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The immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis, is involved in complement evasion.

Seele J, Beineke A, Hillermann LM, Jaschok-Kentner B, von Pawel-Rammingen U, Valentin-Weigand P, Baums CG - Vet. Res. (2015)

Bottom Line: Furthermore, expression of Ide(Ssuis) reduced IgM-triggered complement deposition on the bacterial surface.Bactericidal assays confirmed a positive effect of Ide(Ssuis) expression on bacterial survival in porcine blood in the presence of high titers of specific IgM.In conclusion, this study demonstrates that Ide(Ssuis) is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.

View Article: PubMed Central - PubMed

Affiliation: Institute for Microbiology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, 30173, Hannover, Germany. jana_seele@gmx.de.

ABSTRACT
Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated Ide(Ssuis) . The objective of this study was to characterize the function of Ide(Ssuis) in the host-pathogen interaction. Edman-sequencing revealed that Ide(Ssuis) cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that Ide(Ssuis) is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant Ide(Ssuis) . Furthermore, expression of Ide(Ssuis) reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10Δide(Ssuis). Bactericidal assays confirmed a positive effect of Ide(Ssuis) expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that Ide(Ssuis) is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.

No MeSH data available.


Related in: MedlinePlus

Hemolysis caused by the classical complement activation pathway is abrogated by IdeSsuisin dependence of the protease activity. (A) Purified sheep erythrocytes were incubated with water (defined as 100% hemolysis), physiological sodium chloride solution (NaCl), sera of a piglet drawn before (pre vaccination serum) and 7 days after prime vaccination (post vaccination αEry serum) with ovine erythrocytes. The αEry serum was heat inactivated or treated with EDTA to access the impact of complement activation. To specifically inhibit the classical complement pathway 10 mM EGTA plus 15 mM MgCl2 was used. (B) Illustration of rIdeSsuis and its truncated derivatives. The amino acids of IdeSsuis included in these constructs are superscribed. The region homologous to IdeS is shaded. (C) Complement dependent hemolysis is reduced by pretreatment of the indicated different αEry sera with rIdeSsuis, rIdeSsuis_homologue (rIdeSsuis_h) and rIdeSsuis_C_terminus (rIdeSsuis_C) but not with rMRP and rFBPS. (D) Proteolytic activity of rIdeSsuis and rIdeSsuis_homologue is crucial for complement inhibition. The IdeSsuis constructs were incubated with the cysteine-protease inhibitor iodoacetamide prior to incubation with the αEry sera as indicated. The final dilutions of porcine sera in the hemolysis assay were 1:20 in all cases. Bars and error bars represent mean values and standard deviations, respectively. Significant differences are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).
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Fig2: Hemolysis caused by the classical complement activation pathway is abrogated by IdeSsuisin dependence of the protease activity. (A) Purified sheep erythrocytes were incubated with water (defined as 100% hemolysis), physiological sodium chloride solution (NaCl), sera of a piglet drawn before (pre vaccination serum) and 7 days after prime vaccination (post vaccination αEry serum) with ovine erythrocytes. The αEry serum was heat inactivated or treated with EDTA to access the impact of complement activation. To specifically inhibit the classical complement pathway 10 mM EGTA plus 15 mM MgCl2 was used. (B) Illustration of rIdeSsuis and its truncated derivatives. The amino acids of IdeSsuis included in these constructs are superscribed. The region homologous to IdeS is shaded. (C) Complement dependent hemolysis is reduced by pretreatment of the indicated different αEry sera with rIdeSsuis, rIdeSsuis_homologue (rIdeSsuis_h) and rIdeSsuis_C_terminus (rIdeSsuis_C) but not with rMRP and rFBPS. (D) Proteolytic activity of rIdeSsuis and rIdeSsuis_homologue is crucial for complement inhibition. The IdeSsuis constructs were incubated with the cysteine-protease inhibitor iodoacetamide prior to incubation with the αEry sera as indicated. The final dilutions of porcine sera in the hemolysis assay were 1:20 in all cases. Bars and error bars represent mean values and standard deviations, respectively. Significant differences are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).

Mentions: The classical complement activation pathway can be studied in hemolysis assays using sera containing antibodies directed against erythrocytes. We investigated the impact of IdeSsuis on complement activation in a hemolysis assay including sera drawn from piglets vaccinated with erythrocytes (αEry sera). In accordance with complement activation, treatment of αEry sera with heat, EDTA or EGTA plus MgCl2 completely abolished the hemolytic activity of the αEry sera (Figure 2A). For functional analysis of IdeSsuis, αEry sera drawn after prime and booster vaccination were treated with different rIdeSsuis constructs (Figure 2B) prior incubation with erythrocytes. Incubation of the post-prime αEry serum with rIdeSsuis and rIdeSsuis_homologue (the domain containing the IgM protease), almost completely abolished this hemolysis (Figure 2C). Noteworthy, treatment of the post-prime αEry serum with two recombinant control proteins (MRP and FBPS), did not result in abrogation of hemolysis (Figure 2C). Interestingly, treatment of αEry sera with proteolytic inactive construct rIdeSsuis_C_terminus led also to a significant reduction of hemolysis indicating a separate role of the C-terminus in complement evasion. However, significant differences between inhibition of complement activation through αEry sera drawn after prime and booster vaccination were only observed for the IdeSsuis constructs with IgM protease activity (Figure 2C).Figure 2


The immunoglobulin M-degrading enzyme of Streptococcus suis, IdeSsuis, is involved in complement evasion.

