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

Abstract

Streptococcus canis is a zoonotic agent that causes serious invasive diseases in domestic animals and humans, but knowledge about its pathogenic potential and underlying virulence mechanisms is limited. Here, we report on the ability of certain S. canis isolates to form large bacterial aggregates when grown in liquid broth. Bacterial aggregation was attributed to the presence and the self-binding activity of SCM, the M protein of S. canis, as evaluated by bacterial sedimentation assays, immunofluorescence-and electron microscopic approaches. Using a variety of truncated recombinant SCM fragments, we demonstrated that homophilic SCM interactions occur via the N-terminal, but not the C-terminal part, of the mature M protein. Interestingly, when incubated in human plasma, SCM forms soluble protein complexes comprising its known ligands, immunoglobulin G (IgG) and plasminogen (Plg). Co-incubation studies with purified host proteins revealed that SCM-mediated complex formation is based on the interaction of SCM with itself and with IgG, but not with Plg or fibrinogen (Fbg), well-established constituents of M proteinmediated protein complexes in human-associated streptococci. Notably, these soluble, SCMmediated plasma complexes harbored complement factor C1q, which can induce complement breakdown in the periphery and therefore represent another immune evasion mechanism of SCM.

Novel Models of Streptococcus canis Colonization and Disease Reveal Modest Contributions of M-Like (SCM) Protein

“…The M-protein is a well-characterized virulence factor of S. pyogenes , and the orthologous nature of the SCM protein suggests a potential for a similar role in S. canis pathogenesis. To investigate the role of SCM in S. canis virulence, we utilized a SCM insertional mutant made from the S. canis G361 clinical isolate strain that no longer produces the SCM protein and exhibits reduced aggregate formation in culture [ 32 ]. We confirmed that loss of SCM did not alter in vitro growth of S. canis compared to wildtype S. canis in bacteriologic media (THB, Figure 1 A) or tissue culture media (RPMI-1640, Figure 1 B).…”

“…Thus, loss of SCM in S. canis possibly reduces surface proteins with greater hydrophobicity, leading to the observed decrease in biofilm formation. Alternatively, because SCM activity is associated with S. canis aggregation microcolony formation in vitro [ 56 ], loss of homophilic interactions between SCM in the Δ scm strain may alter the morphology of S. canis biofilms [ 32 , 57 ]. In fact, although there was no difference in the relative fluorescent units (RFU) of WT G361 and Δ scm biofilms in RPMI-1640, the morphology of the biofilms was visibly different.…”

“…S. pyogenes M proteins confer resistance against phagocytosis by inhibiting the alternate complement pathway [ 58 , 59 ], or alternatively, through forming bacterial aggregates with self [ 56 , 60 ] or as a complex with host cells [ 61 , 62 ]. Similarly, in S. canis , this self-aggregation is lost in the absence of SCM [ 32 ]. However, in our assays with canine or human macrophage cell lines, we found that presence of SCM did not alter macrophage-mediated killing of S. canis ( Figure 2 ).…”

“…Bacterial strains used in this study include Streptococcus canis human vaginal wildtype (WT) isolate G361 [ 33 ] and isogenic scm -targeted insertional mutant G361∆ scm [ 32 ], Streptococcus pyogenes human invasive isolate 5448 M1 and isogenic Δ emm1 [ 34 ], and Streptococcus agalactiae human meningeal isolate COH1 (ATCC BAA-1176). All bacterial strains were grown to stationary phase in Todd-Hewitt broth (THB, Hardy Diagnostics), or THB agar plates, at 37 °C without shaking.…”

“…SCM is a fibrillar surface protein [ 26 ] which binds plasminogen [ 29 ] and the Fc region of IgGs from multiple species including human, dog, cat, and mouse [ 31 ]. We recently demonstrated that SCM self-interactions facilitate bacterial aggregation and that SCM interactions with IgG initiate formation of protein complexes in human plasma [ 32 ]. However, the contribution of SCM, and impact of its allelic variability, to S. canis colonization and virulence remains undefined.…”