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Antibacterial activity of the contact and complement systems is blocked by SIC, a protein secreted by Streptococcus pyogenes.

Frick IM, Shannon O, Åkesson P, Mörgelin M, Collin M, Schmidtchen A, Björck L - J. Biol. Chem. (2010)

Bottom Line: Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides.Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein.Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University, SE-221 84 Lund, Sweden. Inga-Maria.Frick@med.lu.se

ABSTRACT
Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype.

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Related in: MedlinePlus

SIC associated with the surface of S. pyogenes enhances the bacterial multiplication in human plasma. A, the AP1 strain and the isogenic AP1 SIC-deficient mutant strain SIC− were cultivated in human plasma (diluted in TH (1:1)) for 8 h at 37 °C. Bacteria were washed with PBS, and bound proteins were eluted at pH 2.0 and subjected to SDS-PAGE followed by Western blotting. Lane 1, proteins eluted from AP1 bacteria; lane 2, proteins eluted from the AP1 mutant SIC−; and lane 3, SIC, 0.5 μg. B, AP1 bacteria (1 × 108 cfu) were incubated with radiolabeled protein SIC (750,000 cpm) in TH or in plasma (diluted 1:5 with TH) at 37 °C for 1 h. The bacteria were washed with PBS and resuspended in SDS sample buffer. Following separation by Tricine-SDS-PAGE, the gel was dried and subjected to autoradiography. Lane 1, AP1 in TH; lane 2, AP1 in plasma (1:5 with TH). C–E, wild-type AP1 bacteria and the isogenic mutant SIC− were grown in human plasma (diluted in TH (1:1)) overnight at 37 °C. The presence of SIC at the bacterial surface was demonstrated by fluorescence microscopy (right panel) using rabbit anti-SIC F(ab′)2 fragments, followed by Alexa Fluor 594-conjugated goat anti-rabbit F(ab′)2 fragments. Scale bar represents 10 μm. C, AP1 bacteria. D, SIC− bacteria. E, AP1 background fluorescence with the secondary goat F(ab′)2 fragments only. F, wild-type AP1 bacteria (black bars) and the isogenic mutant SIC− (gray bars) were grown in human plasma. At indicated time points, bacteria were plated on TH agar plates. Plates were incubated overnight at 37 °C, the number of cfu was determined, and the MF was calculated. The MF of AP1 was set to 1 at each time point in each individual experiment, and the MF of the mutant SIC− was related to this. Mean values ± S.E. of 10 experiments are shown. p values were determined by using the Mann-Whitney U test.
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Figure 6: SIC associated with the surface of S. pyogenes enhances the bacterial multiplication in human plasma. A, the AP1 strain and the isogenic AP1 SIC-deficient mutant strain SIC− were cultivated in human plasma (diluted in TH (1:1)) for 8 h at 37 °C. Bacteria were washed with PBS, and bound proteins were eluted at pH 2.0 and subjected to SDS-PAGE followed by Western blotting. Lane 1, proteins eluted from AP1 bacteria; lane 2, proteins eluted from the AP1 mutant SIC−; and lane 3, SIC, 0.5 μg. B, AP1 bacteria (1 × 108 cfu) were incubated with radiolabeled protein SIC (750,000 cpm) in TH or in plasma (diluted 1:5 with TH) at 37 °C for 1 h. The bacteria were washed with PBS and resuspended in SDS sample buffer. Following separation by Tricine-SDS-PAGE, the gel was dried and subjected to autoradiography. Lane 1, AP1 in TH; lane 2, AP1 in plasma (1:5 with TH). C–E, wild-type AP1 bacteria and the isogenic mutant SIC− were grown in human plasma (diluted in TH (1:1)) overnight at 37 °C. The presence of SIC at the bacterial surface was demonstrated by fluorescence microscopy (right panel) using rabbit anti-SIC F(ab′)2 fragments, followed by Alexa Fluor 594-conjugated goat anti-rabbit F(ab′)2 fragments. Scale bar represents 10 μm. C, AP1 bacteria. D, SIC− bacteria. E, AP1 background fluorescence with the secondary goat F(ab′)2 fragments only. F, wild-type AP1 bacteria (black bars) and the isogenic mutant SIC− (gray bars) were grown in human plasma. At indicated time points, bacteria were plated on TH agar plates. Plates were incubated overnight at 37 °C, the number of cfu was determined, and the MF was calculated. The MF of AP1 was set to 1 at each time point in each individual experiment, and the MF of the mutant SIC− was related to this. Mean values ± S.E. of 10 experiments are shown. p values were determined by using the Mann-Whitney U test.

