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.

Homophilic protein interactions facilitate bacterial aggregation and IgG-dependent complex formation by theStreptococcus canisM protein SCM

“…Notably, single bacteria in the large aggregates of strain G361 were decorated with IgG; no such binding was seen in the SCM − strain G2 and the isogenic SCM-targeted mutant G361Δscm (Figure 2). These results corroborate the role of SCM as the only IgG-binding receptor of S. canis [23].…”

“…We next applied western blot analysis of SCM-mediated protein complexes from human plasma to investigate their composition in more detail. In a first step, we used antibodies against known SCM ligands, such as Plg and IgG [21][22][23]. We were also interested in knowing whether complement factor C1q is incorporated into the protein aggregates, since this might constitute another immune evasion pathway for S. canis.…”

“…In western blot analysis, a specific band of approximately 92 kDa was observed for SCM-(S, asterisk), but not for FOG-mediated protein complexes (F), when antibodies directed against human Plg (α-Plg) were used. Because of the intrinsic IgG binding capability of SCM [23] and FOG [30], protein bands with molecular masses of approximately 160 kDa (S) and 66 kDa (F) appeared, resulting from the non-opsonic interaction between both M proteins and the secondary antibody. To verify the presence of IgG molecules in the protein complexes, we used a secondary antibody against human IgG (α-IgG) that was raised in goats and therefore not recognized by the IgG-binding regions of SCM and FOG [23,30].…”

“…Its anti-phagocytic activity is based on its particular capability to interact with itself (i) directly in a homophilic manner and (ii) indirectly using the hostderived zymogen plasminogen as a bridging molecule [22]. Furthermore, we recently described how SCM binds immunoglobulin G (IgG) in a non-opsonic manner (via the constant IgG-Fc domain) and found that this interaction prevents the deposition of C1q, a primary component of the classical complement activation pathway, on the bacterial surface [23].…”

“…Homophilic SCM-SCMinteractions and concurrent IgG-binding activity are required for protein complex formation. Interestingly, and in contrast to surface-attached SCM [23], the interaction between SCM and IgG in complex enables C1q sequestration and may represent a virulence mechanism to evade complement-mediated opsonization.…”