<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

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

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

Human sera specifically recognize SAP.

<p>(A) Immuno-reactivity of human sera towards recombinant SAP(H+L) and SAP(L). The data were obtained by ELISA and represent the mean±SD of 4 human sera. (B) Results of competitive-inhibition ELISA demonstrating antigenic specificity of human antibodies reacting with plates coated with recombinant SAP. Percent inhibition of binding of human serum by each inhibiting antigen was determined by comparison of absorbance at 492 nm in the presence and absence of inhibitor. White circle labels indicate the mean±SD of the % inhibition by SAP of 4 human sera. Black square labels indicate % inhibition by an unrelated GBS protein (AP-1).</p

Expression of SAP recombinant protein.

<p>(A) SDS-PAGE of the mixture of the high and low molecular weight forms of SAP (left lane) as obtained after affinity chromatography and SAP(L) as obtained after anionic exchange chromatography (right lane). (B) Schematic representation of the recombinant form of SAP. Due to the presence of an alternative translation site the protein is expressed in two forms: SAP(H), the full-length form of the enzyme; SAP(L), the truncated form without the CBMs.</p

In vivo SAP protein expression is modulated by the presence of α-glucans.

<p>(A) RT-PCR and WB analysis of SAP expression in COH1 wt strain and COH1Δsap strain grown in the presence of different sugars. Peptidoglycan-associated protein fraction was separated by 10% (w/v) SDS-PAGE. Blots were overlaid with a mouse anti-SAP polyclonal antibody and stained with HRP-conjugated antibody. (B) Immunogold electron microscopy and confocal imaging of SAP expression in COH1 wild type strain and COH1Δsap strain grown as in (A). For IEM, fixed bacteria were incubated with an anti-SAP serum and then labeled with secondary antibody conjugated to 10-nm gold particles. Scale bars 200 nm. In confocal imaging experiments, bacteria were stained with mouse polyclonal anti-capsular type III antibodies (red) and the SAP protein with rabbit polyclonal anti-SAP antibodies (green). Magnification, ×100.</p

Exposure of SAP on bacterial surface in the presence of different carbohydrates.

*<p>Numbers indicate the delta mean of fluorescence relative to bacteria incubated with a SAP immune serum versus bacteria incubated with a pre-immune serum.</p