Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Dec;80(12):4078-88.
doi: 10.1128/IAI.00069-12. Epub 2012 Sep 10.

Cysteine-scanning mutagenesis supports the importance of Clostridium perfringens enterotoxin amino acids 80 to 106 for membrane insertion and pore formation

Jianwu Chen  1 , James R Theoret, Archana Shrestha, James G Smedley 3rd, Bruce A McClane
Affiliations

Cysteine-scanning mutagenesis supports the importance of Clostridium perfringens enterotoxin amino acids 80 to 106 for membrane insertion and pore formation

Jianwu Chen et al. Infect Immun. 2012 Dec.

Abstract

Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal symptoms of the second most common bacterial food-borne illness. Previous studies suggested that a region named TM1, which has amphipathic characteristics and spans from amino acids 81 to 106 of the native CPE protein, forms a β-hairpin involved in β-barrel pore formation. To further explore the potential role of TM1 in pore formation, the single Cys naturally present in CPE at residue 186 was first altered to alanine by mutagenesis; the resultant rCPE variant, named C186A, was shown to retain cytotoxic properties. Cys-scanning mutagenesis was then performed in which individual Cys mutations were introduced into each TM1 residue of the C186A variant. When those Cys variants were characterized, three variants were identified that exhibit reduced cytotoxicity despite possessing binding and oligomerization abilities similar to those of the C186A variant from which they were derived. Pronase challenge experiments suggested that the reduced cytotoxicity of those two Cys variants, i.e., the F91C and F95C variants, which model to the tip of the β-hairpin, was attributable to a lessened ability of these variants to insert into membranes after oligomerization. In contrast, another Cys variant, i.e., the G103C variant, with impaired cytotoxicity apparently inserted into membranes after oligomerization but could not form a pore with a fully functional channel. Collectively, these results support the TM1 region forming a β-hairpin as an important step in CPE insertion and pore formation. Furthermore, this work identifies the first amino acid residues specifically involved in those two steps in CPE action.

