Human alpha-defensins neutralize toxins of the mono-ADP-ribosyltransferase family

Abstract

Various bacterial pathogens secrete toxins, which are not only responsible for fatal pathogenesis of disease, but also facilitate evasion of host defences. One of the best-known bacterial toxin groups is the mono-ADP-ribosyltransferase family. In the present study, we demonstrate that human neutrophil alpha-defensins are potent inhibitors of the bacterial enzymes, particularly against DT (diphtheria toxin) and ETA (Pseudomonas exotoxin A). HNP1 (human neutrophil protein 1) inhibited DT- or ETA-mediated ADP-ribosylation of eEF2 (eukaryotic elongation factor 2) and protected HeLa cells against DT- or ETA-induced cell death. Kinetic analysis revealed that inhibition of DT and ETA by HNP1 was competitive with respect to eEF2 and uncompetitive against NAD+ substrates. Our results reveal that toxin neutralization represents a novel biological function of HNPs in host defence.

Figure 1. Separation of reaction products of HNP1 after incubation with mono-ARTs

HNP1 was incubated with 50 mM potassium phosphate buffer (pH 7.5) alone, murine ART-1 (mART-1), DT, ETA, CT or PT and then analysed by reverse-phase HPLC. The arrows indicate the peaks corresponding to the ADP-ribose-HNP1.

Figure 2. Effect of HNP1 on DT- or ETA-treated cells

HeLa cells were treated with DT (A) or ETA (B) in the presence of the indicated amounts of HNP1 or LL-37. Bacterial toxins and peptides were treated at the same time and incubated for 24 h. Cell viability was determined by the MTT assay.

Figure 3. Effect of HNP1 on DT or ETA-mediated ADP-ribosylation of eEF2

HeLa cell lysates were incubated with DT (A) or ETA (B) in the presence of the indicated amounts of HNP1 or LL-37. The samples were analysed by SDS/PAGE followed by autoradiography. In the presence of HNP1 at 0.1, 0.5 or 2.5 μM, DT activity decreased to 25, 8 and 0.5% of control DT activity and ETA activity declined to 37, 12 and 1% of control ETA activity respectively.

Figure 4. Binding properties of HNP1 to DT and ETA

The binding curve for HNP1 with DTA (A) or ETAc (B) was determined from the quenching of the intrinsic protein fluorescence. The raw fluorescence quenching results were converted into fractional saturation values [20] and plotted against the HNP1 concentration.

Figure 5. Inhibitory properties of HNP1 against DTA and ETAc

The substrates, ϵ-NAD⁺ and eEF2, in the fluorescence-based ART assay (see the Experimental section) were used for examining the inhibitory properties of HNP1 against DTA (A) and ETAc (B). The IC₅₀ value was determined by non-linear regression curve fitting using Origin 6.1 (OriginLab, Northampton, MA, U.S.A.). The data were fitted to the exponential first-order decay function.

Figure 6. Mode of inhibition of HNP1 against DTA and ETAc

The NAD⁺-dependent DTA (A) and ETAc (B) assays were performed with the fixed amount of eEF2 and variable concentrations of HNP1 and ϵ-NAD⁺. The eEF2-dependent ETAc assay (C) was performed at a fixed concentration of ϵ-NAD⁺ while varying the amounts of eEF2 in the presence of HNP1. The data were analysed by linear regression analysis of the Hanes–Woolf plot. V₀, initial velocity of the progress curves for the ADPRT reaction in units of μM/s.

Figure 7. Protection of infected cells by HNP1

HeLa cells were infected with C. diphtheriae and then treated with the indicated amounts of HNP1. Cytotoxicity was determined by measuring lactate dehydrogenase levels released from HeLa cells.