CnaBE3 domain promotes significant bacterial load reduction in mice challenged with Newman strain and with NCTC8325 strain defective for sdrE gene.

<p>Immunized mice were intravenously infected with either <i>Staphylococcus aureus</i> Newman (N = 54, 4 independent experiments) or NCTC8325 (sdrE negative) strains (N = 48, 3 independent experiments). A) Both, mice immunized with CnaBE3 domain or SdrE protein showed a significant reduction in bacterial load in kidneys when infected with <i>S. aureus</i> Newman strain if compared to adjuvant alone immunized mice used as negative control. B) Mice immunized with CnaBE3 domain show a significant reduction in bacterial load when challenged with NCTC8325 strain as compared to adjuvant alone immunized animals. Each dot represents a single mouse, and geometric means are reported. Statistical analysis was performed using a Mann–Whitney U test (*<i>p</i>≤0.05, **<i>p</i>≤0.01, ns means not significant).</p

Schematic representation of Sdr proteins and amino acid sequence of CnaBC2 CnaBD5 and CnaBE3 domains.

<p>A) Schematic representation of Sdr proteins. A putative leader peptide (LP) sequence and an LPXTG motif are depicted in black. The A domain is reported in light gray, whereas B repeats (two, three, or five, for SdrC, SdrE, and SdrD, respectively) are shown in white, and contain putative CnaB domains shown in dark gray. Finally, at the C-terminus, the SD repeat domain is depicted in dark gray. In addition, boundaries of CnaBC2, CnaBD5 and CnaBE3 domains are reported. B) CnaBC2, CnaBD5 and CnaBE3 domain amino acid sequences are aligned. Identical residues are highlighted, and putative CnaB domains are encompassed by a black box.</p

CnaBE3 domain sequence is highly conserved among phylogenetically distinct strains.

<p>A) The depicted phylogenetic tree was obtained using the Sequence Types (ST) of a panel of 59 epidemiologically relevant <i>S. aureus</i> strains. Clonal complexes 1, 5, 8 and 30 are highlighted. The eleven bacterial strains selected for conservation analysis are in bold. B) The percentages of amino acid sequence identity obtained from the comparison of Sdr proteins and the CnaBC2, D5 and E3 domain amino acid sequences of Newman strain to those of an epidemiologically relevant panel of <i>S. aureus</i> strains are reported. Dark gray color means that proteins are absent, whereas white color means present and conserved with an identity percentage ≥ 90, and light gray color means present but variable with an identity percentage ≥ 75 and ≤ 89, on at least 75% of the amino acid sequence.</p

Anti-CnaBE3 domain antibodies recognize all the three Sdr full length proteins.

<p>Western blot analysis of bacterial cell wall extracts from NCTC8325, Newman, MSSA476, MW2, N315, Mu50, Mu3, USA300 FPR3757, MRSA252, TW20, and MN8 <i>S. aureus</i> strains blotted with anti-CnaBE3 domain antibodies. The Sdr proteins of each strain are highlighted.</p

CnaBE3 domain is recognized by sera of <i>S. aureus</i> infected patients.

<p>The immune reactivity of a panel of 30 human sera collected from <i>S. aureus</i> infected patients was tested against the CnaBE3 domain by ELISA assay. A panel of 46 sera collected from healthy donors was used as control. Anti-CnaBE3 IgG titers of sera collected from the patients were significantly higher than those of sera collected from healthy donors. Each dot represents a single serum, and geometric means are reported. Statistical analysis was performed with a Mann–Whitney U test. Values are expressed in lnAU (natural logarithm of Arbitrary Units), for the calculation method see the material and method section (*<i>p</i>≤0.05).</p

Trypsin digestion of full length SdrE protein and sequence analysis of the 37 kDa resistant fragment.

<p>A) SDS-PAGE analysis of an overnight trypsin digestion of the full length SdrE protein dialyzed either in 1 mM CaCl₂ or in 1 mM EDTA. B) The C-terminal region of the full length SdrE protein is schematically depicted. The N-terminal sequence of the 37kDa resistant fragment obtained by Edman degradation (TPKYSLGDY) is shown, together with the peptides derived by trypsin digestion (black bars), and identified by analyzing the MALDI-TOF MS spectrum reported in panel C. “T” indicates trypsin autocatalytic fragment.</p

Antibodies against CnaBE3 and SdrE mediate opsonophagocytic killing of <i>S. aureus</i>.

<p>Sera of mice immunized either with CnaBE3 domain or with SdrE full length protein at dilution of 1:500, rabbit complement, human promyelocytic leukemia cells HL-60, and the <i>S. aureus</i> strain Newman were incubated for 1 h and plated on TSA for CFU counting. No bacterial killing was observed in the absence of serum, HL-60 cells, complement, or in presence of control serum (preimmune serum), showing the specificity of both CnaBE3 and SdrE antisera in mediating opsonophagocytic killing of the pathogen. Error bars represent standard deviation. Statistical analysis was performed by paired t test (*<i>p</i>≤0.05, ns means not significant). For calculation of the killing percentage see the material and method section.</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

Anti-SslE antibodies impair translocation of <i>E. coli</i> through a mucin-gel matrix.

<p>(A) IHE3034 wild-type, the IHE3034Δ<i>sslE</i> knockout mutant, IHE3034Δ<i>sslE</i>::<i>sslE_</i>WT (complemented with the <i>sslE</i> gene wild-type) and IHE3034Δ<i>sslE</i>::<i>sslE_</i>mut (complemented with <i>sslE</i> gene mutated in the putative metallopeptidase motif), were loaded on the top of a mucin-gel matrix column polymerized in a 1 ml syringe. After 3 hours at 37°C, eluted fractions were collected and plated for CFU counting. The results were reported as percentage of CFU recovered in four different fractions, sequentially eluted from the bottom of the column, with respect to the initial inoculum. (B) Dose-dependent inhibition of IHE3034 translocation through a mucin-gel matrix by anti-SslE antibodies. Serial dilutions (range 1∶50–1∶1350) of antibodies were used for inhibition. Antibodies against the unrelated ExPEC c1275 were used as a negative control at the dilution 1∶50. Translocation was reported as the percentage of CFU recovered with respect to the initial inoculum for three sequentially eluted fractions. P values were determined using a two-tailed unpaired Student's significance test.</p