant. (A) A549 cells were cotransfected with different siRNAs, the hBD2 luciferase reporter gene, and the Renilla luciferase plasmid. Cells either were left uninfected (open bars) or were infected with 52145-Δwca_K2 (filled bars), and hBD2 promoter activation was measured as relative luciferase activity 6 h postinfection. (B) A549 cells were cotransfected with different siRNAs, the hBD3 luciferase reporter gene, and the Renilla luciferase plasmid. Cells either were left uninfected (open bars) or were infected with 52145-Δwca_K2 (filled bars), and hBD3 promoter activation was measured as relative luciferase activity 16 h postinfection. Data are means ± standard deviations (n = 3). Asterisks indicate results that are significantly different (P < 0.05 by a one-tailed t test) from the results for infected cells treated with a control siRNA.

FIG. 6.

CYLD and MKP-1 act as negative regulators for K. pneumoniae CPS mutant-induced expression of human β-defensins. (A) A549 cells were cotransfected with either a CYLD siRNA, an MKP-1 siRNA, or a control siRNA; the hBD2 or hBD3 luciferase reporter gene; and the Renilla luciferase plasmid. Cells either were left uninfected (control [CON]) (open bars) or were infected with 52145-Δwca_K2 (filled bars), and hBD promoter activation was measured as relative luciferase activity. Data are means ± standard deviations. Asterisks indicate results that are significantly different (P < 0.05 by a one-tailed t test) from the results for infected cells treated with a control siRNA. (B) CYLD and MKP-1 mRNA levels in A549-infected cells were assessed by RT-qPCR. Control, noninfected cells. Data are presented as means ± standard deviations (n = 3). *, P < 0.05 for comparison with noninfected cells. (C) Immunoblots showing CYLD, MKP-1, and tubulin levels in extracts of A549 cells infected with Kp52145 or 52145-Δwca_K2 for different times. Results are representative of four independent experiments. (D) A549 cells were cotransfected with either a CYLD siRNA, an MKP-1 siRNA, or a control siRNA; the hBD2 or hBD3 luciferase reporter gene; and the Renilla luciferase plasmid. Cells either were left uninfected (open bars) or were infected with Kp52145 (solid bars), and hBD promoter activation was measured as relative luciferase activity. CON, noninfected cells.

FIG. 7.

Dissection of receptors involved in the induction of CYLD (A) and MKP-1 (B) during infection with the K. pneumoniae CPS mutant. A549 cells were transfected either with a siRNA for TLR2, TLR4, or NOD1 or with a control siRNA, and after 48 h, they were infected with 52145-Δwca_K2 for 90 min. Immunoblots show CYLD or MKP-1 levels (top) or tubulin levels (bottom) in extracts of A549-infected cells. The results are representative of three independent experiments. The relative ratios of CYLD or MKP-1 (means ± standard deviations) obtained after densitometric analysis of gels are given at the bottom (n = 3). Asterisks indicate results that are significantly different (P < 0.05 by a one-tailed t test) from the results for infected cells treated with a control siRNA. CON, noninfected cells.