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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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The holin like TcdE.A. Constructs used for holin expression under the control of the late transcription regulatory (LTR) elements of phage λ. The promoter pR′ and the transcriptional terminator tR′ of the λ LTR region are depicted as a bent arrow and a hairpin structure, respectively. B. TcdE sequence: all possible translational starts are indicated as Met1, Met25 and Met27, the potential Shine-Dalgarno sequences are underlined and mutated nucleotides in the specified constructs are highlighted in bold.
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ppat-1002727-g007: The holin like TcdE.A. Constructs used for holin expression under the control of the late transcription regulatory (LTR) elements of phage λ. The promoter pR′ and the transcriptional terminator tR′ of the λ LTR region are depicted as a bent arrow and a hairpin structure, respectively. B. TcdE sequence: all possible translational starts are indicated as Met1, Met25 and Met27, the potential Shine-Dalgarno sequences are underlined and mutated nucleotides in the specified constructs are highlighted in bold.

Mentions: Since no bona fide system for assaying holin activity exists in C. difficile, we turned to assays of E. coli λ lysogens expressing TcdE. To test whether TcdE has any holin-like activity, we asked whether TcdE could complement a λ lysogen that has a nonsense mutation (Sam7) in its holin gene but has a functional endolysin gene [27]. We confirmed the functionality of this system using plasmids pJN4 and pJN5 (Figure 7A) expressing, respectively, a μιξτυρε of holin S105 and antiholin S107 or S105 alone from the λ late promoter and the λS RBS [28]. As shown in Figure 8A, thermo-induction of E. coli MC1063 λ (cI857Sam7) containing pJN4 or pJN5 led to complete bacterial lysis after 40 or 70 minutes, respectively, which is in good agreement with published values [18]. No lysis was observed after heat induction of a plasmid-free lysogen or a lysogen carrying the empty vector (Figure 8A).


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

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

The holin like TcdE.A. Constructs used for holin expression under the control of the late transcription regulatory (LTR) elements of phage λ. The promoter pR′ and the transcriptional terminator tR′ of the λ LTR region are depicted as a bent arrow and a hairpin structure, respectively. B. TcdE sequence: all possible translational starts are indicated as Met1, Met25 and Met27, the potential Shine-Dalgarno sequences are underlined and mutated nucleotides in the specified constructs are highlighted in bold.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369941&req=5

ppat-1002727-g007: The holin like TcdE.A. Constructs used for holin expression under the control of the late transcription regulatory (LTR) elements of phage λ. The promoter pR′ and the transcriptional terminator tR′ of the λ LTR region are depicted as a bent arrow and a hairpin structure, respectively. B. TcdE sequence: all possible translational starts are indicated as Met1, Met25 and Met27, the potential Shine-Dalgarno sequences are underlined and mutated nucleotides in the specified constructs are highlighted in bold.
Mentions: Since no bona fide system for assaying holin activity exists in C. difficile, we turned to assays of E. coli λ lysogens expressing TcdE. To test whether TcdE has any holin-like activity, we asked whether TcdE could complement a λ lysogen that has a nonsense mutation (Sam7) in its holin gene but has a functional endolysin gene [27]. We confirmed the functionality of this system using plasmids pJN4 and pJN5 (Figure 7A) expressing, respectively, a μιξτυρε of holin S105 and antiholin S107 or S105 alone from the λ late promoter and the λS RBS [28]. As shown in Figure 8A, thermo-induction of E. coli MC1063 λ (cI857Sam7) containing pJN4 or pJN5 led to complete bacterial lysis after 40 or 70 minutes, respectively, which is in good agreement with published values [18]. No lysis was observed after heat induction of a plasmid-free lysogen or a lysogen carrying the empty vector (Figure 8A).

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