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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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Quantification of toxins and lactate dehydrogenase (LDH) activity in parent JIR8094 and tcdE mutant strains.Toxin titers in the culture supernatants (A) and in cytoplasmic proteins (B) were determined by ELISA and the signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. Student's t-test was used for statistical analysis. (*) P-value is < to 0.05. Dot blots with monoclonal anti-TcdA and anti-TcdB are shown in the lower panels. Purified TcdA and TcdB were used as controls. C. LDH activity in the cytoplasmic fraction was determined using Promega CytoTox 96 and was measured as micro units calculated in comparison with standards provided in the kit. D. Cytoplasmic proteins collected from a 16 hours old cultures of parental and tcdE mutant strains were analyzed in dot blots using monoclonal antibodies against L7/L12 ribosomal subunits and the RNA polymerase beta subunit.
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ppat-1002727-g003: Quantification of toxins and lactate dehydrogenase (LDH) activity in parent JIR8094 and tcdE mutant strains.Toxin titers in the culture supernatants (A) and in cytoplasmic proteins (B) were determined by ELISA and the signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. Student's t-test was used for statistical analysis. (*) P-value is < to 0.05. Dot blots with monoclonal anti-TcdA and anti-TcdB are shown in the lower panels. Purified TcdA and TcdB were used as controls. C. LDH activity in the cytoplasmic fraction was determined using Promega CytoTox 96 and was measured as micro units calculated in comparison with standards provided in the kit. D. Cytoplasmic proteins collected from a 16 hours old cultures of parental and tcdE mutant strains were analyzed in dot blots using monoclonal antibodies against L7/L12 ribosomal subunits and the RNA polymerase beta subunit.

Mentions: We used the ClosTron system recently developed by Heap and coworkers [24] to disrupt the tcdE gene by insertion of a group II intron from pMTL007 (see Materials and Methods). To confirm the disruption, the tcdE-specific primers OBD231 and OBD232 were used to amplify a 550 bp PCR product from the parental JIR8094 DNA, corresponding to the wild type tcdE gene, and a 2.5 kbp product from the mutant DNA, corresponding to the tcdE gene with group II intron inserted within it (Figure 2A). When PCR was carried out using intron-specific primers EBS(U) and ERM along with OBD232 and OBD231, PCR products of 1150 bp and 570 bp respectively could be amplified from tcdE mutant DNA but not from the wild-type strain's DNA (Figure 2A) confirming the insertion of the intron in the tcdE gene. Furthermore, Southern blot analysis confirmed that the intron had inserted in only one DNA region in the tcdE mutant (Figure 2B). The wild-type and the tcdE mutant strains grew similarly in TY broth, but a slightly higher OD600 was seen for the mutant strain compared the wild-type after 16 hrs (Figure 2C). In quantitative reverse transcription-PCR assays the levels of tcdA and tcdB transcripts were the same for the parental and mutant strains (data not shown). This result suggests that intron insertion within tcdE exerts no polar effect on the expression of tcdA, consistent with the fact that tcdA transcription occurs primarily from its own promoter (Figure 1A) [12]. We confirmed by dot blot experiments that the quantity of TcdA detected in total crude extracts of both strains was the same (Figure 2D). However, when we analyzed the culture supernatants, we observed a dramatic reduction in the amount of toxin secreted by the mutant when compared to the wild-type strain (Figure 3A). On the other hand, more toxin accumulated in the cytoplasm of the tcdE mutant than in the wild-type strain in stationary phase cells (Figure 3B). Both observations were confirmed by dot blot analysis with monoclonal antibodies against TcdA and TcdB (Figure 3AB) and Vero cell cytotoxicity assays, which is predominantly toxin B-assessed (Figure S1).


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

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

Quantification of toxins and lactate dehydrogenase (LDH) activity in parent JIR8094 and tcdE mutant strains.Toxin titers in the culture supernatants (A) and in cytoplasmic proteins (B) were determined by ELISA and the signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. Student's t-test was used for statistical analysis. (*) P-value is < to 0.05. Dot blots with monoclonal anti-TcdA and anti-TcdB are shown in the lower panels. Purified TcdA and TcdB were used as controls. C. LDH activity in the cytoplasmic fraction was determined using Promega CytoTox 96 and was measured as micro units calculated in comparison with standards provided in the kit. D. Cytoplasmic proteins collected from a 16 hours old cultures of parental and tcdE mutant strains were analyzed in dot blots using monoclonal antibodies against L7/L12 ribosomal subunits and the RNA polymerase beta subunit.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369941&req=5

ppat-1002727-g003: Quantification of toxins and lactate dehydrogenase (LDH) activity in parent JIR8094 and tcdE mutant strains.Toxin titers in the culture supernatants (A) and in cytoplasmic proteins (B) were determined by ELISA and the signal from the test was recorded as absorbance at 450 nm. The data shown are the mean +/− standard error of three replicative samples. Student's t-test was used for statistical analysis. (*) P-value is < to 0.05. Dot blots with monoclonal anti-TcdA and anti-TcdB are shown in the lower panels. Purified TcdA and TcdB were used as controls. C. LDH activity in the cytoplasmic fraction was determined using Promega CytoTox 96 and was measured as micro units calculated in comparison with standards provided in the kit. D. Cytoplasmic proteins collected from a 16 hours old cultures of parental and tcdE mutant strains were analyzed in dot blots using monoclonal antibodies against L7/L12 ribosomal subunits and the RNA polymerase beta subunit.
Mentions: We used the ClosTron system recently developed by Heap and coworkers [24] to disrupt the tcdE gene by insertion of a group II intron from pMTL007 (see Materials and Methods). To confirm the disruption, the tcdE-specific primers OBD231 and OBD232 were used to amplify a 550 bp PCR product from the parental JIR8094 DNA, corresponding to the wild type tcdE gene, and a 2.5 kbp product from the mutant DNA, corresponding to the tcdE gene with group II intron inserted within it (Figure 2A). When PCR was carried out using intron-specific primers EBS(U) and ERM along with OBD232 and OBD231, PCR products of 1150 bp and 570 bp respectively could be amplified from tcdE mutant DNA but not from the wild-type strain's DNA (Figure 2A) confirming the insertion of the intron in the tcdE gene. Furthermore, Southern blot analysis confirmed that the intron had inserted in only one DNA region in the tcdE mutant (Figure 2B). The wild-type and the tcdE mutant strains grew similarly in TY broth, but a slightly higher OD600 was seen for the mutant strain compared the wild-type after 16 hrs (Figure 2C). In quantitative reverse transcription-PCR assays the levels of tcdA and tcdB transcripts were the same for the parental and mutant strains (data not shown). This result suggests that intron insertion within tcdE exerts no polar effect on the expression of tcdA, consistent with the fact that tcdA transcription occurs primarily from its own promoter (Figure 1A) [12]. We confirmed by dot blot experiments that the quantity of TcdA detected in total crude extracts of both strains was the same (Figure 2D). However, when we analyzed the culture supernatants, we observed a dramatic reduction in the amount of toxin secreted by the mutant when compared to the wild-type strain (Figure 3A). On the other hand, more toxin accumulated in the cytoplasm of the tcdE mutant than in the wild-type strain in stationary phase cells (Figure 3B). Both observations were confirmed by dot blot analysis with monoclonal antibodies against TcdA and TcdB (Figure 3AB) and Vero cell cytotoxicity assays, which is predominantly toxin B-assessed (Figure S1).

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