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. 2015 Mar;83(3):986-95.
doi: 10.1128/IAI.02955-14. Epub 2014 Dec 29.

New role for human α-defensin 5 in the fight against hypervirulent Clostridium difficile strains (VSports注册入口)

Lucinda Furci  1 , Rossella Baldan  2 , Valentina Bianchini  2 , Alberto Trovato  2 , Cristina Ossi  3 , Paola Cichero  3 , Daniela M Cirillo  2
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New role for human α-defensin 5 in the fight against hypervirulent Clostridium difficile strains (V体育ios版)

Lucinda Furci et al. Infect Immun. 2015 Mar.

Abstract

Clostridium difficile infection (CDI), one of the most common hospital-acquired infections, is increasing in incidence and severity with the emergence and diffusion of hypervirulent strains. CDI is precipitated by antibiotic treatment that destroys the equilibrium of the gut microbiota. Human α-defensin 5 (HD5), the most abundant enteric antimicrobial peptide, is a key regulator of gut microbiota homeostasis, yet it is still unknown if C. difficile, which successfully evades killing by other host microbicidal peptides, is susceptible to HD5. We evaluated, by means of viability assay, fluorescence-activated cell sorter (FACS) analysis, and electron microscopy, the antimicrobial activities of α-defensins 1 and 5 against a panel of C. difficile strains encompassing the most prevalent epidemic and hypervirulent PCR ribotypes in Europe (012, 014/020, 106, 018, 027, and 078). Here we show that (i) concentrations of HD5 within the intestinal physiological range produced massive C. difficile cell killing; (ii) HD5 bactericidal activity was mediated by membrane depolarization and bacterial fragmentation with a pattern of damage peculiar to C VSports手机版. difficile bacilli, compared to commensals like Escherichia coli and Enterococcus faecalis; and (iii) unexpectedly, hypervirulent ribotypes were among the most susceptible to both defensins. These results support the notion that HD5, naturally present at very high concentrations in the mucosa of the small intestine, could indeed control the very early steps of CDI by killing C. difficile bacilli at their germination site. As a consequence, HD5 can be regarded as a good candidate for the containment of hypervirulent C. difficile strains, and it could be exploited in the therapy of CDI and relapsing C. difficile-associated disease. .

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Figures

FIG 1
FIG 1
HD5 and HNP1 inhibit C. difficile growth in vitro. (A) C. difficile reference strain CD630 (RT012) and clinical epidemic strains CD369 (RT018) and CD349 (RT027). (B) Comparison with α-defensin activity against E. coli ATCC 25922 and E. faecalis ATCC 29212. All strains were exposed for 2 h at 37°C to HD5, HNP1, or unrelated control peptide RL26495 at concentrations ranging from 0.1 to 14 μM. The number of CFU was determined in triplicate and expressed as percentage of the initial inoculum. The means ± SEMs for at least three independent experiments are shown.
FIG 2
FIG 2
Bactericidal effects of HD5 and HNP1 on C. difficile. (A) Transmission electron micrographs of α-defensin-treated C. difficile. Suspensions of C. difficile CD630 (RT012) bacilli were incubated in the absence or in the presence of 7 μM HD5, HNP1, or control peptide RL26495. Arrowheads indicate cell wall detachment or severe leakage of cytoplasmic contents; arrows indicate mesosome-like structures and fibers extending from the cell surface. (B) Dose-dependent bactericidal effects of HD5 and HNPl against C. difficile R20291 (RT027). Bacteria were incubated in the presence or absence of increasing concentrations of α-defensins, and the degrees of membrane depolarization [DiBAC4(3) binding (FLl)] and bacterial fragmentation (FS) were quantified by FACS analysis. Gate A, undamaged bacteria; gate B, depolarized bacteria; gate C, bacterial cell fragments. All plots are representative of at least four independent experiments.
FIG 3
FIG 3
Killing of C. difficile by α-defensins results in a peculiar pattern of damage. (A) Dot blots of C. difficile CD630 (RT012), E. coli ATCC 25922, and E. faecalis ATCC 29212 populations after incubation with 7 μM HD5, HNP1, or control peptide RL26495. Membrane depolarization and cell size were recorded by FACS analysis. (B) Quantification of dose-dependent bacterial damage. Percentages of undamaged bacteria (gate A), depolarized fluorescent bacteria (gate B), and bacterial cell fragments (gate C) were obtained by treatment with increasing concentrations of α-defensins (open symbols) or control peptide RL26495 (solid symbols) and analyzed by FACS. Circles, CD630; squares, E. coli ATCC 25922; triangles, E. faecalis ATCC 29212. Each data point is representative of 4 to 6 independent experiments. Error bars show mean values ± SEMs. *, P < 0.05; ***, P < 0.001 (compared to C. difficile) according to Fisher's exact test.
FIG 4
FIG 4
Susceptibility to α-defensins of C. difficile strains belonging to different PCR ribotypes. Bacterial suspensions were tested against 7 μM HD5 (A) and HNP1 (B) or control peptide (data not shown) for 2 h at 37°C. Values are means ± SEMs for C. difficile hypervirulent and other epidemic PCR ribotypes, E. coli ATCC 25922, and E. faecalis ATCC 29212. P values were calculated by the Wilcoxon Mann-Whitney rank sum 2-tailed test.
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