Limits...
Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα.

Brockmeyer J, Aldick T, Soltwisch J, Zhang W, Tarr PI, Weiss A, Dreisewerd K, Müthing J, Bielaszewska M, Karch H - Environ. Microbiol. (2011)

Bottom Line: In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed.We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors.Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food Chemistry, University of Münster, Corrensstrasse 45, Münster, Germany. jbrockm@uni-muenster.de

Show MeSH

Related in: MedlinePlus

Mass spectrometric analysis of the trypsin-digested Mr ∼82 kDa breakdown fragment. A. Representative MALDI-TOF-MS spectrum of the Mr ∼82 kDa breakdown fragment of EHEC-Hly. Peaks identified as EHEC-Hly-specific (1–14), EspPα-specific (E1–E8) or EHEC-Hly-specific matrix adducts of 189 m/z units (*) are indicated. B. Schematic illustration of the 107 kDa large EHEC-Hly with its structural domains and the proposed cleavage site. The calcium-binding domain is indicated by strips and the hydrophobic domain is marked in dark grey. The EspPα cleavage site is located in the N-terminal end of the hydrophobic domain of EHEC-Hly as indicated by an arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3472028&req=5

fig03: Mass spectrometric analysis of the trypsin-digested Mr ∼82 kDa breakdown fragment. A. Representative MALDI-TOF-MS spectrum of the Mr ∼82 kDa breakdown fragment of EHEC-Hly. Peaks identified as EHEC-Hly-specific (1–14), EspPα-specific (E1–E8) or EHEC-Hly-specific matrix adducts of 189 m/z units (*) are indicated. B. Schematic illustration of the 107 kDa large EHEC-Hly with its structural domains and the proposed cleavage site. The calcium-binding domain is indicated by strips and the hydrophobic domain is marked in dark grey. The EspPα cleavage site is located in the N-terminal end of the hydrophobic domain of EHEC-Hly as indicated by an arrow.

Mentions: To verify the identity of the EHEC-Hly cleavage products and to estimate which domains might be affected, we analysed the immunoreactive breakdown fragments (Mr ∼84 kDa, ∼82 kDa and ∼34 kDa) by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In all of the three breakdown products, EHEC-Hly-specific peptides located in the C-terminal end of the protein were identified, indicating that after the formation of the Mr ∼84 kDa fragment further degradation leads to the generation of the smaller EHEC-Hly breakdown products. As an example, a representative peptide mass spectrum of the Mr ∼82 kDa fragment after tryptic digest is shown in Fig. 3A. In this EHEC-Hly fragment we identified 14 EHEC-Hly-specific peptides ranging in length between 6 and 24 amino acids resulting in a sequence coverage of 14.5% (Table 4, 1–14). The detected EHEC-Hly-specific peptides were located between amino acids 255 and 939 of the 998-amino-acid-large EHEC-Hly molecule (Fig. 3B). Several fragments were chemically modified because of reactions during sample preparation or data acquisition or formed matrix adducts (indicated in Table 4). The potential region of proteolytic cleavage by EspPα was determined to be located in the N-terminal end of EHEC-Hly as indicated by the arrow in the schematic illustration of the toxin (Fig. 3B). This is further underlined by the theoretical mass of 80.3 kDa of the EHEC-Hly fragment ranging from amino acid 255 to 998, which corresponds well to the observed migration pattern in gel electrophoresis (Fig. 1A). Further analysis of non-EHEC-Hly signals revealed the presence of eight peptides specific for EspPα (Table 4, peptides E1–E8). These peptides are entirely located in the N-terminal end of the serine protease and represent the previously described autodegradation product of EspPα (Dutta et al., 2002). This EspPα-specific fragment, which is commonly found in all EspPα isolations, has a Mr of ∼86 kDa and accompanied also the isolated Mr ∼82 kDa breakdown product of EHEC-Hly due to a similar electrophoretic separation property as evidenced by immunoblotting analysis (data not shown).


Enterohaemorrhagic Escherichia coli haemolysin is cleaved and inactivated by serine protease EspPα.

