IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism

Abstract

Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab’)2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.

Survival of Streptococcus suis in Porcine Blood Is Limited by the Antibody- and Complement-Dependent Oxidative Burst Response of Granulocytes

“…(rIde Ssuis _h_95°C) as a negative control as well as the loss-of-function point-mutated variant rIde Ssuis _h_C195S (9). Complete cleavage of IgM in IgG-depleted hyperimmune serum through the addition of rIde Ssuis _h was proven by Western blotting, as demonstrated in Fig.…”

“…Complement is involved in oxidative burst induction of porcine granulocytes in response to S. suis. To investigate the involvement of complement in the induction of the oxidative burst and killing of streptococci, we used vaccinia virus complement control protein (VCP), which was previously described to be an effective complement inhibitor in porcine blood (9,20). Bactericidal and oxidative burst assays with S. suis strain 10 were conducted in the presence of VCP, apocynin, or the combination of both inhibitors.…”

“…To further characterize the role of IgM and the possible IgM-complement-oxidative burst axis that might mediate the killing of S. suis, we used IgG-depleted hyperimmune serum but now in combination with Ide Ssuis , a protease highly specific for porcine IgM, derived from S. suis itself (21). Noteworthy, previous bactericidal assays showed that the expression of Ide Ssuis by S. suis is not sufficient to cleave most of the IgM in porcine blood (9). Consequently, we conducted bactericidal and oxidative burst experiments with IgG-depleted hyperimmune serum after cleaving IgM with a recombinant Ide Ssuis homologue (rIde Ssuis _h) (21), a truncated variant of the enzyme containing the active center.…”

“…capsule or the D-alanylation of lipoteichoic acid, have been investigated with regard to their potential to prevent phagocytosis by neutrophils (6)(7)(8), few studies have been conducted to elucidate the mechanisms by which these important immune cells actually kill S. suis. Specific antibodies and complement have been shown to promote the killing of S. suis in whole blood or by isolated neutrophils (6,9). Neutrophils are known to produce reactive oxygen species (ROS) in a process called the oxidative or respiratory burst (10).…”

Involvement of Various Enzymes in the Physiology and Pathogenesis of Streptococcus suis

“…Copper efflux [20] PmtA Ferrous iron and cobalt efflux, tolerance to hydrogen peroxide-induced oxidative stress [21] MsmK Utilization of multiple carbohydrates, pathogenesis [22,23] Ide Ssuis Degradation of porcine IgM, complement evasion [24][25][26] IgdE Degradation of porcine IgG [27] IgA1 protease/ZmpC Degradation of human IgA1, pathogenesis 1 [28][29][30][31] eSTK Maintaining bacterial morphology, tolerance to stresses, pathogenesis [32][33][34] eSTP Virulence, cell adhesion, and immune evasion 2 [35,36] SspA-1 Virulence, trigger of proinflammatory cytokines [37,38] SspA-2 Pathogenesis, proinflammatory response in macrophages [39][40][41] Superoxide dismutase Oxidative stress resistance, virulence [42,43] NADH oxidase Tolerance to oxidative stress, virulence [44,45] SsnA Degradation of human and porcine neutrophil extracellular traps, pathogenesis [46][47][48] EndAsuis Degradation of neutrophil extracellular traps [49] Enolase Binding of extracellular matrix components, pathogenesis [50][51][52][53][54][55][56][57][58] LuxS Growth, biofilm formation, capsule synthesis, hydrogen peroxide resistance, resistance to fluoroquinolones, pathogenesis [59]…”