PubMed Disclaimer

Figures

Fig 1
Fig 1
Comparison of biochemical characteristics of the C186A variant versus rCPE. (A) Cellular morphological damage. To initially assess the cytotoxic properties of the C186A variant, a morphological-damage assay (33) was performed. Confluent Caco-2 cell monolayers were treated at 37°C for 60 min with HBSS or HBSS containing 2.5 μg/ml of nCPE, rCPE, or the C186A variant. After this treatment, Caco-2 cells were photomicrographed at a total magnification of ×400. To test the specificity of cytotoxicity caused by these toxins, the C186A variant or rCPE was preincubated at 37°C with CPE polyclonal antiserum for 15 min; those mixtures were then applied to confluent Caco-2 cells in 6-well plates. Shown are representative results from three repetitions of this experiment. Ab, antibody. (B) Large-complex formation by the C186A variant and rCPE. Confluent Caco-2 cells were treated with 2.5 μg/ml of either rCPE species for 60 min at 37°C. The treated cells were then loaded onto a 4.25% polyacrylamide gel containing SDS. Large-complex formation was evaluated by Western blotting of the gel using rabbit polyclonal anti-CPE serum. “CH1” and “CH2” represent the two CPE large complexes, which migrate anomalously as ∼155- and 200-kDa proteins, respectively, on these gels (26). (C) Limited trypsin digestion assay. To assess gross conformational changes in the C186A variant, both rCPE and the C186A variant (500 ng) were incubated with trypsin (2 μg) at 25°C for the periods indicated above the lanes. After samples were stopped by the addition of Laemmli buffer and boiling for 5 min, proteins in those samples were separated by electrophoresis on an SDS-containing 10% polyacrylamide gel, followed by Western blotting with rabbit anti-CPE polyclonal antibody.
Fig 2
Fig 2
Location and membrane topology model of putative TM1 transmembrane domain in CPE. (A) Location of the TM1 region (red) in the CPE monomer. (Reprinted from reference with permission from Elsevier.) (B) Analysis of the CPE primary sequence predicted that the TM1 region between amino acids 90 and 106 comprises a β-hairpin with alternating hydrophilic and hydrophobic amino acids (2, 15, 33). A β-hairpin is often used by β-PFTs for membrane insertion and pore formation (23, 34). Circled amino acids were found in this study to be important for CPE cytotoxicity (see Results).
Fig 3
Fig 3
86Rb release experiments. (A) Cytotoxicity screening of rCPE mutants by an 86Rb release assay. Confluent Caco-2 cells in 24-well plates were radiolabeled with 4 μCi/well of 86RbCl for 4 h at 37°C and then treated at 37°C with 10 μg/ml of the specified purified 6× His-tagged rCPE species. After 15 min, 86Rb released into the medium was collected and quantified with a Cobra Quantum gamma counter. Data from this experiment were converted into the percentage of maximal 86Rb release (see Materials and Methods). The bars shown in red correspond to significantly attenuated mutants (P < 0.05). Duplicate samples of each variant were tested in three independent experiments. Error bars depict the standard errors. (B) Dose effects of rCPE species on 86Rb release. To further characterize the Cys variants showing reduced 86Rb release in panel A, 86RbCl-labeled Caco-2 cells were treated at 37°C with increasing amounts of the specified rCPE species. Data shown represent the means of three independent experiments, and the error bars show the standard deviations. (C) Cellular morphological damage caused by rCPE species. To confirm the results from the86Rb release experiments, a morphological damage assay was performed as described for Fig. 1. Confluent Caco-2 cell monolayers were treated at 37°C for 60 min with 2.5 μg/ml of each specified rCPE species, including nCPE, rCPE, the C186A variant, the F87C variant, the Q97C variant, and the D48A variant. As a control, some Caco-2 cells were treated with HBSS buffer alone (cHBSS; no added rCPE species).
Fig 4
Fig 4
Limited trypsin digestion analysis of rCPE species. To assess gross conformational changes among the Cys variants, 500 ng of the F87C variant (A), the F91C variant (B), the F95C variant (C), or the G103C variant (D) was digested at RT with trypsin (2 μg) for the specified times. The digestion pattern was then detected by Western blotting using rabbit anti-CPE polyclonal antibody.
Fig 5
Fig 5
Large-complex formation by rCPE species. After confluent Caco-2 cells were treated for 60 min at 37°C with 2.5 μg/ml of each specified rCPE species, samples were electrophoresed on an SDS-containing 4.25% polyacrylamide gel. Complexes were then electrotransferred onto a nitrocellulose membrane and detected by CPE Western blotting. The “lysate” lane corresponds to Caco-2 cells treated with a mock affinity enrichment preparation with lysates from E. coli cells transformed with the pTrcHis A empty vector.
Fig 6
Fig 6
Pronase degradation resistance of large complexes formed by rCPE species. Confluent Caco-2 cells were treated for 1 h at RT with 2.5 μg of each purified rCPE species, including the C186A variant (A), the F87C variant (B), the F91C variant (C), the F95C variant (D), and the G103C variant (E), and then washed to remove unbound toxin. Membrane fractions collected from those cells were then incubated with the concentrations of pronase indicated above each lane at RT for the indicated amounts of time. After digestion, membranes were pelleted and resuspended in Laemmli buffer. The sample proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane, followed by Western blotting with rabbit polyclonal anti-CPE antiserum. Note that CH-2 levels formed by each variant varied from experiment to experiment, as did the formation of bands running below CH-1; similar highly variable bands running below CH-1 have been observed previously in CPE-treated cells (28) and could represent either breakdown of CH-1 or CH-2 or an intermediate in formation of CH-1 or CH-2. Results shown are representative of three repetitions of this experiment.
Fig 7
Fig 7
Phenotypic rescue of Cys substitution mutants. Residues F91 and F95 of the C186A variant were changed to tryptophan or tyrosine, while residue G103C of the C186A variant was changed to alanine or serine. Confluent Caco-2 cells in 24-well plates were radiolabeled with 4 μCi/well of 86RbCl for 4 h at 37°C and then treated at 37°C with 10 μg/ml of purified 6× His-tagged rCPE species. After 15 min, 86Rb released into the medium was collected and quantified with a Cobra Quantum gamma counter. Data from this experiment were converted into percentage of maximal 86Rb release. The experiment was performed three times, each with duplicate samples. Error bars depict the standard errors. *, significant (P < 0.05) difference between the CPE variant and C186A, as determined using the Student t test; **, significant (P < 0.05) difference between the alternative substitution variant and the equivalent Cys substitution variant.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bos J, et al. 2005. Fatal necrotizing enteritis following a foodborne outbreak of enterotoxigenic Clostridium perfringens type A infection. Clin. Infect. Dis. 15:78–83 - PubMed
    1. Briggs DC, et al. 2011. Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins. J. Mol. Biol. 413:138–149 - PMC - PubMed
    1. Carman RJ. 1997. Clostridium perfringens in spontaneous and antibiotic-associated diarrhoea of man and other animals. Rev. Med. Microbiol. 8(Suppl 1):S43–S45
    1. CDC 2011, posting date Clostridium perfringens. CDC, Atlanta, GA: http://www.cdc.gov/foodborneburden/clostridium-perfringens.html
    1. Chakrabarti G, McClane BA. 2005. The importance of calcium influx, calpain, and calmodulin for the activation of CaCo-2 cell death pathways by Clostridium perfringens enterotoxin. Cell. Microbiol. 7:129–146 - PubMed

Publication types