Modification of

<p>β<b>-defensin by selected ADP-ribosyltransferases.</b> (<b>A</b>) HBD1 is ADP-ribosylated by CTA and LTA. HBD1 (3 µg, 38.18 µM)) was incubated with CTA (2.5 U) or LTA (8.9 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h (Toxin). Reactions were also performed in the presence of 2 mM NAD (Toxin + NAD) or 2 mM ADP-ribose (Toxin + ADP-ribose). Control reactions performed with heat-inactivated CTA or LTA (HI-Toxin) or in the absence of toxins (-Toxin) are also shown. The ADP-ribosylated peptides were separated by SDS-PAGE in a 10% NuPAGE gel and transferred to nitrocellulose. The membrane was treated as previously described, incubated with streptavidin-HRP conjugated (1∶10000 dilution) before visualization of the biotin-ADP-ribose labeled bands by chemiluminescence. (<b>B</b>) ADP-ribosylation of HBD1 by ART1. HBD1 (3 µg, 38.18 µM) was incubated with 6.8 U of ART1 (ART1) or heat-inactivated ART1 (HI-ART1) and 10 µM biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h. (<b>C</b>) HBD1 is ADP-ribosylated in a dose response fashion. HBD1 at the concentration shown in the Figure was incubated with CTA (2.5 U) or LTA (8.9 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h. (<b>D</b>) Time dependent ADP-ribosylation of HBD1. HBD1 (3 µg) was incubated with CTA (1.25 U) or LTA (4.45 U) using the same conditions above described. Times of incubation are indicated in the Figure. Molecular markers are on the left. Data shown are representative of two independent experiments.</p

Modification of HNP-1 by selected ADP-ribosyltransferases.

<p>(<b>A</b>) HNP-1 is ADP-ribosylated by CTA and LTA but only weakly by NarE. HNP-1 (3 µg, 43.56 µM) was incubated with CTA (2.5 U), LTA (8.9 U) or NarE (2 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h (Toxin). The same reactions were performed with heat-inactivated toxins (HI-Toxin), in the presence of 2 mM NAD (Toxin + NAD), or 2 mM ADP-ribose (Toxin + ADP-ribose). The ADP-ribosylated peptides were resolved by SDS-PAGE in a 10% NuPAGE gel, using MES as running buffer and transferred to nitrocellulose. After blocking with 5% BSA in PBS containing 0.05% Tween-20 (PBS-T) for 1 h, the blot was incubated with streptavidin-HRP conjugated (1∶10000 dilution) for 1 h at RT in the same buffer. The biotin-ADP-ribose labeled bands were visualized by chemiluminescence. (<b>B</b>) ART1 ADP-ribosylated HNP-1. HNP-1 (3 µg, 43.56 µM) was incubated with ART1 (6.8 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate, pH 7.5 at 30°C for 1 h (ART1). A control reaction with heat-inactivated ART1 is also shown (HI-ART1). (<b>C</b>) SDS-PAGE analysis of the purification grade of 2 µg each of CTA, LTA and NarE. (<b>D</b>) HNP-1 is ADP-ribosylated in a dose and response dependent manner by CTA and LTA. HNP-1 at the concentration shown in the Figure was incubated with CTA (2.5 U), or LTA (8.9 U) and 10 µM of biotin-NAD in 50 mM potassium phosphate buffer, pH 7.5, at 30°C for 1 h. (<b>E</b>) HNP-1 is ADP-ribosylated in time dependent fashion. HNP-1 (3 µg) was incubated with CTA (1.25 U) or LTA (4.45 U) using the same conditions above described for the times of incubation indicated in the Figure. Molecular markers are on the left. Data shown are representative of several experiments performed in the same conditions.</p

MALDI-TOF mass spectra of HNP-1 reaction with CTA.

