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IgG protease Mac/IdeS is not essential for phagocyte resistance or mouse virulence of M1T1 group A Streptococcus.

Okumura CY, Anderson EL, Döhrmann S, Tran DN, Olson J, von Pawel-Rammingen U, Nizet V - MBio (2013)

Bottom Line: We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence.In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse.These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of California, San Diego, La Jolla, California, USA. okumura@oxy.edu

ABSTRACT

Unlabelled: The Mac/IdeS protein of group A Streptococcus (GAS) is a secreted cysteine protease with cleavage specificity for IgG and is highly expressed in the GAS serotype M1T1 clone, which is the serotype most frequently isolated from patients with life-threatening invasive infections. While studies of Mac/IdeS with recombinant protein have shown that the protein can potentially prevent opsonophagocytosis of GAS by neutrophils, the role of the protein in immune evasion as physiologically produced by the living organism has not been studied. Here we examined the contribution of Mac/IdeS to invasive GAS disease by generating a mutant lacking Mac/IdeS in the hyperinvasive M1T1 background. While Mac/IdeS was highly expressed and proteolytically active in the hyperinvasive strain, elimination of the bacterial protease did not significantly influence GAS phagocytic uptake, oxidative-burst induction, cathelicidin sensitivity, resistance to neutrophil or macrophage killing, or pathogenicity in pre- or postimmune mouse infectious challenges. We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence. Given the conservation of Mac/IdeS and homologues across GAS strains, it is possible that Mac/IdeS serves another important function in GAS ecology or contributes to virulence in other strain backgrounds.

Importance: Group A Streptococcus (GAS) causes human infections ranging from strep throat to life-threatening conditions such as flesh-eating disease and toxic shock syndrome. Common disease-associated clones of GAS can cause both mild and severe infections because of a characteristic mutation and subsequent change in the expression of several genes that develops under host immune selection. One of these genes encodes Mac/IdeS, a protease that has been shown to cleave antibodies important to the immune defense system. In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse. These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.

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Mac is upregulated in the AP M1T1 GAS strain and cleaves IgG. (A) mac gene expression is significantly upregulated in log- and stationary-phase cultures, while speB is down-regulated in AP M1T1 GAS compared to the original WT strain prior to AP. Data are shown as the mean ± the standard error of the mean of three independent experiments. n.s., not significant; **, P < 0.01; ***, P < 0.001 (one-way ANOVA with Tukey’s multiple-comparison posttest). (B) Western blot analysis of Mac/IdeS expression in log- and stationary-phase cultures following targeted mutagenesis, AP, and complementation analysis in the M1T1 GAS background. Cell-free culture supernatants were probed with polyclonal anti-Mac antiserum. Mac/IdeS was not detected in fourfold-concentrated supernatants from the WT M1T1 GAS strain. (C and D) Cleavage of human serum Ig (C) or purified human IgG (D) by cell-free culture supernatants from log- and stationary-phase cultures of AP, Δmac mutant, and complemented strains. Blots were probed with anti-human IgG antibodies. Heavy and light chains of Igs are visible, and the arrow indicates the cleavage product. In panels B to D, results of an experiment representative of at least three independent experiments are shown. The values to the left of panels C and D are molecular sizes in kilodaltons.
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fig1: Mac is upregulated in the AP M1T1 GAS strain and cleaves IgG. (A) mac gene expression is significantly upregulated in log- and stationary-phase cultures, while speB is down-regulated in AP M1T1 GAS compared to the original WT strain prior to AP. Data are shown as the mean ± the standard error of the mean of three independent experiments. n.s., not significant; **, P < 0.01; ***, P < 0.001 (one-way ANOVA with Tukey’s multiple-comparison posttest). (B) Western blot analysis of Mac/IdeS expression in log- and stationary-phase cultures following targeted mutagenesis, AP, and complementation analysis in the M1T1 GAS background. Cell-free culture supernatants were probed with polyclonal anti-Mac antiserum. Mac/IdeS was not detected in fourfold-concentrated supernatants from the WT M1T1 GAS strain. (C and D) Cleavage of human serum Ig (C) or purified human IgG (D) by cell-free culture supernatants from log- and stationary-phase cultures of AP, Δmac mutant, and complemented strains. Blots were probed with anti-human IgG antibodies. Heavy and light chains of Igs are visible, and the arrow indicates the cleavage product. In panels B to D, results of an experiment representative of at least three independent experiments are shown. The values to the left of panels C and D are molecular sizes in kilodaltons.

