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

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

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

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

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

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