Bacterial Periplasmic Oxidoreductases Control the Activity of Oxidized Human Antimicrobial β-Defensin 1

Abstract

The antimicrobial peptide human β-defensin 1 (hBD1) is continuously produced by epithelial cells in many tissues. Compared to other defensins, hBD1 has only minor antibiotic activity in its native state. After reduction of its disulfide bridges, however, it becomes a potent antimicrobial agent against bacteria, while the oxidized native form (hBD1ox) shows specific activity against Gram-negative bacteria. We show that the killing mechanism of hBD1ox depends on aerobic growth conditions and bacterial enzymes. We analyzed the different activities of hBD1 using mutants of Escherichia coli lacking one or more specific proteins of their outer membrane, cytosol, or redox systems. We discovered that DsbA and DsbB are essential for the antimicrobial activity of hBD1ox but not for that of reduced hBD1 (hBD1red). Furthermore, our results strongly suggest that hBD1ox uses outer membrane protein FepA to penetrate the bacterial periplasm space. In contrast, other bacterial proteins in the outer membrane and cytosol did not modify the antimicrobial activity. Using immunogold labeling, we identified the localization of hBD1ox in the periplasmic space and partly in the outer membrane of E. coli However, in resistant mutants lacking DsbA and DsbB, hBD1ox was detected mainly in the bacterial cytosol. In summary, we discovered that hBD1ox could use FepA to enter the periplasmic space, where its activity depends on presence of DsbA and DsbB. HBD1ox concentrates in the periplasm in Gram-negative bacteria, which finally leads to bleb formation and death of the bacteria. Thus, the bacterial redox system plays an essential role in mechanisms of resistance against host-derived peptides such as hBD1.

Keywords: antimicrobial peptides; defensins; hBD1; innate host defense; periplasmic oxidoreductases DsbA and DsbB; redox regulation.

FIG 1

The antimicrobial activity of hBD1ox is specific for Gram-negative bacteria. HBD1ox and hBD1red were incubated with Gram-negative bacteria in an aerobic (A) or anaerobic (B) environment in the radial diffusion assay. Antimicrobial activity was measured by analyzing the diameter of the inhibition zone. A diameter of 2.5 mm (dotted line) is the diameter of the punched well. Data are presented as mean ± SEM from three independent experiments.

FIG 2

Antimicrobial activity against bacteria with mutations in the genes of interest. (A) Schematic overview of located membrane and periplasmic proteins in bacteria. (B to D) Two micrograms of hBD1ox and hBD1red was tested against E. coli strains with different protein knockouts in the outer membrane (B), cytosol (C), and periplasmic space (D) by radial diffusion assay. (D) HBD1ox shows a decreased antimicrobial activity against bacteria without DsbA or inner membrane proteins DsbB and TonB in comparison to the WT E. coli MC1000. The diameter of the inhibition zone was measured in the radial diffusion assay to determine the antimicrobial activity. A diameter of 2.5 mm (dotted line) is the diameter of the punched well. Results from experiments with wild-type E. coli MC1000 were pooled (n = 18) and used as the control for all the times when E. coli MC1000 mutants (n = 3) are assessed. All data are presented as mean ± SEM.

FIG 3

Bacterial oxidoreductases DsbA and DsbB are essential for the activity of hBD1ox. (A) Two micrograms of hBD1ox and hBD1red was tested against E. coli with protein double knockouts in the redox system and outer membrane. Bacteria without both oxidoreductases obtain resistance against hBD1ox (**, P = 0.0044; ****, P < 0.0001). (B) Membrane depolarization assay of E. coli MC1000 ΔdsbA ΔdsbB incubated with reduced and oxidized hBD1 for 1 h. HBD3ox (50 μg/ml) was used as positive control (+), and the negative control (−) was incubated without any peptide. HBD1ox is not able to significantly affect the bacterial membrane (**, P = 0.0067). (C) Four micrograms of hBD1ox was used in the plasmid induction experiment with either E. coli MC1000 ΔdsbA ΔdsbB, E. coli MC1000 ΔdsbA ΔdsbB harboring plasmids pQE60::dsbB and pBAD33::dsbA to generate the wild-type phenotype, or, as a control, E. coli MC1000 ΔdsbA ΔdsbB with empty plasmids pQE60 and pBAD33. Plasmid expression was during the 2.5 h of incubation, with addition of 0.4% l-arabinose and 2 mM IPTG every 30 min. HBD1ox shows antimicrobial activity against the mutant containing plasmids pQE60::dsbB and pBAD33::dsbA (**, P = 0.0036; ****, P < 0.0001). The diameter of the inhibition zone was measured in a radial diffusion assay to determine the antimicrobial activity. Results of representative radial diffusion assays are shown. A diameter of 2.5 mm (dotted line) is the diameter of the punched well. Data are presented as mean ± SEM from at least three independent biological replicates. Student's t test was used for comparison of normally distributed data.

FIG 4

HBD1ox induces bleb formation in E. coli. (A) WT E. coli MC1000 and E. coli MC1000 ΔdsbA ΔdsbB were treated with hBD1ox for 2 h. The samples were fixed in Karnovsky's reagent, and the morphology was analyzed with scanning electron microscopy. Electron microscopy pictures from one representative experiment are shown. Scale bar, 2 μm. (B) Visual analysis of electron microscopy pictures. Outer membrane vesicles (blebs) were counted and determined by four different experts. Data are presented as mean ± SEM from the analyzed bacteria (n = 10). The statistical significance was evaluated by using Student's t test (**, P = 0.0031).

FIG 5

Localization of hBD1ox in bacterial compartments. WT E. coli MC1000 and E. coli MC1000 ΔdsbA ΔdsbB were treated with hBD1ox for 2 h. The samples were fixed and incubated with antibodies against the hBD1ox. The secondary antibodies were conjugated to 6-nm gold particles (black points) and visualized by electron microscopy. (A) Electron microscopy pictures from one representative experiment. Scale bar, 0.2 μm. (B) Visual analysis of electron microscopy pictures. Gold-labeled hBD1ox was counted in bacterial periplasm or cytosol per bacterium. Gold particles per bacterium (percent) are represented (WT, n = 39; ΔdsbA ΔdsbB mutant, n = 30) as determined by four independent individuals.

FIG 6

Schematic model describing a potential mechanism of hBD1ox activity. (A) Proposed model for cell death in case of an intact DsbA/DsbB complex. HBD1ox can diffuse in the periplasm by using FepA or FhuA, where it can interact directly or indirectly with DsbA/DsbB. Subsequently, the accumulation of hBD1ox in the periplasm induces bleb formation and finally bacterial cell lysis by an unknown mechanism. (B) Hypothetical model for hBD1ox resistance in bacteria without a traditional DsbA/DsbB complex. Without DsbA/DsbB, hBD1ox diffuses into the cytosol and is finally degraded.