Date Published: August 7, 2012
Publisher: Public Library of Science
Author(s): Marta Castagnini, Monica Picchianti, Eleonora Talluri, Massimiliano Biagini, Mariangela Del Vecchio, Paolo Di Procolo, Nathalie Norais, Vincenzo Nardi-Dei, Enrico Balducci, Michel R. Popoff. http://doi.org/10.1371/journal.pone.0041417
Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and β- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and β- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues.
Human defensins are cationic multifunctional arginine-rich peptides (molecular masses ranging from 3.5 to 6 kDa) characterized by three intramolecular disulfide bridges that stabilize their structure –. Defensins display microbicidal activity against a wide spectrum of Gram-negative and Gram-positive bacteria, fungi and viruses . They are also cytotoxic for epithelial cells and chemotactic for T-cells. Based on the presence of six conserved cysteine residues and sequence homology, human defensins are grouped into α- and β- defensins. The first group (α-defensins) includes human neutrophil peptides (HNP)-1 to 4, major components of the azurophilic granules of neutrophils, and two enteric human defensins, HD-5 and HD-6, isolated from the granules of Paneth cells in the small intestine, . The second group (β-defensins), is mainly expressed in epithelial cells of various organs –. It has been shown that ADP-ribosylation of HNP-1 on arginine 14 reduces its antimicrobial and cytotoxic activities . Mono ADP-ribosylation consists in the enzymatic transfer of the single ADP-ribose moiety of NAD to specific amino-acid residues of acceptor proteins coupled to the release of nicotinamide (nam) . In mammals this reaction is catalyzed by a family of ADP-ribosyltransferases (ART1-5) , , while the best studied ADP-ribosylation reactions are those catalyzed by bacterial ADP-ribosylating toxins. The ADP-ribosylation of a large panel of host proteins catalyzed by bacterial toxins leads to the interruption of cellular metabolic and regulatory pathways causing severe diseases . Vibrio cholerae toxin (CT) , Escherichia coli heat labile enterotoxin (LT) , Pseudomonas aeruginosa exoenzyme S (ExoS)  and the recently discovered NarE, a toxin-like protein from Neisseria meningitidis, recognize arginine as an ADP-ribose acceptor in a similar fashion to ART1 and ART5 , . Arginine specificity is conferred to ARTs by the presence of the R-S-EXE triad signature in the active site . Recent studies indicated that α-defensins display a novel biological function consisting in the ability to neutralize the activity of potent bacterial toxins like lethal factor, a metalloprotease produced by Bacillus anthracis, and toxin B produced by Clostridium difficile. Moreover it has been shown that HNP1-3 neutralize the cytotoxic effects exerted by diphtheria toxin (DT) and Pseudomonas aeruginosa exotoxin A (ETA), while they were inactive on CT and pertussis toxin (PT) . The neutralization of toxins with selected amino-acid specificity prompted us to hypothesize that mono ADP-ribosylation of specific amino-acids may block defensin ability to inhibit the activities of toxins. Therefore, we evaluated whether HNP-1 could be recognized by arginine-specific bacterial ARTs. In the present paper we provide evidence that CT and LT ADP-ribosylated α- and β- defensins, which thus represent novel substrates for these bacterial ARTs. On the other hand, NarE and ExoS did not modify either α- or β- defensins. Interestingly, unmodified HNP-1 exerted inhibition on NarE transferase activity suggesting a regulatory role. While the ADP-ribosyltransferase activity was inhibited by HNP-1, the NAD-glycohydrolase (NADase) activity remained unaltered. Furthermore, HNP-1 strongly enhanced the auto-ADP-ribosylation of NarE, a recently discovered catalytic activity of this toxin. Overall, our data highlight the interplay between ADP-ribosylating toxins and human defensins.
