IdeS, a highly specific immunoglobulin G (IgG)-cleaving enzyme from Streptococcus pyogenes, is inhibited by specific IgG antibodies generated during infection

Abstract

IdeS, a recently discovered cysteine proteinase secreted by the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G. The fact that the enzyme targets one of the key molecules of the adapted immune response raised the question of whether an antibody response against IdeS could inhibit, i.e., neutralize, enzyme activity. Paired acute- and convalescent-phase serum samples from patients with pharyngotonsillitis (n = 10), bacteremia (n = 7), and erysipelas (n = 4) were analyzed. Antibodies with the ability to neutralize IdeS enzymatic activity were already found in two-thirds of acute-phase sera. However, patients who seroconverted to IdeS, in particular patients with pharyngotonsillitis and erysipelas, developed specific antibodies during convalescence with an increased capability to efficiently neutralize the enzymatic activity of IdeS. Also, the presence of neutralizing antibodies decreased the ability of IdeS to mediate bacterial survival in human immune blood. In patients with bacteremia, several acute-phase sera contained neutralizing antibodies, but no correlation was found to severity or outcome of invasive infections. Still, the fact that the human immune response targets the enzymatic activity of IdeS supports the view that the enzyme plays an important role during streptococcal infection.

FIG. 1.

Alignment of complex II IdeS proteins analyzed in this study. The previously published sequence of complex II IdeS is designated CII (8). Amino acids 212 to 277 are shown. Sequence differences are boldfaced and variations indicated below the alignment. The additional cysteine residues in M28 serotypes are boxed.

FIG. 2.

Inactivation of IdeS enzymatic activity is mediated by antibody binding. (A) IdeS was added to the convalescent-phase serum sample from patient PS2 to determine neutralizing serum activity. ¹²⁵I-labeled myeloma IgG1 was used as the substrate, and enzyme activity was monitored by determination of the extent of ¹²⁵I-IgG1 cleavage. PS2A, acute-phase serum sample (bar 1); PS2C, convalescent-phase serum sample (bar 2). IgG was removed from PS2C, and the serum sample was resupplemented with either unspecific polyclonal IgG (bar 3) or the original convalescent-phase serum sample IgG (bar 4). (B) Convalescent-phase serum was preabsorbed with inactive IdeS variants prior to incubation with active enzyme. IdeS variants used are indicated above each lane. PBS (lane 1) and GST (lane 2) were used as controls. The position of the diagnostic 31-kDa cleavage product is indicated. (C) Graphic representation of IdeS variants used to absorb neutralizing antibodies.

FIG. 3.

Effect of convalescent-phase or acute-phase serum samples on IdeS activity towards myeloma IgG. Dilutions of patient serum samples were incubated with constant amounts of recombinant IdeS and ¹²⁵I-labeled myeloma IgG1 as the substrate. Sera from patients PT15 and PS2 had increases of >100% in ELISA index, and their patient PS5 showed a 19% increase in ELISA index. Inhibitions of IdeS activity in convalescent-phase serum samples at a 100-fold serum dilution were 83%, 83%, and 23%, respectively. Open symbols are acute-phase samples; filled symbols are convalescent-phase samples. Enzyme activity is relative to activity in serum depleted of IgG.

FIG. 4.

Neutralizing antibodies interfere with IdeS-mediated survival in human blood. Whole human immune blood was treated with IdeS preincubated with polyclonal IgG (IgG) or purified neutralizing IgG (nIgG). S. pyogenes strain AP1 was incubated with treated blood, and the bacterial survival rate is shown as the number of CFU at 60 min compared to the number of CFU of the IgG control, which arbitrarily was set to 100%. The absolute survival of the IgG control varied between 78 and 84% of that of the inoculum. IdeS^C94S was used as a negative control. *, P < 0.05 compared to the control by Student's t test.