Brockmeyer J, Aldick T, Soltwisch J, Zhang W, Tarr PI, Weiss A, Dreisewerd K, Müthing J, Bielaszewska M, Karch H - Environ. Microbiol. (2011)

Mass spectrometric analysis of the trypsin-digested Mr ∼82 kDa breakdown fragment. A. Representative MALDI-TOF-MS spectrum of the Mr ∼82 kDa breakdown fragment of EHEC-Hly. Peaks identified as EHEC-Hly-specific (1–14), EspPα-specific (E1–E8) or EHEC-Hly-specific matrix adducts of 189 m/z units (*) are indicated. B. Schematic illustration of the 107 kDa large EHEC-Hly with its structural domains and the proposed cleavage site. The calcium-binding domain is indicated by strips and the hydrophobic domain is marked in dark grey. The EspPα cleavage site is located in the N-terminal end of the hydrophobic domain of EHEC-Hly as indicated by an arrow.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472028&req=5

fig03: Mass spectrometric analysis of the trypsin-digested Mr ∼82 kDa breakdown fragment. A. Representative MALDI-TOF-MS spectrum of the Mr ∼82 kDa breakdown fragment of EHEC-Hly. Peaks identified as EHEC-Hly-specific (1–14), EspPα-specific (E1–E8) or EHEC-Hly-specific matrix adducts of 189 m/z units (*) are indicated. B. Schematic illustration of the 107 kDa large EHEC-Hly with its structural domains and the proposed cleavage site. The calcium-binding domain is indicated by strips and the hydrophobic domain is marked in dark grey. The EspPα cleavage site is located in the N-terminal end of the hydrophobic domain of EHEC-Hly as indicated by an arrow.
Mentions: To verify the identity of the EHEC-Hly cleavage products and to estimate which domains might be affected, we analysed the immunoreactive breakdown fragments (Mr ∼84 kDa, ∼82 kDa and ∼34 kDa) by peptide mass fingerprinting using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In all of the three breakdown products, EHEC-Hly-specific peptides located in the C-terminal end of the protein were identified, indicating that after the formation of the Mr ∼84 kDa fragment further degradation leads to the generation of the smaller EHEC-Hly breakdown products. As an example, a representative peptide mass spectrum of the Mr ∼82 kDa fragment after tryptic digest is shown in Fig. 3A. In this EHEC-Hly fragment we identified 14 EHEC-Hly-specific peptides ranging in length between 6 and 24 amino acids resulting in a sequence coverage of 14.5% (Table 4, 1–14). The detected EHEC-Hly-specific peptides were located between amino acids 255 and 939 of the 998-amino-acid-large EHEC-Hly molecule (Fig. 3B). Several fragments were chemically modified because of reactions during sample preparation or data acquisition or formed matrix adducts (indicated in Table 4). The potential region of proteolytic cleavage by EspPα was determined to be located in the N-terminal end of EHEC-Hly as indicated by the arrow in the schematic illustration of the toxin (Fig. 3B). This is further underlined by the theoretical mass of 80.3 kDa of the EHEC-Hly fragment ranging from amino acid 255 to 998, which corresponds well to the observed migration pattern in gel electrophoresis (Fig. 1A). Further analysis of non-EHEC-Hly signals revealed the presence of eight peptides specific for EspPα (Table 4, peptides E1–E8). These peptides are entirely located in the N-terminal end of the serine protease and represent the previously described autodegradation product of EspPα (Dutta et al., 2002). This EspPα-specific fragment, which is commonly found in all EspPα isolations, has a Mr of ∼86 kDa and accompanied also the isolated Mr ∼82 kDa breakdown product of EHEC-Hly due to a similar electrophoretic separation property as evidenced by immunoblotting analysis (data not shown).

Bottom Line: In a cellular infection system, the cytolytic potential of EHEC-Hly-secreting recombinant strains was abolished when EspPα was coexpressed.We propose the concept of bacterial effector molecule interference (BEMI), reflecting the concerted interplay of virulence factors.Interference between effector molecules might be an additional way to regulate virulence functions and increases the complexity of monomolecular phenotypes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Food Chemistry, University of Münster, Corrensstrasse 45, Münster, Germany. jbrockm@uni-muenster.de

Show MeSH
Related in: MedlinePlus