<p>Mass spectra analysis confirmed the mono-ADP-ribosylation of HNP-1 by CTA after incubation at 30°C for 1h in the presence of 2 mM NAD. Upper panels (left side: spectrum of <i>m/z</i> 2500 – 5300, right side: zoomed spectrum of <i>m/z</i> 3800 – 4300) show the mass of the control reaction, i.e. HNP-1 incubated only with NAD without toxin (<i>m/z</i> 3442.12). Lower panels (left side: spectrum of <i>m/z</i> 2500 – 5300, right side: zoomed spectrum of <i>m/z</i> 3800–4300) represent the unmodified HNP-1 peptide and the product of ADP-ribosylation peptide by CTA (<i>m/z</i> 3983.15). Stars (*) correspond to Sinapinic Acid adducts (+206 Da).</p

HNP-1 is ADP-ribosylated at R14.

<p>HNP-1 R14K and HNP-1 R15K protein variants (3 µg, 43.85 µM) were incubated with CTA (2.5 U), LTA (8.9 U) or ART1 (5.5 U) in the presence of 10 µM biotin-NAD, in 50 mM potassium phosphate buffer, pH 7.5 at 30°C for 1 h. The ADP-ribosylated peptides were separated by SDS-PAGE in a 10% NuPAGE gel and transferred to nitrocellulose. Membranes treated as previously described, were incubated with streptavidin-HRP conjugated (1∶10000 dilution) before visualization of the biotin-ADP-ribose labeled bands by chemiluminescence. Here shown in comparison with the modification of HNP-1 wild-type in the same reaction conditions.</p

Human defensins.

<p>Human defensins.</p

HNP-1 enhanced the auto-ADP-ribosylation of NarE.

<p>Purified NarE (0.4 µg) was auto-ADP-ribosylated with 10 µM biotin-NAD in 50 mM potassium phosphate pH 7.5 at 30°C for 3 and 6 h in the presence of the indicated concentrations of HNP-1. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. To detect biotin-ADP-ribosylated NarE, the membrane was incubated with streptavidin-HRP (upper panel). For western blotting the nitrocellulose membrane was incubated with rabbit polyclonal α-NarE (1∶10000 dilution) and with α-rabbit HRP-conjugated (lower panel). Labeled bands were detected by chemioluminescence. Data shown are representative of two independent experiments.</p

SslE Elicits Functional Antibodies That Impair <i>In Vitro</i> Mucinase Activity and <i>In Vivo</i> Colonization by Both Intestinal and Extraintestinal <i>Escherichia coli</i> Strains

<div><p>SslE, the <u>S</u>ecreted and <u>s</u>urface-associated <u>l</u>ipoprotein from <i><u>E</u>scherichia coli</i>, has recently been associated to the M60-like extracellular zinc-metalloprotease sub-family which is implicated in glycan recognition and processing. SslE can be divided into two main variants and we recently proposed it as a potential vaccine candidate. By applying a number of <i>in vitro</i> bioassays and comparing wild type, knockout mutant and complemented strains, we have now demonstrated that SslE specifically contributes to degradation of mucin substrates, typically present in the intestine and bladder. Mutation of the zinc metallopeptidase motif of SslE dramatically impaired <i>E. coli</i> mucinase activity, confirming the specificity of the phenotype observed. Moreover, antibodies raised against variant I SslE, cloned from strain IHE3034 (SslE_IHE3034), are able to inhibit translocation of <i>E. coli</i> strains expressing different variants through a mucin-based matrix, suggesting that SslE induces cross-reactive functional antibodies that affect the metallopeptidase activity. To test this hypothesis, we used well-established animal models and demonstrated that immunization with SslE_IHE3034 significantly reduced gut, kidney and spleen colonization by strains producing variant II SslE and belonging to different pathotypes. Taken together, these data strongly support the importance of SslE in <i>E. coli</i> colonization of mucosal surfaces and reinforce the use of this antigen as a component of a broadly protective vaccine against pathogenic <i>E. coli</i> species.</p></div

The <i>sslE</i> promoter is functional in an intestinal model of colonization.