Mentions: When AP1 bacteria were grown in human plasma, only trace amounts of SIC could be detected in the plasma by Western blot or ELISA following 8 h of growth. This observation indicated that the SIC secreted by the bacteria in plasma environment could be bound to the bacterial surface where it blocks binding of HK or the activity of generated antibacterial HK fragments. The SIC-deficient isogenic mutant strain SIC− derived from AP1 (29) was included as a negative control in these experiments. Due to the poor growth of S. pyogenes in 100% plasma, the bacteria were cultivated in 50% plasma to enhance the number of bacterial cells. Following growth, bacteria were carefully washed, and proteins associated with the surface were released by low pH and subjected to Western blot analysis using antibodies against SIC. A prominent band of 35 kDa, released from AP1 bacteria, reacted with the SIC antibody (Fig. 6A, lane 1). No such band was identified in the material eluted from the SIC− mutant (Fig. 6A, lane 2). SIC eluted from AP1 bacteria appeared slightly smaller than the SIC control isolated from AP1 growth medium (Fig. 6A, lane 3), indicating that the surface-bound SIC represents a processed form of the protein. In addition, immunoreactive bands of ∼31, 50, and 55 kDa, respectively, were present in the material eluted from both strains. AP1 bacteria bind several abundant plasma proteins, including albumin, fibrinogen, and IgG, via the surface proteins H and M1 (43, 44). Thus, the band migrating at 55 kDa corresponds to IgG heavy chains interacting with the HRP-conjugated protein A used in the assay. M1 protein is released from the bacterial surface (44), and the 50-kDa band represents M1 protein interacting with the rabbit antibody against SIC through nonimmune IgGFc binding (44). AP1 bacteria secretes IdeS, a proteolytic enzyme that cleaves IgG heavy chains in the hinge region (45). Surface-bound IgG is cleaved by IdeS, and under reducing conditions, heavy chain fragments of 31 kDa are generated, which bind the secondary protein A reagent in the Western blot assay. NH2-terminal sequencing of this band confirmed it to be the 31-kDa IgG fragment mentioned above.


Antibacterial activity of the contact and complement systems is blocked by SIC, a protein secreted by Streptococcus pyogenes.

Frick IM, Shannon O, Åkesson P, Mörgelin M, Collin M, Schmidtchen A, Björck L - J. Biol. Chem. (2010)

