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Secretion of Clostridium difficile toxins A and B requires the holin-like protein TcdE.

Govind R, Dupuy B - PLoS Pathog. (2012)

Bottom Line: A C. difficile tcdE mutant strain grew at the same rate as the wild-type strain, but accumulated a dramatically reduced amount of toxin proteins in the medium.In addition, TcdE did not overtly affect membrane integrity of C. difficile in the presence of TcdA/TcdB.TcdE appears to be the first example of a bacterial protein that releases toxins into the environment by a phage-like system.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France.

ABSTRACT
The pathogenesis of Clostridium difficile, the major cause of antibiotic-associated diarrhea, is mainly associated with the production and activities of two major toxins. In many bacteria, toxins are released into the extracellular environment via the general secretion pathways. C. difficile toxins A and B have no export signature and their secretion is not explainable by cell lysis, suggesting that they might be secreted by an unusual mechanism. The TcdE protein encoded within the C. difficile pathogenicity locus (PaLoc) has predicted structural features similar to those of bacteriophage holin proteins. During many types of phage infection, host lysis is driven by an endolysin that crosses the cytoplasmic membrane through a pore formed by holin oligomerization. We demonstrated that TcdE has a holin-like activity by functionally complementing a λ phage deprived of its holin. Similar to λ holin, TcdE expressed in Escherichia coli and C. difficile formed oligomers in the cytoplamic membrane. A C. difficile tcdE mutant strain grew at the same rate as the wild-type strain, but accumulated a dramatically reduced amount of toxin proteins in the medium. However, the complemented tcdE mutant released the toxins efficiently. There was no difference in the abundance of tcdA and tcdB transcripts or of several cytoplasmic proteins in the mutant and the wild-type strains. In addition, TcdE did not overtly affect membrane integrity of C. difficile in the presence of TcdA/TcdB. Thus, TcdE acts as a holin-like protein to facilitate the release of C. difficile toxins to the extracellular environment, but, unlike the phage holins, does not cause the non-specific release of cytosolic contents. TcdE appears to be the first example of a bacterial protein that releases toxins into the environment by a phage-like system.

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Localization of TcdE in E. coli and C. difficile.A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+tcdE-6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+tcdE-6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.
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ppat-1002727-g009: Localization of TcdE in E. coli and C. difficile.A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+tcdE-6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+tcdE-6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.

Mentions: In E. coli, the λS protein exists in an oligomeric state in the inner membrane [31]. To localize TcdE, we expressed TcdE with a C-terminal 6xHis Tag (pCD463) in E. coli strain MC1063 λCmrΔ(SR), which does not express holin or endolysin (see Materials and Methods). This version of TcdE complemented the λ (cI857Sam7) lysogen (Figure 8C). After induction of TcdE expression, cells were collected and the cytosolic and the inner membrane proteins were separated by SDS-PAGE and analyzed by Western blot with antibodies against the 6xHis Tag or ribosomal proteins L7/L12 or the membrane-bound β subunit of E. coli ATP synthase (Figure 9A). As expected, the ribosomal subunits were detected only in the cytoplasmic fraction and the ATPase β subunit was detected primarily in the membrane fraction. The anti-6xHis Tag antibodies detected TcdE only in the membrane fraction (Figure 9A), indicating that TcdE is exclusively membrane-bound in E. coli.


Secretion of Clostridium difficile toxins A and B requires the holin-like protein TcdE.

Govind R, Dupuy B - PLoS Pathog. (2012)

Localization of TcdE in E. coli and C. difficile.A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+tcdE-6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+tcdE-6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369941&req=5

ppat-1002727-g009: Localization of TcdE in E. coli and C. difficile.A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+tcdE-6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+tcdE-6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.
Mentions: In E. coli, the λS protein exists in an oligomeric state in the inner membrane [31]. To localize TcdE, we expressed TcdE with a C-terminal 6xHis Tag (pCD463) in E. coli strain MC1063 λCmrΔ(SR), which does not express holin or endolysin (see Materials and Methods). This version of TcdE complemented the λ (cI857Sam7) lysogen (Figure 8C). After induction of TcdE expression, cells were collected and the cytosolic and the inner membrane proteins were separated by SDS-PAGE and analyzed by Western blot with antibodies against the 6xHis Tag or ribosomal proteins L7/L12 or the membrane-bound β subunit of E. coli ATP synthase (Figure 9A). As expected, the ribosomal subunits were detected only in the cytoplasmic fraction and the ATPase β subunit was detected primarily in the membrane fraction. The anti-6xHis Tag antibodies detected TcdE only in the membrane fraction (Figure 9A), indicating that TcdE is exclusively membrane-bound in E. coli.

Bottom Line: A C. difficile tcdE mutant strain grew at the same rate as the wild-type strain, but accumulated a dramatically reduced amount of toxin proteins in the medium.In addition, TcdE did not overtly affect membrane integrity of C. difficile in the presence of TcdA/TcdB.TcdE appears to be the first example of a bacterial protein that releases toxins into the environment by a phage-like system.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France.

ABSTRACT
The pathogenesis of Clostridium difficile, the major cause of antibiotic-associated diarrhea, is mainly associated with the production and activities of two major toxins. In many bacteria, toxins are released into the extracellular environment via the general secretion pathways. C. difficile toxins A and B have no export signature and their secretion is not explainable by cell lysis, suggesting that they might be secreted by an unusual mechanism. The TcdE protein encoded within the C. difficile pathogenicity locus (PaLoc) has predicted structural features similar to those of bacteriophage holin proteins. During many types of phage infection, host lysis is driven by an endolysin that crosses the cytoplasmic membrane through a pore formed by holin oligomerization. We demonstrated that TcdE has a holin-like activity by functionally complementing a λ phage deprived of its holin. Similar to λ holin, TcdE expressed in Escherichia coli and C. difficile formed oligomers in the cytoplamic membrane. A C. difficile tcdE mutant strain grew at the same rate as the wild-type strain, but accumulated a dramatically reduced amount of toxin proteins in the medium. However, the complemented tcdE mutant released the toxins efficiently. There was no difference in the abundance of tcdA and tcdB transcripts or of several cytoplasmic proteins in the mutant and the wild-type strains. In addition, TcdE did not overtly affect membrane integrity of C. difficile in the presence of TcdA/TcdB. Thus, TcdE acts as a holin-like protein to facilitate the release of C. difficile toxins to the extracellular environment, but, unlike the phage holins, does not cause the non-specific release of cytosolic contents. TcdE appears to be the first example of a bacterial protein that releases toxins into the environment by a phage-like system.

Show MeSH
Related in: MedlinePlus