Plasmid-encoded toxin (Pet) of Escherichia coli cleaves complement system proteins and inhibits complement-mediated lysis in vitro


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Article
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Description
The serine protease Pet is an autotransporter protein of the SPATEs family, important in the pathogenicity of Escherichia coli. The pet gene was initially found in the E. coli virulence plasmid, pAA2. Although this virulence factor was initially described in an intestinal pathotype, pet may also be present in other E. coli pathotypes, including extraintestinal (ExPEC). The complement system is an important defense mechanism of the immune system that can be activated by invading pathogens. Proteases produced by pathogenic bacteria, such as SPATEs, have proteolytic activity and can cleave components of the complement system, promoting bacterial resistance to human serum. Considering these factors, the proteolytic activity of Pet and its role in evading the complement system were investigated. Proteolytic assays were performed by incubating purified components of the complement system with Pet and Pet S260I (a catalytic site mutant) proteins. Pet, but not Pet S260I, could cleave C3, C5 and C9 components, and also inhibited the natural formation of C9 polymers. Furthermore, a dose-dependent inhibition of ZnCl2-induced C9 polymerization in vitro was observed. E. coli DH5α survived incubation with human serum pre-treated with Pet. Therefore, Pet can potentially interfere with the alternative and the terminal pathways of the complement system. In addition, by cleaving C9, Pet may inhibit membrane attack complex (MAC) formation on the bacterial outer membrane. Thus, our data are suggestive of a role of Pet in resistance of Escherichia coli to human serum.
Reference
. Plasmid-encoded toxin (Pet) of Escherichia coli cleaves complement system proteins and inhibits complement-mediated lysis in vitro. .
Link to cite this reference
https://repositorio.butantan.gov.br/handle/butantan/5126
Issue Date
2023


Files in This Item:

Ensaio Proteolítico - 5-24h - C3.tif
Description: Figure 1a - Degradation of C3 by Pet, in 5 and 24 hours of incubation. By Immunoblotting using specific antibodies, degradation products (red asterisk) of the molecule were observed only in the presence of the concentrated supernatant containing Pet.
Size: 24.31 MB
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Ensaio Proteolítico - 5-24h - C5.tif
Description: Figure 1b - Degradation of C5 by Pet, in 5 and 24 hours of incubation. By Immunoblotting using specific antibodies, degradation products (red asterisk) of the molecule were observed only in the presence of the concentrated supernatant containing Pet
Size: 24.31 MB
Format: TIFF
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Ensaio Proteolítico - 5-24h - C9.tif
Description: Figure 1c - Degradation of C9 by Pet, in 5 and 24 hours of incubation. By Immunoblotting using specific antibodies, degradation products (red asterisk) of the molecule were observed only in the presence of the concentrated supernatant containing Pet
Size: 24.31 MB
Format: TIFF
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Ensaio Proteolítico - 30m-1h - C3.tif
Description: Figure 2a - Degradation of C3 by Pet, in 30min and 1 hour of incubation. By Immunoblotting using specific antibodies, degradation products (red asterisk) of the molecule were observed only in the presence of the concentrated supernatant containing Pet and with 1 hour of incubation
Size: 48.62 MB
Format: TIFF
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Ensaio Proteolítico - 30m-1h - C5.tif
Description: Figure 2b - Degradation of C5 by Pet, in 30min and 1 hour of incubation. By Immunoblotting using specific antibodies, degradation products (red asterisk) of the molecule were observed only in the presence of the concentrated supernatant containing Pet and with 1 hour of incubation
Size: 24.31 MB
Format: TIFF
View/Open
Ensaio Proteolítico - 30m-1h - C9.tif
Description: Figure 2c - Degradation of C9 by Pet, in 30min and 1 hour of incubation. By Immunoblotting using specific antibodies, degradation products (red asterisk) of the molecule were observed only in the presence of the concentrated supernatant containing Pet and with 1 hour of incubation.
Size: 24.31 MB
Format: TIFF
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Ensaio PoliC9 - ZnCl2.tif
Description: Figure 3a and 3b - Dose-dependent degradation of C9 molecules and C9 polymers by Pet. In red asterisks, a dose-dependent degradation of C9 molecules and inhibition of the formation of C9 polymers were observed only in the presence of the supernatant containing Pet. In addition, in green asterisk, by firstly inducing the formation of C9 polymers and posteriorly incubating with Pet, degradation of both the C9 molecules and C9 polymers were observed.
Size: 24.31 MB
Format: TIFF
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DH5a human serum resistance assay - CFU data (Fig. 4) (1).xls
Description: Figure 4 - DH5 alpha Colony Forming Units count after serum resitance assay
Size: 23.5 kB
Format: Microsoft Excel
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DH5a human serum resistance assay - Two-way ANOVA data (Fig. 4) (3).xls
Description: Figure 4 - Statistical anlysis of DH5 alpha human serum resistance
Size: 42.5 kB
Format: Microsoft Excel
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DH5a human serum resistance assay - Graph (Fig. 4) (2).pptx
Description: Figure 4 - Resistance to the bactericidal activity of human serum assay. Bacteria survived incubations with the IHS and Pet-HS groups. There was no bacterial survival in the group treated with normal human serum (NHS) and in the group treated with human serum pre-treated with supernatant of the negative control (HB101-HS).
Size: 189.89 kB
Format: Microsoft Powerpoint XML
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Hypotetical model.tif
Description: Figure 5 - Hypothetical model of how the serine protease Pet could reach the bloodstream and degrade complement components
Size: 1.29 MB
Format: TIFF
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