The IL-8 protease SpyCEP is detrimental for Group A Streptococcus host-cells interaction and biofilm formation

Abstract

SpyCEP-mediated chemokine degradation translates into more efficient spreading and increased severity of invasive Group A Streptococcus (GAS) infections, due to impaired neutrophil recruitment to the site of infection. SpyCEP is markedly up-regulated in invasive as compared to colonizing GAS isolates raising the question whether SpyCEP expression hinders bacterial attachment and thus colonization of the host. To address this question we used a molecular approach involving the use of homologous GAS strains either expressing or not SpyCEP or expressing an enzymatically inactive variant of SpyCEP. We found that expression of enzymatically functional SpyCEP lowered GAS adherence and invasion potential toward various epithelial and endothelial cells. SpyCEP also blunted biofilm formation capacity. Our data indicate that expression of SpyCEP decreases colonization and thus might be detrimental for the spreading of GAS.

Keywords: Group A Streptococcus; IL-8 protease; SpyCEP; biofilms; colonization; virulence factors.

Figure 1

Enhanced GAS adherence to and invasion of epithelial and endothelial cells in the absence of SpyCEP. The epithelial cell lines Detroit-562 and A549 and the endothelial cells Ea.hy926 and HUVECs were challenged with the GAS strains GAS WT, GAS ΔcepA, and GAS ΔcepA compl. MOI of 1 was used for adherence assays and MOI of 10 was used for invasion assays. To assess adherence and invasion an antibiotic protection assay was used and adhering/invading bacteria enumerated by plating and colony counting. (A) GAS adherence to and (B) invasion of the epithelial and endothelial cells were assessed after 30 min and 2 h respectively. The absence of SpyCEP significantly increased adherence to and invasion of epithelial and endothelial cells by GAS. Each experiment was carried out in triplicate; the figure shows one representative experiment. Error bars represent the standard error, statistical analysis was carried out using a two-tailed t-test (ns = p > 0.05, ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p < 0.0001).

Figure 2

SpyCEP activity requirement for GAS adherence to and invasion of epithelial and endothelial cells. Representative epithelial (Detroit-562) and endothelial (EA.hy926) cell lines were challenged with GAS WT, GAS ΔcepA, GAS ΔcepA compl and GAS ΔcepA compl^*. (A) Adherence and (B) invasion assays were carried out as described in Figure 1. GAS ΔcepA compl^* behaved like GAS ΔcepA in terms of adherence and invasion to Detroit-562 and EA.hy926 cells. Each experiment was carried out in triplicate and the graphs show one representative experiment. Error bars represent the standard error, statistical analysis was carried out using a two-tailed t-test (ns = p > 0.05, ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p < 0.0001). The p-values are relative to GAS WT.

Figure 3

Decreased GAS cell surface attachment and biofilm formation in the presence of SpyCEP. (A) Microscopy analysis of GAS attached to the surface of human pharyngeal cells. GAS strains and Detroit-562 cells were stained with CFSE (green) and DAPI (blue) respectively and attachment was assessed using a fluorescence confocal laser scanning microscope. The scale bar in the confocal images represents 20 μm. (B) Integrated densities of attached bacteria obtained from confocal images were evaluated using ImageJ. The integrated density is measured in arbitrary units (AU). Data were pooled from three independent experiments done in triplicate, mean ± SD; ^*p < 0.01, compared to GAS WT. (C) Biofilm formation was assessed for GAS WT, GAS ΔcepA, GAS ΔcepA compl and GAS ΔcepA compl^*. The absence of SpyCEP or the presence of an inactive SpyCEP form resulted in increased biofilm formation. The graph represents pooled data from three independent experiments; the error bars represent the standard deviation. Statistical analysis was carried out using a two-tailed t-test (ns = p > 0.05, ^*p ≤ 0.05, ^**p ≤ 0.01). The p-values are relative to GAS WT.

Figure 4

Assessment of SpyCEP cleavage and hyaluronic acid content. (A) SpyCEP-mediated cleavage of proteins involved in bacteria-host cells interactions was assessed. After overnight incubation in the presence of recombinant SpyCEP (rSpyCEP, 100 ng/ml), the samples were loaded on a polyacrylamide gel and cleavage assessed by coomassie blue staining. IL-8 was used as a positive control for the cleavage reaction. (B) β-1 integrin expression on EA.hy926 cells surface after exposure to buffer only (control), rSpyCEP (10 μg/ml) or an unrelated protein (S1LG, 10 μg/ml) purified using the same method. The graph represents two pooled experiments and error bars represent the standard deviation. (C) Hyaluronic acid (HA) content quantification in the GAS strains. Error bars represent the standard deviation, statistical analysis was carried out using a two-tailed t-test (ns = p > 0.05). The p-values are relative to GAS WT.