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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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Complementation of TcdE mutant.A Growth curve of parent, the TcdE mutant and the complemented TcdE mutant strains. A. The inducer ATc (20 ng/ml) was added to bacterial cultures at 4 hrs after inoculation, indicated by an arrow. The star * indicates the time point when the cultures were harvested for toxin release analysis. B. Toxins were quantified by ELISA from supernatants of bacterial cultures induced by 20 ng/ml ATc for 2 hours. The signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. C. Dot blots of culture supernatants of the parental, the TcdE mutant and the complemented TcdE mutant, induced or not induced by ATc (0 and 20 ng/ml), with monoclonal anti-TcdA. D. Dot blots of samples in B with monoclonal antibodies against L7/L12 ribosomal subunits.
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ppat-1002727-g004: Complementation of TcdE mutant.A Growth curve of parent, the TcdE mutant and the complemented TcdE mutant strains. A. The inducer ATc (20 ng/ml) was added to bacterial cultures at 4 hrs after inoculation, indicated by an arrow. The star * indicates the time point when the cultures were harvested for toxin release analysis. B. Toxins were quantified by ELISA from supernatants of bacterial cultures induced by 20 ng/ml ATc for 2 hours. The signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. C. Dot blots of culture supernatants of the parental, the TcdE mutant and the complemented TcdE mutant, induced or not induced by ATc (0 and 20 ng/ml), with monoclonal anti-TcdA. D. Dot blots of samples in B with monoclonal antibodies against L7/L12 ribosomal subunits.

Mentions: To confirm that the defect in toxin secretion was due to the disruption of TcdE, we complemented the tcdE mutant with the wild type tcdE gene. Expression of TcdE from its own promoter using a multicopy plasmid was observed to be lethal to C. difficile (see below). Hence we expressed TcdE in the mutant strain using a tightly controlled expression system [25]. The tcdE ORF with a C-terminal 6xHis Tag was cloned downstream of a tetracycline-inducible promoter in the vector pRPF185 to create pRG60 (see Materials and Methods). We first showed that TcdE-6His is expressed in C. difficile cultures induced with 20 ng/ml to 50 ng/ml of ATc (Anhydrotetracycline), a non-antibiotic analog of tetracycline, without affecting cell growth (data not shown). Thus, we induced the C. difficile cultures with ATc (20 ng/ml) to test the effect of TcdE-6His on toxin release in the complemented mutant strain. The tcdE mutant carrying pRG60 along with the control strains JIR8094 and the tcdE mutant carrying the vector pRPF185 were grown for 4 hours to an OD600 of 0.3 in TY broth and induced with ATc. No difference in growth of the three strains could be observed during the first two hours after induction (Figure 4A). However, after more than two hours, the growth rate of the complemented tcdE mutant began to decrease (Figure 4A). Hence, using ELISA, we measured toxin proteins in the supernatant fluid of cultures two hours after induction with ATc. The culture supernatant of the complemented tcdE mutant had a higher concentration of toxin proteins than did the parent strain JIR8094 or the tcdE mutant (Figure 4B). We confirmed by a dot blot experiment with monoclonal antibodies against TcdA that the tcdE gene on pRG60 complements the tcdE mutant (Figure 4C). Thus, these data imply that TcdE is directly required for toxin release.


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

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

Complementation of TcdE mutant.A Growth curve of parent, the TcdE mutant and the complemented TcdE mutant strains. A. The inducer ATc (20 ng/ml) was added to bacterial cultures at 4 hrs after inoculation, indicated by an arrow. The star * indicates the time point when the cultures were harvested for toxin release analysis. B. Toxins were quantified by ELISA from supernatants of bacterial cultures induced by 20 ng/ml ATc for 2 hours. The signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. C. Dot blots of culture supernatants of the parental, the TcdE mutant and the complemented TcdE mutant, induced or not induced by ATc (0 and 20 ng/ml), with monoclonal anti-TcdA. D. Dot blots of samples in B with monoclonal antibodies against L7/L12 ribosomal subunits.
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ppat-1002727-g004: Complementation of TcdE mutant.A Growth curve of parent, the TcdE mutant and the complemented TcdE mutant strains. A. The inducer ATc (20 ng/ml) was added to bacterial cultures at 4 hrs after inoculation, indicated by an arrow. The star * indicates the time point when the cultures were harvested for toxin release analysis. B. Toxins were quantified by ELISA from supernatants of bacterial cultures induced by 20 ng/ml ATc for 2 hours. The signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. C. Dot blots of culture supernatants of the parental, the TcdE mutant and the complemented TcdE mutant, induced or not induced by ATc (0 and 20 ng/ml), with monoclonal anti-TcdA. D. Dot blots of samples in B with monoclonal antibodies against L7/L12 ribosomal subunits.
Mentions: To confirm that the defect in toxin secretion was due to the disruption of TcdE, we complemented the tcdE mutant with the wild type tcdE gene. Expression of TcdE from its own promoter using a multicopy plasmid was observed to be lethal to C. difficile (see below). Hence we expressed TcdE in the mutant strain using a tightly controlled expression system [25]. The tcdE ORF with a C-terminal 6xHis Tag was cloned downstream of a tetracycline-inducible promoter in the vector pRPF185 to create pRG60 (see Materials and Methods). We first showed that TcdE-6His is expressed in C. difficile cultures induced with 20 ng/ml to 50 ng/ml of ATc (Anhydrotetracycline), a non-antibiotic analog of tetracycline, without affecting cell growth (data not shown). Thus, we induced the C. difficile cultures with ATc (20 ng/ml) to test the effect of TcdE-6His on toxin release in the complemented mutant strain. The tcdE mutant carrying pRG60 along with the control strains JIR8094 and the tcdE mutant carrying the vector pRPF185 were grown for 4 hours to an OD600 of 0.3 in TY broth and induced with ATc. No difference in growth of the three strains could be observed during the first two hours after induction (Figure 4A). However, after more than two hours, the growth rate of the complemented tcdE mutant began to decrease (Figure 4A). Hence, using ELISA, we measured toxin proteins in the supernatant fluid of cultures two hours after induction with ATc. The culture supernatant of the complemented tcdE mutant had a higher concentration of toxin proteins than did the parent strain JIR8094 or the tcdE mutant (Figure 4B). We confirmed by a dot blot experiment with monoclonal antibodies against TcdA that the tcdE gene on pRG60 complements the tcdE mutant (Figure 4C). Thus, these data imply that TcdE is directly required for toxin release.

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