<p>(A) 2D <i>in vivo</i> imaging at 24 hours of mice intragastrically infected with GL53-P<i>neg</i>-<i>luxCDABE</i> (promoterless control vector), with the bioluminescent derivative GL53-P<i>sslE</i>-<i>luxCDABE</i> and with the GL53-P<i>em7-luxCDABE</i> (positive control). (B) 3D image reconstruction showing <i>ssIE</i>-promoter driven luciferase expression in <i>E. coli</i> localized in the intestinal tract. (C) RT-PCR of RNA purified from: <i>in vitro</i> lab-grown GL53 bacteria (lane 2, positive control); caecum tract of uninfected mice (lane 3, negative control); GL53 bacteria recovered from infected mice (lane 4); GL53 bacteria recovered from infected mice without the RT step (lane 5). 1 Kb Plus DNA Ladder (Life Technologies) is shown in lane 1.</p

SslE_IHE3034 induces cross-protection in intestinal colonization, UTI and sepsis models.

<p>(A) Thirty CD1 mice were intranasally immunized with 30 µg of SslE_IHE3034 at days 1, 21 and 35. Saline was used in the negative control groups. Challenge was done by oral gavage with 5×10⁷ CFU of strain GL53 at day 49. Serial dilutions of the homogenized intestinal caecum tract were plated and the CFU number was enumerated. Statistical significance of protection was obtained using the Mann Whitney test. (B) SslE_IHE3034 prevents the spread of the UPEC strain 536 into the kidneys and spleen in an ascending model of urinary tract infection. Thirty mice were immunized intranasally with 10 µg cholera toxin (CT) alone or with 100 µg of SslE_IHE3034 at a 10∶1 ratio of antigen:CT (day 1). After two boosts of 25 µg antigen (10∶1 ratio of antigen to CT) or CT alone (day 7 and 14), mice were transurethrally challenged with 10⁸ CFU of strain 536 at day 21. After 48 h, bladder, kidneys and spleen were harvested and homogenized. Bacteria in urine and in the tissue homogenates were enumerated by plating serial dilutions. Symbols represent CFU/g tissue or CFU/ml urine of individual mice, and bars indicate median values. P values were determined using the nonparametric Mann-Whitney significance test. (C) SslE_IHE3034 protects against the SEPEC strain IN1S in a sepsis mouse model. CD1 out-bred mice were immunized by subcutaneous injections at day 1, 21, and 35 with 20 µg of recombinant SslE_IHE3034 formulated with alum or alum alone. Immunized animals were challenged at day 49 with a sublethal dose of heterologous strain IN1S and survival was monitored for up to 4 days. The results are indicated as percentage of survival out of a total number of 40 mice. P values were determined using the nonparametric Mann-Whitney significance test.</p

SslE mucinolytic activity.

<p>(A) Mucin lysis (clear plates) was assessed by amido black staining. IHE3034 wild-type, IHE3034Δ<i>sslE</i> knockout mutant, IHE3034Δ<i>sslE</i>::<i>sslE_</i>WT (complemented with the <i>sslE</i> wild-type gene), and IHE3034Δ<i>sslE</i>::<i>sslE_</i>mut (complemented with the <i>sslE</i> gene mutated in the putative metallopeptidase motif), were grown on plates containing 0.5% bovine submaxillary mucin (SIGMA) and stained with 0.1% (wt/vol) amido black in 3.5 M acetic acid for 30 min and destained with 1.2 M acetic acid. (B) The four strains were engineered for constitutive luciferase expression (p<i>lux</i> operon) and mucinolytic activity was detected by the <i>In Vivo</i> Imaging System (IVIS) technology. Bacterial migration in the mucin-agar plates is shown, from the point of inoculum (time zero; t0) to growth at 24 hours (24 h).</p