SCM, the M Protein of Streptococcus canis Binds Immunoglobulin G

Abstract

The M protein of Streptococcus canis (SCM) is a virulence factor and serves as a surface-associated receptor with a particular affinity for mini-plasminogen, a cleavage product of the broad-spectrum serine protease plasmin. Here, we report that SCM has an additional high-affinity immunoglobulin G (IgG) binding activity. The ability of a particular S. canis isolate to bind to IgG significantly correlates with a scm-positive phenotype, suggesting a dominant role of SCM as an IgG receptor. Subsequent heterologous expression of SCM in non-IgG binding S. gordonii and Western Blot analysis with purified recombinant SCM proteins confirmed its IgG receptor function. As expected for a zoonotic agent, the SCM-IgG interaction is species-unspecific, with a particular affinity of SCM for IgGs derived from human, cats, dogs, horses, mice, and rabbits, but not from cows and goats. Similar to other streptococcal IgG-binding proteins, the interaction between SCM and IgG occurs via the conserved Fc domain and is, therefore, non-opsonic. Interestingly, the interaction between SCM and IgG-Fc on the bacterial surface specifically prevents opsonization by C1q, which might constitute another anti-phagocytic mechanism of SCM. Extensive binding analyses with a variety of different truncated SCM fragments defined a region of 52 amino acids located in the central part of the mature SCM protein which is important for IgG binding. This binding region is highly conserved among SCM proteins derived from different S. canis isolates but differs significantly from IgG-Fc receptors of S. pyogenes and S. dysgalactiae sub. equisimilis, respectively. In summary, we present an additional role of SCM in the pathogen-host interaction of S. canis. The detailed analysis of the SCM-IgG interaction should contribute to a better understanding of the complex roles of M proteins in streptococcal pathogenesis.

Keywords: Immunoglobulin G; M protein; Streptococcus canis; anti-phagocytic activity; zoonosis.

Figure 1

IgG binding to S. canis. (A) Binding of iodinated IgG to 21 different scm-positive (black bars) and scm-negative (gray bars) S. canis isolates was analyzed by gamma-counting. Data represent triplicates of three independent experiments and are presented as mean values ± standard deviation. IgG-binding to scm-negative S. canis G2 and G2424/96, and G17, and to SCM-expressing S. canis G361 and G15 (B) as well as to S. gordonii bacteria (SGO) and heterologously SCM-expressing S. gordonii bacteria (C) was analyzed by flow cytometry using polyclonal IgG raised in rabbit and fluorescence—conjugated secondary antibody. IgG binding was detected for SCM-expressing S. canis strains and also for SCM-expressing S. gordonii. Histograms of representative results are shown. (D) Field Transmission Electron microscopic visualization of the bacterial strains G361, G2424/96, G2, G15, and G17. Bound IgG was detected with protein A-gold nanoparticles. FESEM analysis of S. canis G2 and G2424/96 revealed only marginal background binding, whereas strong IgG binding is shown for G361 and moderate binding was visualized on the surface of G15 and G17 (white spheres). Bars represent 200 nm.

Figure 2

Plasminogen and IgG binding of SCM. In radioactive inhibition studies binding of iodinated plasminogen (A) or iodinated IgG (B) to S. canis G361 was determined after incubation with non-labeled IgG (1.0 μM) or plasminogen (1.0 μM). Data represent triplicates of three independent experiments and are presented as mean values ± standard deviation. Significance was calculated using an one-way ANOVA followed by Dunnett's post-test. ^**P-values < 0.01, ^***P-values < 0.001.

Figure 3

Identification of the specific IgG-binding site in SCM of S. canis G361. (A) Summary of ELISA and quantitative Dot blot assays to study IgG-binding activity by various C-terminal, N-terminal, and truncated SCM fragments. Positions of first and last amino acid of the fragments are indicated. IgG binding activity is indicated with “+.” (B) The truncated N-terminal SCM fragments N-173 and N225, the C-terminal fragment C-173, and a fragment lacking the putative IgG-binding region ranging from amino acid 173 to 225 were tested for IgG-binding activity by ELISA. (C,D) Interactions of soluble human IgG-Fc with immobilized SCM-WT or KO173225 were analyzed by surface plasmon resonance spectroscopy. The representative sensograms show a dose-dependent binding of IgG-Fc to SCM-WT whereas no binding was detectable for KO173225. The association and dissociation was observed, each of 300 s. Values of the control flow cells were subtracted from each sensogram. The K_D value is indicated.

Figure 4

Nucleotide sequence alignment and phylogenetic analysis of IgG-binding site sequences derived from SCM and other M proteins of S. canis strains and their respective references obtained from the nucleotide database of NCBI. The consensus nucleotide sequence is displayed on top of the alignment followed by the respective amino acid translation. Within the alignment sequence, similarities are displayed with dots and single nucleotide polymorphisms (SNPs) are shown with the regarding nucleotide and amino acid in one letter code, if the SNP is a non-synonymous one. Overall within the displayed IgG-binding region of SCM only three SNPs were detected, with one non-synonymous SNP that is represented by only a single strain and the associated reference sequence.

Figure 5

Analogous rather than homologous interaction of SCM and FOG with IgG-Fc and C1q. (A) Inhibition of IgG binding to SCM was determined by in ELISA after incubation of 10 μg of M protein FOG, SCM, and the truncated SCM protein fragment KO173225 (SCM-KO). Results represent triplicates of three independent experiments and are presented as mean values ± standard deviation. Significance was calculated using one-way ANOVA followed by a Dunnett's post-test. ^***P-values < 0.001, ns, not significant. (B) Different streptococcal strains harboring either a SCM- (G361) or FOG- (G45) positive or a SCM- (G2) and FOG- (G89) negative genotype, respectively, were co-incubated with IgG, C1q, or IgG and C1q in combination. After eluting host proteins form the bacterial surface, immobilization of C1q was determined in Western blot analysis using antibodies directed against C1q. Recombinant C1q served as a positive control (co).