The fibrinogen-binding M1 protein reduces pharyngeal cell adherence and colonization phenotypes of M1T1 group A Streptococcus

Abstract

Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis ("strep throat") to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.

Keywords: Bacterial Adhesion; Bacterial Pathogenesis; Fibrinogen; Streptococcus pyogenes; Virulence Factors.

FIGURE 1.

M1 protein is the major Fg-binding factor of M1T1 GAS strain 5448. A, flow cytometric analysis of WT and ΔM1 mutant GAS with FITC-labeled Fg: WT (red), WT + FITC-Fg (blue), ΔM1 + FITC-Fg (green), and complemented mutant (Comp) + FITC-Fg (orange).The bar graph shows quantification of the flow cytometry results. B, quantification of Fg binding by L. lactis strains expressing WT and Fg binding-deficient mutant versions of the M protein, expressed as a percentage of the original inoculum recovered bound to an Fg-coated plate. C, quantification of the GAS inoculum after binding to 8 μg/ml Fg. Assays were performed in triplicate and repeated three independent times. ***, p < 0.005 (one-way analysis of variance). n.s., not significant.

FIGURE 2.

M1 protein-Fg binding reduces adherence to pharyngeal keratinocytes. Shown are the adherence to (A) and invasion of (B) OKP7 pharyngeal keratinocytes by logarithmic phase WT and ΔM1 mutant GAS in the presence or absence of 8 μg/ml Fg, normalized to the inoculum. C, bright-field and fluorescence microscopy of FITC-labeled GAS adherence to OKP7 cells. FITC channel images were merged with bright-field images in Photoshop. Adherence/invasion assays were done in triplicate using triplicate sample wells. **, p < 0.01; ***, p < 0.005 (Student's t test). Comp, complemented mutant.

FIGURE 3.

M1 protein is sufficient to promote bacterial Fg binding and pharyngeal cell adherence, but differential effects are absent in stationary phase GAS. Shown are the adherence to (A) and invasion of (B) HaCaT skin keratinocytes by logarithmic phase WT and ΔM1 mutant GAS. Also shown are the adherence to (C) and invasion of (D) OKP7 pharyngeal epithelial cells by logarithmic phase L. lactis expressing M1 protein constructs in the presence or absence of 8 μg/ml Fg, normalized to the inoculum. Adherence/invasion assays were performed in triplicate and repeated independently three times. *, p < 0.05; **, p < 0.01; ***, p < 0.005 (Student's t test). ns, not significant.

FIGURE 4.

SpeB cleavage of the M1 protein counteracts M1- and Fg-mediated reductions in pharyngeal epithelial cell adherence. Shown are the adherence to (A) and invasion of (B) OKP7 pharyngeal keratinocytes by stationary phase WT and ΔM1 mutant GAS in the presence or absence of 8 μg/ml Fg (adherence only), normalized to the inoculum. C, left, flow cytometric analysis of stationary phase M1T1 GAS 5448 strains with FITC-labeled Fg: WT (red), WT + FITC-Fg (blue), ΔM1 + FITC-Fg (green), and ΔSpeB + FITC-Fg (orange). Right, quantification of flow cytometry with the GAS 5448 strains. D, adherence of stationary phase ΔSpeB mutant GAS to OKP7 pharyngeal epithelial cells in the presence or absence of 8 μg/ml Fg, normalized to the inoculum. Shown are the adherence to (E) and invasion of (F) logarithmic phase GAS to OKP7 cells in the presence or absence of 8 μg/ml Fg (adherence only) and 10 μg/ml recombinant SpeB, normalized to the inoculum. Fg binding assays were performed in triplicate and repeated three independent times. ***, p < 0.005 (one-way analysis of variance). Adherence and invasion assays were performed in triplicate and repeated three independent times. *, p < 0.05; **, p < 0.01; ***, p < 0.005 (Student's t test).

FIGURE 5.

M1 protein reduces NALT colonization in a murine infection model. A, schematic of the infection model. Mice were nasally inoculated with a 1:1 mixture of two GAS strains, and NALT tissue was harvested at 24 h post-infection, homogenized, and plated on selective media. Cm, chloramphenicol; Em, erythromycin; abx, antibiotics. B, histological section of GAS-infected NALT tissue (Gram-stained). The arrows point to clusters of GAS. C, histological section of uninfected NALT tissue (Gram-stained). D, plot of the ratio of recovered ΔM1 GAS to WT GAS. ○, samples in which WT GAS was below detectable levels (limit of detection used for ratio calculation). The dashed line indicates a 1:1 ratio. E, plot of the ratio of recovered ΔM1 GAS to complemented mutant GAS. The dashed line indicates a 1:1 ratio.