<em>Streptococcus pyogenes</em> SpyCEP Influences Host-Pathogen Interactions during Infection in a Murine Air Pouch Model

<div><p><em>Streptococcus pyogenes</em> is a major human pathogen worldwide, responsible for both local and systemic infections. These bacteria express the subtilisin-like protease SpyCEP which cleaves human IL-8 and related chemokines. We show that localization of SpyCEP is growth-phase and strain dependent. Significant shedding was observed only in a strain naturally overexpressing SpyCEP, and shedding was not dependent on SpyCEP autoproteolytic activity. Surface-bound SpyCEP in two different strains was capable of cleaving IL-8. To investigate SpyCEP action <em>in vivo</em>, we adapted the mouse air pouch model of infection for parallel quantification of bacterial growth, host immune cell recruitment and chemokine levels <em>in situ</em>. In response to infection, the predominant cells recruited were neutrophils, monocytes and eosinophils. Concomitantly, the chemokines KC, LIX, and MIP-2 <em>in situ</em> were drastically increased in mice infected with the SpyCEP knockout strain, and growth of this mutant strain was reduced compared to the wild type. SpyCEP has been described as a potential vaccine candidate against <em>S. pyogenes</em>, and we showed that surface-associated SpyCEP was recognized by specific antibodies. <em>In vitro</em>, such antibodies also counteracted the inhibitory effects of SpyCEP on chemokine mediated PMN recruitment. Thus, α-SpyCEP antibodies may benefit the host both directly by enabling opsonophagocytosis, and indirectly, by neutralizing an important virulence factor. The animal model we employed shows promise for broad application in the study of bacterial pathogenesis.</p> </div

Activity of rSpyCEP and PMN transmigration.

<p>(<b>A</b>) Comparison of rSpyCEP and native SpyCEP activity on IL-8. SDS-PAGE (18% Tris-Glycine) and silver staining after 2 h of digestion with 5 ng, 1 ng, 0.2 ng, 0.04 ng or 0 ng (ctr lane) of rSpyCEP+3348Δ<i>spyCEP</i> extracts (left). Cell wall extracts containing comparable amounts of native SpyCEP from 3348 extracts (right). Full and trace arrowheads indicate intact and cleaved IL-8, respectively. Lower panel: control Western blot showing relative SpyCEP amounts compared to 5 ng of rSpyCEP (ctr). (<b>B,C</b>) SpyCEP effect on PMN transmigration. (<b>B</b>) Murine or (<b>C</b>) human PMN migration in response to KC or IL-8 (white), respectively, in the presence of rSpyCEP (black) or rSpyCEP* (grey). (<b>D</b>) Counteraction of rSpyCEP activity by specific antibodies. Human PMN migration using IL-8 (white); IL-8, rSpyCEP and α-SpyCEP (black); and IL-8, rSpyCEP, α-Spy0269 (grey). Data represent means plus SEM of one representative experiment using triplicates. Statistical significance was tested by unpaired Student T, (*) <i>P</i><0.05, (**) <i>P</i><0.01, (***), <i>P</i><0.001.</p

<i>S. Pyogenes</i> infection in a murine air pouch model.

<p>(<b>A</b>) Schematic of the <i>in vivo</i> air pouch model. For each experiment, 8 CD1 mice per group were infected with 1×10⁷ CFU of 3348, 3348Δ<i>spyCEP</i> or PBS. After air pouch inflation and bacterial infection, the lavage material was collected and fractionated for bacterial viable counts, leukocyte counts and chemokine analysis for each animal. (<b>B</b>) Bacterial multiplication factor (total CFU/inoculum CFU) in the lavage from individual mice. Time points 4 and 24 h represent aggregate data from two experiments. Horizontal bars are geometric means. Statistical significance (*) was tested by Mann-Whitney U, <i>P</i> = 0.014).</p

Specific antibodies recognize SpyCEP on bacteria.

<p>Exponential (<b>A</b>) and stationary (<b>B</b>) phase bacteria were labeled with pooled mouse α-Alum (white) or mouse α-SpyCEP (gray) sera. Secondary antibodies were rabbit α-mouse phycoerythrin conjugates, and the fluorescence (PE-A) is shown on the x axis. Strains 3348 (w.t.), 3348Δ<i>spyCEP</i> (Δ) and 3348<i>spyCEP</i>* (*) are displayed on the z axis.</p

Localization and activity of SpyCEP.

<p>(<b>A</b>) Schematic representation of SF370 wild type and genetically manipulated <i>spy0416/spyCEP</i> loci. The same mutagenesis was conducted in the 3348 strain. The base substitution 452A>C is indicated (*). (<b>B</b>) Western blot analysis of cell wall extracts or supernatants from the 3348 and SF370 w.t. and mutant strains. SpyCEP was detected using rabbit polyclonal α-SpyCEP serum. 10 ng of rSpyCEP or rSpyCEP* were added as controls (ctr). (<b>C–D</b>) IL-8 cleavage assay with washed live bacteria. SDS-PAGE and silver staining were performed after digestion of IL-8 (10 µg/ml) in the presence of 10 µg/ml of chloramphenicol. Uncleaved and cleaved IL-8 are indicated by full and trace arrowhead respectively. Control lane (ctr) is IL-8 alone. (<b>C</b>) Strains were grown to exponential or stationary phase, resuspended in PBS at comparable bacterial densities (∼10⁹ CFU), and then incubated with IL-8. (<b>D</b>) Exponential phase bacteria 3348 w.t. (10⁷ CFU) and SF370 w.t. (10⁸ CFU) were serially diluted in PBS using 4 fold steps before incubation with IL-8.</p

Cell recruitment <i>in situ</i>.