SIC associated with the surface of S. pyogenes enhances the bacterial multiplication in human plasma. A, the AP1 strain and the isogenic AP1 SIC-deficient mutant strain SIC− were cultivated in human plasma (diluted in TH (1:1)) for 8 h at 37 °C. Bacteria were washed with PBS, and bound proteins were eluted at pH 2.0 and subjected to SDS-PAGE followed by Western blotting. Lane 1, proteins eluted from AP1 bacteria; lane 2, proteins eluted from the AP1 mutant SIC−; and lane 3, SIC, 0.5 μg. B, AP1 bacteria (1 × 108 cfu) were incubated with radiolabeled protein SIC (750,000 cpm) in TH or in plasma (diluted 1:5 with TH) at 37 °C for 1 h. The bacteria were washed with PBS and resuspended in SDS sample buffer. Following separation by Tricine-SDS-PAGE, the gel was dried and subjected to autoradiography. Lane 1, AP1 in TH; lane 2, AP1 in plasma (1:5 with TH). C–E, wild-type AP1 bacteria and the isogenic mutant SIC− were grown in human plasma (diluted in TH (1:1)) overnight at 37 °C. The presence of SIC at the bacterial surface was demonstrated by fluorescence microscopy (right panel) using rabbit anti-SIC F(ab′)2 fragments, followed by Alexa Fluor 594-conjugated goat anti-rabbit F(ab′)2 fragments. Scale bar represents 10 μm. C, AP1 bacteria. D, SIC− bacteria. E, AP1 background fluorescence with the secondary goat F(ab′)2 fragments only. F, wild-type AP1 bacteria (black bars) and the isogenic mutant SIC− (gray bars) were grown in human plasma. At indicated time points, bacteria were plated on TH agar plates. Plates were incubated overnight at 37 °C, the number of cfu was determined, and the MF was calculated. The MF of AP1 was set to 1 at each time point in each individual experiment, and the MF of the mutant SIC− was related to this. Mean values ± S.E. of 10 experiments are shown. p values were determined by using the Mann-Whitney U test.
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Figure 6: SIC associated with the surface of S. pyogenes enhances the bacterial multiplication in human plasma. A, the AP1 strain and the isogenic AP1 SIC-deficient mutant strain SIC− were cultivated in human plasma (diluted in TH (1:1)) for 8 h at 37 °C. Bacteria were washed with PBS, and bound proteins were eluted at pH 2.0 and subjected to SDS-PAGE followed by Western blotting. Lane 1, proteins eluted from AP1 bacteria; lane 2, proteins eluted from the AP1 mutant SIC−; and lane 3, SIC, 0.5 μg. B, AP1 bacteria (1 × 108 cfu) were incubated with radiolabeled protein SIC (750,000 cpm) in TH or in plasma (diluted 1:5 with TH) at 37 °C for 1 h. The bacteria were washed with PBS and resuspended in SDS sample buffer. Following separation by Tricine-SDS-PAGE, the gel was dried and subjected to autoradiography. Lane 1, AP1 in TH; lane 2, AP1 in plasma (1:5 with TH). C–E, wild-type AP1 bacteria and the isogenic mutant SIC− were grown in human plasma (diluted in TH (1:1)) overnight at 37 °C. The presence of SIC at the bacterial surface was demonstrated by fluorescence microscopy (right panel) using rabbit anti-SIC F(ab′)2 fragments, followed by Alexa Fluor 594-conjugated goat anti-rabbit F(ab′)2 fragments. Scale bar represents 10 μm. C, AP1 bacteria. D, SIC− bacteria. E, AP1 background fluorescence with the secondary goat F(ab′)2 fragments only. F, wild-type AP1 bacteria (black bars) and the isogenic mutant SIC− (gray bars) were grown in human plasma. At indicated time points, bacteria were plated on TH agar plates. Plates were incubated overnight at 37 °C, the number of cfu was determined, and the MF was calculated. The MF of AP1 was set to 1 at each time point in each individual experiment, and the MF of the mutant SIC− was related to this. Mean values ± S.E. of 10 experiments are shown. p values were determined by using the Mann-Whitney U test.
Mentions: When AP1 bacteria were grown in human plasma, only trace amounts of SIC could be detected in the plasma by Western blot or ELISA following 8 h of growth. This observation indicated that the SIC secreted by the bacteria in plasma environment could be bound to the bacterial surface where it blocks binding of HK or the activity of generated antibacterial HK fragments. The SIC-deficient isogenic mutant strain SIC− derived from AP1 (29) was included as a negative control in these experiments. Due to the poor growth of S. pyogenes in 100% plasma, the bacteria were cultivated in 50% plasma to enhance the number of bacterial cells. Following growth, bacteria were carefully washed, and proteins associated with the surface were released by low pH and subjected to Western blot analysis using antibodies against SIC. A prominent band of 35 kDa, released from AP1 bacteria, reacted with the SIC antibody (Fig. 6A, lane 1). No such band was identified in the material eluted from the SIC− mutant (Fig. 6A, lane 2). SIC eluted from AP1 bacteria appeared slightly smaller than the SIC control isolated from AP1 growth medium (Fig. 6A, lane 3), indicating that the surface-bound SIC represents a processed form of the protein. In addition, immunoreactive bands of ∼31, 50, and 55 kDa, respectively, were present in the material eluted from both strains. AP1 bacteria bind several abundant plasma proteins, including albumin, fibrinogen, and IgG, via the surface proteins H and M1 (43, 44). Thus, the band migrating at 55 kDa corresponds to IgG heavy chains interacting with the HRP-conjugated protein A used in the assay. M1 protein is released from the bacterial surface (44), and the 50-kDa band represents M1 protein interacting with the rabbit antibody against SIC through nonimmune IgGFc binding (44). AP1 bacteria secretes IdeS, a proteolytic enzyme that cleaves IgG heavy chains in the hinge region (45). Surface-bound IgG is cleaved by IdeS, and under reducing conditions, heavy chain fragments of 31 kDa are generated, which bind the secondary protein A reagent in the Western blot assay. NH2-terminal sequencing of this band confirmed it to be the 31-kDa IgG fragment mentioned above.

Bottom Line: Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides.Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein.Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation.

View Article: PubMed Central - PubMed

Affiliation: Division of Infection Medicine, Department of Clinical Sciences, Lund University, SE-221 84 Lund, Sweden. Inga-Maria.Frick@med.lu.se

ABSTRACT
Recent studies have shown that activation of complement and contact systems results in the generation of antibacterial peptides. Streptococcus pyogenes, a major bacterial pathogen in humans, exists in >100 different serotypes due to sequence variation in the surface-associated M protein. Cases of invasive and life-threatening S. pyogenes infections are commonly associated with isolates of the M1 serotype, and in contrast to the large majority of M serotypes, M1 isolates all secrete the SIC protein. Here, we show that SIC interferes with the activation of the contact system and blocks the activity of antibacterial peptides generated through complement and contact activation. This effect promotes the growth of S. pyogenes in human plasma, and in a mouse model of S. pyogenes sepsis, SIC enhances bacterial dissemination, results which help explain the high frequency of severe S. pyogenes infections caused by isolates of the M1 serotype.

Show MeSH
Related in: MedlinePlus