M1 protein of Streptococcus pyogenes increases production of the antibacterial CXC chemokine MIG/CXCL9 in pharyngeal epithelial cells
Introduction
Streptococcus pyogenes have a predilection to adhere to the mucosal layer of the soft palate, where they may cause pharyngitis. In most cases, the infection heals without the use of antibiotics, indicating that robust local host defense mechanisms normally eradicate the bacteria [1].
Among more than one hundred S. pyogenes serotypes, the M1 serotype is the most prevalent and also causes the most severe infections. M protein is one of the classical virulence determinants of S. pyogenes, appearing as hair-like fibrils at the bacterial surface [2]. Some of the 50 kDa α-helical coiled-coil M1 protein is spontaneously shed from the surface [3] but some is also actively released through a cysteine protease (SpeB) secreted by the bacterium itself [4]. M1 protein contains fibrinogen-, IgG- and albumin-binding regions, properties that are important for its virulence [3], [5]. In addition, M1 protein is important for the adhesion of S. pyogenes to epithelial cells via glucosaminoglycans (GAGs) and sialic acid containing cell surface proteins [6], [7]. After an injury to the mucosal layer, it is of critical importance that local host defense mechanisms can eradicate the bacteria to avoid deep severe streptococcal disease such as septicemia, necrotizing fasciitis and streptococcal toxic shock syndrome [8]. In the case an inflammatory response evolves, this is likely to be dependent on sensing of the bacteria and their products by dendritic cells, macrophages, and T cells that reside in submucosal tissues. The inflammatory response results in the production of Th1 cytokines, where IFN-γ is a key cytokine [9], activating epithelial cells of the mucosal layer, which results in an inflamed phenotype [10], [11]. The inflamed epithelium can elaborate antibacterial peptides, among them chemokines, and may thus play an important role during early stages of streptococcal infection [12]. Chemokines comprise a large family of peptides containing conserved cysteine motifs in their NH2-terminus; XC, CC, CXC, and CX3C, respectively. They share GAG-binding properties and the capability to recruit and activate subsets of leukocytes at sites of inflammation [13]. IFN-γ is crucial for expression of the ELR-negative (lacking a glutamate-leucine-arginine-sequence preceding the cysteines) chemokine MIG/CXCL9 (Monokine Induced by IFN-Gamma) [14]. This chemokine can activate CXCR3 a receptor expressed on T cells, NK cells, eosinophils, and endothelial cells [13], resulting in important biological responses such as leukocyte recruitment and angiostasis [13]. In addition to chemotactic activity, some chemokines possess antibacterial activity [15], [16]. MIG has potent antibacterial activity and recently we found high MIG production and MIG-dependent antibacterial activity in stimulated pharyngeal epithelial cells [12], [15].
Here we show that stimulated pharyngeal epithelial cells can recognize M1 protein and S. pyogenes, resulting in increased production of antibacterial MIG. The findings suggest that pharyngeal epithelial cells can modulate their antibacterial response during streptococcal pharyngitis.
Section snippets
M1 protein of S. pyogenes enhances MIG synthesis and expression
M1 protein is released from the surface of S. pyogenes and is known to act as a soluble virulence factor [3], [4], [5]. Therefore, a potentially enhancing effect on MIG production in IFN-γ stimulated pharyngeal epithelial cells was investigated. Addition of recombinant M1 protein caused a dose-dependent increase of MIG production by Detroit cells as detected by ELISA after 24 h of incubation (Fig. 1A).
During pharyngitis, plasma proteins are likely to be present due to capillary leakage caused by
Discussion
In this study, we show that stimulated pharyngeal epithelial cells recognize both whole S. pyogenes bacteria and its classical virulence factor, the M protein, resulting in increased production of the antibacterial CXC chemokine MIG.
Recently, we showed that stimulated pharyngeal epithelial cells possess antibacterial activity and that MIG is an important part of this activity. In addition, we found high levels of MIG in tonsil fluid obtained from patients suffering from streptococcal
Chemicals and reagents
Recombinant human IFN-γ, TNF-α, affinity-purified polyclonal rabbit antibodies against MIG, and isotype-matched irrelevant rabbit antibodies were from Peprotech, Rocky Hill, NJ. IL-1β, mouse monoclonal neutralizing anti-human TNF-α antibody (clone 1825), human IL-1 receptor antagonist (IL-1ra), and an ELISA for the detection of MIG were purchased from R&D Systems, Abingdon, United Kingdom. Rabbit polyclonal antibodies against NF-κB p65 and mouse monoclonal antibodies recognizing p38
Acknowledgments
This project was supported by grants from the Swedish Council (Grant # 7480), the Swedish Heart and Lung Foundation, The Foundations of Bergh, Crafoord, Gröneberg, Julin, Kock, Österlund, Hansa Medical AB, EU project “Antimicrobials by Immune Stimulation”, and funds at Lund University.
We gratefully acknowledge the Swegene Microarray Resource Center at Lund University for help with microarray processing and analysis and the skilful technical assistance of Pia Andersson.
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