To establish whether arginine-specific bacterial ARTs can ADP-ribosylate HNP-1, we incubated HNP-1 with the catalytic A subunit of CT (CTA), LT (LTA), ExoS or NarE individually. As shown in Fig 1A, CTA and LTA catalyzed the transfer of the biotin-ADP-ribose from biotin-NAD to HNP-1 with an efficiency that was comparable to that of ART1 (Fig. 1 B). This incorporation was strongly reduced after heat-inactivation of the toxins (Fig. 1 A). CTA and LTA have both transferase, and NADase activity , . The latter produces ADP-ribose that can react non-enzymatically with lysine residues in proteins . However, since the incorporation of biotin-ADP-ribose on HNP-1 was strongly reduced in the presence of 2 mM unlabelled NAD (200-fold excess) but not with 2 mM ADP-ribose, we could rule out that the reaction was non-enzymatic. The enzymatic nature of the reaction was further confirmed in the dose dependent (Fig. 1 D) and time-course experiments (Fig. 1 E), showing that the increase of modified peptide is dependent on the level of free substrate and by the incubation time. In this respect the purification grade of the toxins (Fig. 1 C) is shown, to exclude the possibility of a blockage of the peptide by contaminating proteins. Under the same conditions, HNP-1 was a poor substrate for NarE (Fig. 1 A) compared to ART1 (Fig. 1 B). ExoS was completely inactive towards HNP-1 (data not shown), in agreement with a previous report . ADP-ribosylation of antimicrobials by CT and LT is not restricted to HNP-1. Also HBD1, which contains only one arginine at position 29 and is constitutively expressed by epithelial cells in the airway , was ADP-ribosylated (Fig. 2 A). As for HNP-1, labelling did not occur in the presence of heat-inactivated toxins. The addition of an excess of unlabelled NAD to the reaction mixture decreased the incorporation of an ADP-ribose moiety on HBD1, while the incorporation of biotin-ADP-ribose on HBD1 was not reduced by the presence of 2 mM ADP-ribose. Dose-dependent reactions and time course experiments support the enzymatic nature of the modification also in the case of HBD1 (Fig. 2 C, D). NarE and ExoS did not modify HBD1 (data not shown). In contrast with a previous report , HBD1 was modified by ART1 to the same extent of HNP-1 (Fig. 2 B). To confirm that the observed modifications corresponded to the addition of the ADP-ribose unit, the products of the reaction of CTA with HNP-1 in the presence of NAD were identified by MALDI-TOF MS. As shown in Fig. 3, these included a peptide of 3442.12 Da, consistent with unmodified HNP-1 (theoretical mass: 3442.1 Da) and a peptide of 3983.15 Da. Although the amount of the modified peptide was low, we can conclude that the reaction is specific since we observed a mass increase consistent with mono ADP-ribosylated HNP-1 (theoretical mass: 3983.1 Da). Similar results were obtained with the LT catalyzed reaction (data not shown). To identify the preferred arginine residue of HNP-1 modified by CTA and LTA, we used two variants of HNP-1 in which a lysine replaced the arginines at positions 14 (HNP-1-R14K) or 15 (HNP-1-R15K). We found that CTA and LTA selectively ADP-ribosylated HNP-1 at R14 (Fig. 4). Recent studies have shown that when HNP-1 is not recognized as a substrate, it is able to inhibit the ART activity of bacterial toxins such as ETA and DT  and also the eukaryotic ART5 multiple catalytic activities . Therefore, since HNP-1 is only weakly modified by NarE, we investigated whether HNP-1 exerts a similar effect on NarE activities. The addition of HNP-1 to the reaction mixture seems to reduce the ADP-ribosyltransferase activity in a concentration dependent fashion (Fig. 5, grey bars) while the NADase activity was not greatly affected (Fig. 5, white bars). In contrast, HNP-1 enhanced the auto-ADP-ribosylation of NarE (Fig. 6 upper panel), a recently discovered activity of this toxin (Picchianti et al. manuscript in preparation).
ADP-ribosylating toxins are usually secreted by bacterial pathogens in the host environment. Some of them, which possess arginine-specificity, could recognize arginine-rich peptides such as α- and β- defensins as substrates. Both α- and β- defensins are released by neutrophils and epithelial cells respectively in high amounts at inflammatory sites. In this report we present evidence that synthetic HNP-1 and HBD1 are ADP-ribosylated in vitro by CTA and LTA. In contrast they are not recognized as substrates by ExoS and only poorly by NarE, suggesting specificity for both bacterial toxins and substrates. The artificial kemptide (PKA peptide substrate), which contains a di-arginine motif, was modified by CT on the first arginine of the motif while a mammalian ART recognized the second arginine within the R-R motif , . In contrast, our data indicate R14 as the preferred modification site, since the HNP-1-R14K was not ADP-ribosylated by the ADP-ribosylating toxins used in this study. Our findings are in contrast with studies performed by other groups, which failed to show toxin-catalyzed incorporation of the ADP-ribose unit on defensins , . Others evaluated the presence of the ADP-ribosylated-HNP-1 by monitoring the absorbance of the modified peptide in reversed-phase chromatography, but not being successful in identifying it , . Therefore we chose a chemiluminescence assay to detect ADP-ribosylation because of the higher sensitivity, allowing the detection of small amounts of modified HNP-1.