Mentions: Animal passage (AP) by mouse subcutaneous infection allows the selection of M1T1 strains with covRS mutation, hyperencapsulation, SpeB inactivation, and virulence factor upregulation, similar to bacteria found in invasive infections (13–15). To confirm that mac/ideS transcript levels were upregulated in our prototypical M1T1 strain (5448, wild type [WT]) and its AP form, we analyzed gene expression in log- and stationary-phase cultures (Fig. 1A). We found that mac/ideS expression was significantly greater in the AP (spontaneous covS mutant) cultures than in the parent M1T1 strain cultures, corroborating previously published results (13). Similar high mac/ideS expression levels were present during both the log and stationary phases of AP strain growth (Fig. 1A). Conversely, cysteine protease gene speB expression levels were much higher in the WT strain than in the AP variant, with maximal speB expression in stationary-phase cultures (Fig. 1A), also in agreement with earlier data (14). To corroborate our gene expression data, we performed Western blot analyses of secreted Mac/IdeS protein expression levels in cell-free culture supernatants from WT and AP GAS strains. Mac/IdeS protein was below the detection limit in WT M1T1 GAS culture supernatants, even when they were concentrated 4-fold, but was readily detected in both log- and stationary-phase culture supernatants of the AP strain (Fig. 1B). Combined, these data show that Mac/IdeS is expressed at relatively low levels by the WT M1T1 strain but that significant levels of protein are produced as the bacteria undergo the genetic switch, suggesting that Mac/IdeS could play a role in the virulence of the hyperinvasive AP form. As the expression of SpeB, which has been reported to have IgG protease activity in vitro (23–25), is markedly down-regulated in the AP strain (Fig. 1A), the effects of Mac/IdeS on IgG cleavage can be studied independently. Thus, we focused the remainder of our studies on the hypervirulent AP M1T1 strain.


IgG protease Mac/IdeS is not essential for phagocyte resistance or mouse virulence of M1T1 group A Streptococcus.

Okumura CY, Anderson EL, Döhrmann S, Tran DN, Olson J, von Pawel-Rammingen U, Nizet V - MBio (2013)