<p>(<b>A</b>) Gating strategy to identify neutrophils (NPh; Ly6G^high, GR1^high), dendritic cells (DC; CD11c⁺, MHC-II⁺), macrophages (MPh; CD11b⁺, F4/80^high), monocytes (Mo; CD11b^high, CD11c⁻, Ly6C^high, GR1⁺) and eosinophils (Eos; Ly6G^int, F4/80^int, SSC^high) 4 h post-infection. (<b>B</b>) Counts of each cell population identified in the lavage 4 h post-infection with 3348 (w.t.), 3348Δ<i>spyCEP</i> (Δ) or PBS. Data shown are means plus SEM of one representative experiment using 8 mice per group, except for the PBS control (n = 3). Statistical significance was tested by Mann-Whitney U: <i>P</i><0.05 (*), <i>P</i><0.01 (**).</p

Structure and Assembly of Group B Streptococcus Pilus 2b Backbone Protein

<div><p>Group B <i>Streptococcus</i> (GBS) is a major cause of invasive disease in infants. Like other Gram-positive bacteria, GBS uses a sortase C-catalyzed transpeptidation mechanism to generate cell surface pili from backbone and ancillary pilin precursor substrates. The three pilus types identified in GBS contain structural subunits that are highly immunogenic and are promising candidates for the development of a broadly-protective vaccine. Here we report the X-ray crystal structure of the backbone protein of pilus 2b (BP-2b) at 1.06Å resolution. The structure reveals a classical IgG-like fold typical of the pilin subunits of other Gram-positive bacteria. The crystallized portion of the protein (residues 185-468) encompasses domains D2 and D3 that together confer high stability to the protein due to the presence of an internal isopeptide bond within each domain. The D2+D3 region, lacking the N-terminal D1 domain, was as potent as the entire protein in conferring protection against GBS challenge in a well-established mouse model. By site-directed mutagenesis and complementation studies in GBS knock-out strains we identified the residues and motives essential for assembly of the BP-2b monomers into high-molecular weight complexes, thus providing new insights into pilus 2b polymerization.</p></div

Biochemical characterization of different BP-2b constructs.

<p><b>(A)</b> Time course of the trypsin-proteolysis reactions at 37°C of BP-2b full length and fragments, analyzed by SDS-PAGE. Different digestion patterns can be observed for the different constructs. Asterisks indicate the not-digested proteins. (<b>B)</b> Differential Scanning Fluorimetry (DSF) analysis of BP-2b proteins (D1+D2+D3, D2+D3 and single domains D1, D2, D3) in presence of Sypro orange showed different thermal stabilities. Graph shows the fluorescence intensity <i>vs</i>. the temperature for the unfolding different BP-2b constructs. (<b>C)</b> Correlation of BP-2b melting temperature with the concentration of Ca²⁺.</p

Structural comparisons of BP-2b_D2+D3 with other pilin backbone proteins.

<p>(<b>A)</b> BP-2b (blue cartoon) is shown overlaid onto: the pilus backbone protein RrgB (pdb 2x9x, red cartoon, left), the major pilin protein GBS80 (pdb 3pf2, green cartoon, middle), and on the major pilin protein BP-2a (pdb 2xtl, pink cartoon, right). (<b>B)</b> Domain architecture of GBS backbone proteins from pilus 1 (BP-1), pilus 2a (BP-2a) and pilus 2b (BP-2b). The proteins are comprised of a signal peptide (SP) at the N-terminus and a C-terminal LPXTG-like motif (in red) linked to the transmembrane domain (TM). BP-1 and BP-2b contain three domains, while BP-2a four domains. The pilin motif involved in pilus polymerization is located near the D1–D2 domain linker while the E-box is located close to the sorting signal. Residues involved in isopeptide bonds are indicated by black bars. Domains present in the crystal structures are included into the box outlined with dashed lines.</p

Lys 175, Glu 423 and the sorting motif LPSTG are involved in BP-2b polymerization in GBS.

<p>Immunoblot analysis of total protein extracts from GBS mutant strain lacking the pilus 2b backbone protein gene (Δ<i>BP-2b</i>) complemented with plasmids expressing the wild-type BP-2b protein (WT) or BP-2b mutants carrying a deletion of the C-terminal sorting signal (BP-2b_ΔLPXTG), alanine substitutions of the putative pilin motif lysine (BP-2b_K175A, BP-2b_K118A BP-2b_K82A) or of the E-box E423 residue (BP-2b_E423A). Nitrocellulose membrane was probed with a mouse antiserum raised against the recombinant BP-2b protein (α-BP–2b).</p