Clostridium perfringens Beta2 toxin forms highly cation-selective channels in lipid bilayers

Abstract

Clostridium perfringens is a potent producer of a variety of toxins. Well studied from these are five toxins (alpha, Beta (CPB), epsilon, iota and CPE) that are produced by seven toxinotype strains (A-G) of C. perfringens. Besides these toxins, C. perfringens produces also another toxin that causes necrotizing enterocolitis in piglets. This toxin termed consensus Beta2 toxin (cCPB2) has a molecular mass of 27,620 Da and shows only little homology to CPB and no one to the other toxins of C. perfringens. Its primary action on cells remained unknown to date. cCPB2 was heterogeneously expressed as fusion protein with GST in Escherichia coli and purified to homogeneity. Although cCPB2 does not exhibit the typical structure of beta-stranded pore-forming proteins and contains no indication for the presence of amphipathic alpha-helices we could demonstrate that cCPB2 is a pore-forming component with an extremely high activity in lipid bilayers. The channels have a single-channel conductance of about 700 pS in 1 M KCl and are highly cation-selective as judged from selectivity measurements in the presence of salt gradients. The high cation selectivity is caused by the presence of net negative charges in or near the channel that allowed an estimate of the channel size being about 1.4 nm wide. Our measurements suggest that the primary effect of cCPB2 is the formation of cation-selective channels followed by necrotic enteritis in humans and animals. We searched in databases for homologs of cCPB2 and constructed a cladogram representing the phylogenetic relationship to the next relatives of cCPB2.

Keywords: Cation-selectivity; Channel formation; Consensus Clostridium perfringens Beta2 toxin (cCPB2); Lipid bilayer membrane; Pore-forming toxins; Propidium iodide uptake.

Fig. 1

A 10% SDS–PAGE of purified consensus GST-CPB2, and consensus CPB2. Lane 1 shows 2 µg of recombinant consensus GST-CPB2 dissolved in 10 µL sample buffer according to Laemmli (1970); lane 2 µg thrombin cleaved GST-CPB2. The left lane of the SDS–PAGE shows the location of the molecular mass markers: 17, 26, 34, 43, 55, and 72 kDa. The gel was stained with Coomassie brilliant blue. B Graphical presentation of the amino acid sequence coverage after tryptic in-gel digestion of GST-cCPB2 excised from protein band in A, lane 1. Amino acid residues (single letter code) shown in black indicate the partial sequence of GST and those in blue that of cCPB2, which were represented by matching peptide ion signals. Amino acid residues shown in red point to partial sequences for which no matching peptide ion signals were observed by nanoLC-ESI-MS^E analysis. Amino acid position numberings are given at the left

Fig. 2

Specific membrane conductance as a function of time after the addition of GST, GST-cCPB2, and cCPB2 to membranes formed of DiPhPC/n-decane. The arrow indicates the addition of the three compounds in a concentration of 3 µg/mL to the aqueous phase bathing individual black bilayers. The data points show the mean ± SD of three experiments. The aqueous phase contained 1 M KCl, pH 6. The applied voltage was 20 mV; T = 20 °C

Fig. 3

Single-channel recording of a DiPhPC/n-decane membrane in the presence of cCPB2 of C. perfringens cleaved with thrombin (A) and GST-cCPB2 (B). The aqueous phase contained unbuffered 1 M KCl, pH 6.0, and about 30 ng/mL cCPB2 (A) and about 40 ng/mL GST-cCPB2 (B). The applied membrane potential was 20 mV and the temperature was 20 °C. The recording was performed with the strip chart recorder at a time resolution of 10 Hz

Fig. 4

Histogram of the probability P(G) for the occurrence of a given conductivity unit in DiPhPC/n-decane membranes in the presence of cleaved cCPB2 and GST-cCPB2 of C. perfringens. The probability of channel occurrence was calculated by dividing the number of fluctuations with a given conductance unit by the total number of conductance fluctuations in the presence of cCPB2 and GST-cCPB2, respectively. A The mean value of the single-channel conductance of all conductance fluctuations was 643 pS for 320 single-channel events collected from different individual membranes. Aqueous phase contained 1 M KCl, pH6, and about 30 ng/mL cCPB2 cleaved with thrombin. The applied voltage was 20 mV and the temperature was 20 °C. The solid line shows a fit of the histogram with a Gaussian distribution. The maximum of the distribution is at a probability of 0.321 ± 0.082 and the conductance is 740 ± 71 pS for all single events taken from nine individual membranes. B The mean value of the single-channel conductance of all conductance fluctuations with GST-cCPB2 was 530 pS for 168 single-channel events collected from different individual membranes. The aqueous phase contained 1 M KCl, pH6, and about 40 ng/mL GST-cCPB2. The applied voltage was 20 mV and the temperature was 20 °C. The solid line shows a fit of the histogram with a Gaussian distribution. The maximum of the distribution is at a probability of 0.412 ± 0.061 and the conductance is 636 ± 66 pS for all single events taken from five individual membranes

Fig. 5

Cytotoxicity of GST-cCPB2 (A) and CPB (B) in HUVEC cells monitored by PI entry. HUVEC cells in DMEM medium containing 5 μg/mL were incubated with GST-cCPB2 (A) or CPB (B) for 1–22 h at 37 °C. PI entry was monitored by spectrofluorometry. The data are expressed as percentages of fluorescence compared to the control consisting of cells treated with 0.2% Triton X-100. Data were collected from three experiments and means and standard deviation were calculated

Fig. 6

Amino acid sequence alignment of cCPB2 and aCPB2 of C. perfringens. The alignment was performed using the NCBI Reference protein sequences WP_096517132 and AAC27654, respectively, and Pole Bioinformatique Lyonnaise Network Protein Sequence Analysis (https://npsa.lyon.inserm.fr/cgi-bin/align_clustalw.pl). Amino acids identical in the two proteins are highlighted in red (*), strongly similar amino acids (:) are given in green and weakly similar ones (.) in blue. The mature amino acid sequence starts for both proteins with amino acid 31

Fig. 7

Analysis of the secondary structure of the mature form of cCPB2 (without signal sequence) for β-strands using PRED-TMBB (http://bioinformatics.biol.uoa.gr/PRED-TMBB/input.jsp). Amino acid stretches that could form transmembrane β-strands are colored in red. Stretches in blue or in green are exposed to different sides of the membrane to the aqueous phases. Some of the green stretches could form turns between two beta-strands

Fig. 8

Single-channel conductance of the cCPB2 channel of C. perfringens as a function of the KCl concentration in the aqueous phase. The broken line represents the fit of the single-channel conductance data (filled circles ± SD, taken from Table 1) with Eqs. (1−4), using 1.5 negative point charges (q = − 2.4 × 10^–19 As) and a channel diameter of about 1.4 nm as fit parameters. The straight line shows the single-channel conductance of consensus CPB2 that would be expected without point charges assuming a single-channel conductance of 450 pS/M. It corresponds to a linear graph between channel conductance and bulk aqueous. The number of single events used to calculate the average single-channel conductance (and SD) is given in Table 1, column 4

Fig. 9

Cladogram representing the phylogenetic relationships of Beta2 toxin like proteins from different Firmicutes. The tree was generated using protein sequences downloaded from the NCBI protein database with the indicated identifiers. The multiple sequence alignment was calculated with Multiple Sequence Alignment (MUSCLE) (https://www.ebi.ac.uk/Tools/msa/muscle/). The tree was obtained using the program MEGA7 (Kumar et al. 2016)