Mac is upregulated in the AP M1T1 GAS strain and cleaves IgG. (A) mac gene expression is significantly upregulated in log- and stationary-phase cultures, while speB is down-regulated in AP M1T1 GAS compared to the original WT strain prior to AP. Data are shown as the mean ± the standard error of the mean of three independent experiments. n.s., not significant; **, P < 0.01; ***, P < 0.001 (one-way ANOVA with Tukey’s multiple-comparison posttest). (B) Western blot analysis of Mac/IdeS expression in log- and stationary-phase cultures following targeted mutagenesis, AP, and complementation analysis in the M1T1 GAS background. Cell-free culture supernatants were probed with polyclonal anti-Mac antiserum. Mac/IdeS was not detected in fourfold-concentrated supernatants from the WT M1T1 GAS strain. (C and D) Cleavage of human serum Ig (C) or purified human IgG (D) by cell-free culture supernatants from log- and stationary-phase cultures of AP, Δmac mutant, and complemented strains. Blots were probed with anti-human IgG antibodies. Heavy and light chains of Igs are visible, and the arrow indicates the cleavage product. In panels B to D, results of an experiment representative of at least three independent experiments are shown. The values to the left of panels C and D are molecular sizes in kilodaltons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig1: Mac is upregulated in the AP M1T1 GAS strain and cleaves IgG. (A) mac gene expression is significantly upregulated in log- and stationary-phase cultures, while speB is down-regulated in AP M1T1 GAS compared to the original WT strain prior to AP. Data are shown as the mean ± the standard error of the mean of three independent experiments. n.s., not significant; **, P < 0.01; ***, P < 0.001 (one-way ANOVA with Tukey’s multiple-comparison posttest). (B) Western blot analysis of Mac/IdeS expression in log- and stationary-phase cultures following targeted mutagenesis, AP, and complementation analysis in the M1T1 GAS background. Cell-free culture supernatants were probed with polyclonal anti-Mac antiserum. Mac/IdeS was not detected in fourfold-concentrated supernatants from the WT M1T1 GAS strain. (C and D) Cleavage of human serum Ig (C) or purified human IgG (D) by cell-free culture supernatants from log- and stationary-phase cultures of AP, Δmac mutant, and complemented strains. Blots were probed with anti-human IgG antibodies. Heavy and light chains of Igs are visible, and the arrow indicates the cleavage product. In panels B to D, results of an experiment representative of at least three independent experiments are shown. The values to the left of panels C and D are molecular sizes in kilodaltons.
Mentions: Animal passage (AP) by mouse subcutaneous infection allows the selection of M1T1 strains with covRS mutation, hyperencapsulation, SpeB inactivation, and virulence factor upregulation, similar to bacteria found in invasive infections (13–15). To confirm that mac/ideS transcript levels were upregulated in our prototypical M1T1 strain (5448, wild type [WT]) and its AP form, we analyzed gene expression in log- and stationary-phase cultures (Fig. 1A). We found that mac/ideS expression was significantly greater in the AP (spontaneous covS mutant) cultures than in the parent M1T1 strain cultures, corroborating previously published results (13). Similar high mac/ideS expression levels were present during both the log and stationary phases of AP strain growth (Fig. 1A). Conversely, cysteine protease gene speB expression levels were much higher in the WT strain than in the AP variant, with maximal speB expression in stationary-phase cultures (Fig. 1A), also in agreement with earlier data (14). To corroborate our gene expression data, we performed Western blot analyses of secreted Mac/IdeS protein expression levels in cell-free culture supernatants from WT and AP GAS strains. Mac/IdeS protein was below the detection limit in WT M1T1 GAS culture supernatants, even when they were concentrated 4-fold, but was readily detected in both log- and stationary-phase culture supernatants of the AP strain (Fig. 1B). Combined, these data show that Mac/IdeS is expressed at relatively low levels by the WT M1T1 strain but that significant levels of protein are produced as the bacteria undergo the genetic switch, suggesting that Mac/IdeS could play a role in the virulence of the hyperinvasive AP form. As the expression of SpeB, which has been reported to have IgG protease activity in vitro (23–25), is markedly down-regulated in the AP strain (Fig. 1A), the effects of Mac/IdeS on IgG cleavage can be studied independently. Thus, we focused the remainder of our studies on the hypervirulent AP M1T1 strain.

Bottom Line: We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence.In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse.These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, University of California, San Diego, La Jolla, California, USA. okumura@oxy.edu

ABSTRACT

Unlabelled: The Mac/IdeS protein of group A Streptococcus (GAS) is a secreted cysteine protease with cleavage specificity for IgG and is highly expressed in the GAS serotype M1T1 clone, which is the serotype most frequently isolated from patients with life-threatening invasive infections. While studies of Mac/IdeS with recombinant protein have shown that the protein can potentially prevent opsonophagocytosis of GAS by neutrophils, the role of the protein in immune evasion as physiologically produced by the living organism has not been studied. Here we examined the contribution of Mac/IdeS to invasive GAS disease by generating a mutant lacking Mac/IdeS in the hyperinvasive M1T1 background. While Mac/IdeS was highly expressed and proteolytically active in the hyperinvasive strain, elimination of the bacterial protease did not significantly influence GAS phagocytic uptake, oxidative-burst induction, cathelicidin sensitivity, resistance to neutrophil or macrophage killing, or pathogenicity in pre- or postimmune mouse infectious challenges. We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence. Given the conservation of Mac/IdeS and homologues across GAS strains, it is possible that Mac/IdeS serves another important function in GAS ecology or contributes to virulence in other strain backgrounds.

Importance: Group A Streptococcus (GAS) causes human infections ranging from strep throat to life-threatening conditions such as flesh-eating disease and toxic shock syndrome. Common disease-associated clones of GAS can cause both mild and severe infections because of a characteristic mutation and subsequent change in the expression of several genes that develops under host immune selection. One of these genes encodes Mac/IdeS, a protease that has been shown to cleave antibodies important to the immune defense system. In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse. These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.

Show MeSH
Related in: MedlinePlus