Seele J, Beineke A, Hillermann LM, Jaschok-Kentner B, von Pawel-Rammingen U, Valentin-Weigand P, Baums CG - Vet. Res. (2015)

Hemolysis caused by the classical complement activation pathway is abrogated by IdeSsuisin dependence of the protease activity. (A) Purified sheep erythrocytes were incubated with water (defined as 100% hemolysis), physiological sodium chloride solution (NaCl), sera of a piglet drawn before (pre vaccination serum) and 7 days after prime vaccination (post vaccination αEry serum) with ovine erythrocytes. The αEry serum was heat inactivated or treated with EDTA to access the impact of complement activation. To specifically inhibit the classical complement pathway 10 mM EGTA plus 15 mM MgCl2 was used. (B) Illustration of rIdeSsuis and its truncated derivatives. The amino acids of IdeSsuis included in these constructs are superscribed. The region homologous to IdeS is shaded. (C) Complement dependent hemolysis is reduced by pretreatment of the indicated different αEry sera with rIdeSsuis, rIdeSsuis_homologue (rIdeSsuis_h) and rIdeSsuis_C_terminus (rIdeSsuis_C) but not with rMRP and rFBPS. (D) Proteolytic activity of rIdeSsuis and rIdeSsuis_homologue is crucial for complement inhibition. The IdeSsuis constructs were incubated with the cysteine-protease inhibitor iodoacetamide prior to incubation with the αEry sera as indicated. The final dilutions of porcine sera in the hemolysis assay were 1:20 in all cases. Bars and error bars represent mean values and standard deviations, respectively. Significant differences are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4404118&req=5

Fig2: Hemolysis caused by the classical complement activation pathway is abrogated by IdeSsuisin dependence of the protease activity. (A) Purified sheep erythrocytes were incubated with water (defined as 100% hemolysis), physiological sodium chloride solution (NaCl), sera of a piglet drawn before (pre vaccination serum) and 7 days after prime vaccination (post vaccination αEry serum) with ovine erythrocytes. The αEry serum was heat inactivated or treated with EDTA to access the impact of complement activation. To specifically inhibit the classical complement pathway 10 mM EGTA plus 15 mM MgCl2 was used. (B) Illustration of rIdeSsuis and its truncated derivatives. The amino acids of IdeSsuis included in these constructs are superscribed. The region homologous to IdeS is shaded. (C) Complement dependent hemolysis is reduced by pretreatment of the indicated different αEry sera with rIdeSsuis, rIdeSsuis_homologue (rIdeSsuis_h) and rIdeSsuis_C_terminus (rIdeSsuis_C) but not with rMRP and rFBPS. (D) Proteolytic activity of rIdeSsuis and rIdeSsuis_homologue is crucial for complement inhibition. The IdeSsuis constructs were incubated with the cysteine-protease inhibitor iodoacetamide prior to incubation with the αEry sera as indicated. The final dilutions of porcine sera in the hemolysis assay were 1:20 in all cases. Bars and error bars represent mean values and standard deviations, respectively. Significant differences are indicated (* p < 0.05; ** p < 0.01; *** p < 0.001).
Mentions: The classical complement activation pathway can be studied in hemolysis assays using sera containing antibodies directed against erythrocytes. We investigated the impact of IdeSsuis on complement activation in a hemolysis assay including sera drawn from piglets vaccinated with erythrocytes (αEry sera). In accordance with complement activation, treatment of αEry sera with heat, EDTA or EGTA plus MgCl2 completely abolished the hemolytic activity of the αEry sera (Figure 2A). For functional analysis of IdeSsuis, αEry sera drawn after prime and booster vaccination were treated with different rIdeSsuis constructs (Figure 2B) prior incubation with erythrocytes. Incubation of the post-prime αEry serum with rIdeSsuis and rIdeSsuis_homologue (the domain containing the IgM protease), almost completely abolished this hemolysis (Figure 2C). Noteworthy, treatment of the post-prime αEry serum with two recombinant control proteins (MRP and FBPS), did not result in abrogation of hemolysis (Figure 2C). Interestingly, treatment of αEry sera with proteolytic inactive construct rIdeSsuis_C_terminus led also to a significant reduction of hemolysis indicating a separate role of the C-terminus in complement evasion. However, significant differences between inhibition of complement activation through αEry sera drawn after prime and booster vaccination were only observed for the IdeSsuis constructs with IgM protease activity (Figure 2C).Figure 2

Bottom Line: Furthermore, expression of Ide(Ssuis) reduced IgM-triggered complement deposition on the bacterial surface.Bactericidal assays confirmed a positive effect of Ide(Ssuis) expression on bacterial survival in porcine blood in the presence of high titers of specific IgM.In conclusion, this study demonstrates that Ide(Ssuis) is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.

View Article: PubMed Central - PubMed

Affiliation: Institute for Microbiology, Centre for Infection Medicine, University of Veterinary Medicine Hannover, 30173, Hannover, Germany. jana_seele@gmx.de.

ABSTRACT
Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated Ide(Ssuis) . The objective of this study was to characterize the function of Ide(Ssuis) in the host-pathogen interaction. Edman-sequencing revealed that Ide(Ssuis) cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that Ide(Ssuis) is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant Ide(Ssuis) . Furthermore, expression of Ide(Ssuis) reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10Δide(Ssuis). Bactericidal assays confirmed a positive effect of Ide(Ssuis) expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that Ide(Ssuis) is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.

No MeSH data available.


Related in: MedlinePlus