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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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FACS analysis of C. difficile cells for membrane permeability through propidium iodide (PI) and SYTO staining.A. The viability standard samples containing the heat killed and actively growing C. difficile cells at 1/100, 50/50 and 100/1 ratio, respectively. B. The tcdE mutant and the parent JIR8094 cells collected from the overnight (16 h) cultures, were subjected to FACS analysis following propidium iodide (PI) and SYTO staining.
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ppat-1002727-g005: FACS analysis of C. difficile cells for membrane permeability through propidium iodide (PI) and SYTO staining.A. The viability standard samples containing the heat killed and actively growing C. difficile cells at 1/100, 50/50 and 100/1 ratio, respectively. B. The tcdE mutant and the parent JIR8094 cells collected from the overnight (16 h) cultures, were subjected to FACS analysis following propidium iodide (PI) and SYTO staining.

Mentions: To test whether TcdE mediates toxin release via cell lysis, we compared the levels of several known cytosolic marker proteins in the tcdE mutant and wild-type strains. No significant difference was seen between the parent strain and the mutant in their levels of cytosolic LDH activity (Figure 3C). In addition, dot blots of cytosolic fractions of overnight cultures (16 hrs) with antibodies to ribosomal subunits (L7/L12) and the RNA polymerase β subunit (see Materials and Methods) showed similar levels of these proteins in the cytosol of the mutant and wild-type strains (Figure 3D). In the complemented tcdE mutant, the amounts of L7/L12 proteins in the culture supernatants were very low and equal to those detected for the parental and tcdE mutant strains (Figure 4D). Finally, we looked for an effect of TcdE expression on C. difficile membrane integrity. We used FACS analysis of cells exposed to the fluorescent nucleotide binding dyes SYTO9 and propidium iodide (PI). SYTO9 is commonly used as a stain for live cells, whereas PI is excluded by the intact cell membrane, thus staining a cell only when the integrity of the membrane has been compromised. When used in combination, cells with intact membranes are labeled green by SYTO9 while membrane-permeablized cells are labeled red by PI. FACS analysis of control samples, containing mixtures of heat-killed and actively growing C. difficile cells, was consistent with the different ratios (1/100, 50/50 and 100/1 of killed/live, respectively) that we used (Figure 5A). When we analysed the mutant and parental strains harvested 16 hrs after inoculation, no significant difference could be observed in the intact vs. membrane-permeable cell populations (Figure 5B). In both strains, the membrane-permeable fraction was negligible indicating minimal cell lysis. Interestingly, cell lysis did occur in a C. difficile tcdA tcdB double mutant strain [26] that produces TcdE, as shown by the loss of absorbance at 600 nm (Figure 6A) and the appearance of red fluorescence (Figure 6B) when compared to the parental and PaLoc negative strains (see the Discussion).


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

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

FACS analysis of C. difficile cells for membrane permeability through propidium iodide (PI) and SYTO staining.A. The viability standard samples containing the heat killed and actively growing C. difficile cells at 1/100, 50/50 and 100/1 ratio, respectively. B. The tcdE mutant and the parent JIR8094 cells collected from the overnight (16 h) cultures, were subjected to FACS analysis following propidium iodide (PI) and SYTO staining.
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Related In: Results  -  Collection

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

ppat-1002727-g005: FACS analysis of C. difficile cells for membrane permeability through propidium iodide (PI) and SYTO staining.A. The viability standard samples containing the heat killed and actively growing C. difficile cells at 1/100, 50/50 and 100/1 ratio, respectively. B. The tcdE mutant and the parent JIR8094 cells collected from the overnight (16 h) cultures, were subjected to FACS analysis following propidium iodide (PI) and SYTO staining.
Mentions: To test whether TcdE mediates toxin release via cell lysis, we compared the levels of several known cytosolic marker proteins in the tcdE mutant and wild-type strains. No significant difference was seen between the parent strain and the mutant in their levels of cytosolic LDH activity (Figure 3C). In addition, dot blots of cytosolic fractions of overnight cultures (16 hrs) with antibodies to ribosomal subunits (L7/L12) and the RNA polymerase β subunit (see Materials and Methods) showed similar levels of these proteins in the cytosol of the mutant and wild-type strains (Figure 3D). In the complemented tcdE mutant, the amounts of L7/L12 proteins in the culture supernatants were very low and equal to those detected for the parental and tcdE mutant strains (Figure 4D). Finally, we looked for an effect of TcdE expression on C. difficile membrane integrity. We used FACS analysis of cells exposed to the fluorescent nucleotide binding dyes SYTO9 and propidium iodide (PI). SYTO9 is commonly used as a stain for live cells, whereas PI is excluded by the intact cell membrane, thus staining a cell only when the integrity of the membrane has been compromised. When used in combination, cells with intact membranes are labeled green by SYTO9 while membrane-permeablized cells are labeled red by PI. FACS analysis of control samples, containing mixtures of heat-killed and actively growing C. difficile cells, was consistent with the different ratios (1/100, 50/50 and 100/1 of killed/live, respectively) that we used (Figure 5A). When we analysed the mutant and parental strains harvested 16 hrs after inoculation, no significant difference could be observed in the intact vs. membrane-permeable cell populations (Figure 5B). In both strains, the membrane-permeable fraction was negligible indicating minimal cell lysis. Interestingly, cell lysis did occur in a C. difficile tcdA tcdB double mutant strain [26] that produces TcdE, as shown by the loss of absorbance at 600 nm (Figure 6A) and the appearance of red fluorescence (Figure 6B) when compared to the parental and PaLoc negative strains (see the Discussion).

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