Human alpha-defensins neutralize anthrax lethal toxin and protect against its fatal consequences

Abstract

Anthrax caused by Bacillus anthracis represents a major bioterroristic threat. B. anthracis produces lethal toxin (LeTx), a combination of lethal factor (LF) and protective antigen that plays a major role in anthrax pathogenesis. We demonstrate that human neutrophil alpha-defensins are potent inhibitors of LF. The inhibition of LF by human neutrophil protein (HNP-1) was noncompetitive. HNP-1 inhibited cleavage of a mitogen-activated protein kinase kinase and restored impaired mitogen-activated protein kinase signaling in LeTx-treated macrophages. HNP-1 rescued murine macrophages from B. anthracis-induced cytotoxicity, and in vivo treatment with HNP-1-3 protected mice against the fatal consequences of LeTx.

Fig. 1.

HNP-1 protects macrophages against B. anthracis-induced cell death. (a) RAW 264.7 cells were infected with B. anthracis spores and then treated with the indicated amounts of HNP-1. Cytotoxicity was determined by measuring released lactate dehydrogenase levels. (b)An in vitro killing assay was performed against spores in the presence or absence of 1 μM HNP-1. After the indicated incubation times, colony-forming units (CFU) were determined.

Fig. 2.

Human α-defensins protect macrophages against cytolysis by anthrax LeTx. (a) RAW 264.7 cells were treated with LeTx (400 ng/ml LF and 1,600 ng/ml PA) in the presence of the indicated amounts of HNP-1, HNP-2, or LL-37. Cell viability was determined by methyl thiazole tetrazolium (MTT) assay. (b) RAW 264.7 cells were treated with LeTx (400 ng/ml LF and 1,600 ng/ml PA) in the presence of 7 μM HNP-1 or LL-37. Five hours after treatment, cells were stained with trypan blue. (c) Viability of RAW 264.7 cells was determined by MTT assay after treatment with LeTx (400 ng/ml LF and 1,600 ng/ml PA) and various concentrations of HNP-1. This assay was performed in RPMI medium 1640 supplemented with 5% FCS.

Fig. 4.

HNP-1 is an inhibitor of LF. (a) In vitro translated MKK3b was incubated for 1 h with the indicated amounts of LF and either HNP-1 or magainin I. Cleavage of MKK3b was analyzed by SDS/PAGE and autoradiography. (b) Collagen was incubated with MMP-1 in the presence of the indicated amounts of HNP-1 or MMP inhibitor. (c) PA was incubated with furin in the presence of HNP-1 or hexa-d-arginine (D6R). (d) RAW 264.7 cells were incubated with HNP-1 at 37°C. After 1 h, the medium was removed and replaced with fresh medium containing LeTx (400 ng/ml LF and 1,600 ng/ml PA). Cells were incubated further at 37°C for 5 h. Viability was determined by MTT assay.

Fig. 6.

HNP-1-3 protects BALB/c mice from LeTx intoxication. Mice were injected with 50 μg of LF plus 50 μg of PA per mouse through one tail vein and then were immediately injected with PBS, 500 μg of purified HNP-1-3, or 500 μg of LL-37 through the other tail vein. Three animals per group were used for this experiment. In another set of experiments, administration of 500 μg of HNP-1-3 achieved 100% protection (data not shown).

Fig. 3.

HNP-1 inhibits proteolysis of MKK in LeTx-treated macrophages. (a) LeTx was added to RAW 264.7 macrophages with (+) or without (-) HNP-1. At the indicated time points, cell lysates were prepared and assessed by Western blotting with an anti-MKK3 antibody. (b) RAW 264.7 cells were treated (+) with LeTx (200 ng/ml LF and 1,600 ng/ml PA) and HNP-1 (30 μM). Two hours after treatment, cells were stimulated with 10 μg/ml B. subtilis lipoteichoic acid (LTA) for 30 min, and the lysates were assessed by immunoblotting with antibodies against MAPKs (Total) and their phosphorylated forms (Phospho). ERK, extracellular signal-regulated kinase.

Fig. 5.

Characterization of LF inhibition by HNP-1. (a) HNP-1 inhibited 50% of LF activity at a concentration of 190 ± 33 nM. (b) Lineweaver-Burk plot indicates noncompetitive inhibition mode. (c) A plotting of V_max versus concentrations of LF confirms that HNP-1 is a reversible noncompetitive inhibitor. dE, release of p-Nitroaniline. (d) DTT-treated HNP-1 did not show any significant effect on LF.