Variations in TcdB Activity and the Hypervirulence of Emerging Strains of Clostridium difficile

Hypervirulent strains of Clostridium difficile have emerged over the past decade, increasing the morbidity and mortality of patients infected by this opportunistic pathogen. Recent work suggested the major C. difficile virulence factor, TcdB, from hypervirulent strains (TcdBHV) was more cytotoxic in vitro than TcdB from historical strains (TcdBHIST). The current study investigated the in vivo impact of altered TcdB tropism, and the underlying mechanism responsible for the differences in activity between the two forms of this toxin. A combination of protein sequence analyses, in vivo studies using a Danio rerio model system, and cell entry combined with fluorescence assays were used to define the critical differences between TcdBHV and TcdBHIST. Sequence analysis found that TcdB was the most variable protein expressed from the pathogenicity locus of C. difficile. In line with these sequence differences, the in vivo effects of TcdBHV were found to be substantially broader and more pronounced than those caused by TcdBHIST. The increased toxicity of TcdBHV was related to the toxin's ability to enter cells more rapidly and at an earlier stage in endocytosis than TcdBHIST. The underlying biochemical mechanism for more rapid cell entry was identified in experiments demonstrating that TcdBHV undergoes acid-induced conformational changes at a pH much higher than that of TcdBHIST. Such pH-related conformational changes are known to be the inciting step in membrane insertion and translocation for TcdB. These data provide insight into a critical change in TcdB activity that contributes to the emerging hypervirulence of C. difficile

Clostridium difficile 027/BI/NAP1 encodes a hypertoxic and antigenically variable form of TcdB.

The Clostridium difficile exotoxin, TcdB, which is a major virulence factor, varies between strains of this pathogen. Herein, we show that TcdB from the epidemic BI/NAP1/027 strain of C. difficile is more lethal, causes more extensive brain hemorrhage, and is antigenically variable from TcdB produced by previously studied strains of this pathogen (TcdB003). In mouse intoxication assays, TcdB from a ribotype 027 strain (TcdB027) was at least four fold more lethal than TcdB003. TcdB027 caused a previously undescribed brain hemorrhage in mice and this correlated with a heightened sensitivity of brain microvascular endothelial cells to the toxin. TcdB003 and TcdB027 also differed in their antigenic profiles and did not share cross-neutralizing epitopes in a major immunogenic region of the protein. Solid phase humoral mapping of epitopes in the carboxy-terminal domains (CTD) of TcdB027 and TcdB003 identified 11 reactive epitopes that varied between the two forms of TcdB, and 13 epitopes that were shared or overlapping. Despite the epitope differences and absence of neutralizing epitopes in the CTD of TcdB027, a toxoid form of this toxin primed a strong protective response. These findings indicate TcdB027 is a more potent toxin than TcdB003 as measured by lethality assays and pathology, moreover the sequence differences between the two forms of TcdB alter antigenic epitopes and reduce cross-neutralization by antibodies targeting the CTD

Comparison of the timing of cell entry between TcdB_HV and TcdB_HIST.

<p>CHO cells were pretreated with TcdB_HIST or TcdB_HV and the lysosomotropic inhibitor, ammonium chloride, was added at the indicated time points. Cytopathic effects were determined at 2 h (A) and 12 h (B), and black bars represent cells treated with TcdB_HV while gray bars represent TcdB_HIST. The error bars mark the standard deviation from the mean. C, untreated control. I, inhibitor alone. T, TcdB alone.</p

Representation of sequence variation between TcdB_HIST and TcdB_HV.

<p>The illustration depicts TcdB_HIST (top) and TcdB_HV (bottom) divided into functional domains: glucosyltransferase (A), cysteine protease (B), translocation (C), and receptor binding (D). (A) Trp 102 and the DXD motif of the glucosyltransferase domain are conserved between TcdB_HIST and TcdB_HV. The amino acids making up the substrate recognition region (SR) show 99% similarity between the strains, and the overall amino acid identity of the domain is 96%. (B) The catalytic triad of the cysteine protease domain remains unchanged between TcdB_HIST and TcdB_HV, and the overall identity of the domain is 96%. TcdB_HIST contains a cysteine at residue 870, while TcdB_HV contains a tyrosine at residue 870. (C) Amino acid identity of the translocation domain is 91%, with a 97% sequence identity occurring in the hydrophobic region (HR). TcdB_HV contains two cysteines in this domain, which are not found in this region of TcdB_HIST. (D) TcdB_HIST and TcdB_HV share an identity of 88% in the putative receptor binding domain. Gray boxes symbolize the CROP (clostridial repetitive oligopeptide) regions, 4 large repeats and 18 small repeats. White boxes indicate TcdB_HV CROPs that have less than 80% similarity to TcdB_HIST. (E) Coomassie stained SDS-PAGE analysis of 1 µg of each TcdB_HIST and TcdB_HV.</p

TNS analysis of pH-induced hydrophobic transitions in TcdB_HIST and TcdB_HV.

<p>TcdB_HIST or TcdB_HV was incubated with TNS for 20 min at 37°C. Samples were analyzed for changes in TNS fluorescence, and the emission profile of each pH is shown and labeled. Panels (A) and (B) represent pH 4.0 to pH 7.0 and panels (C) and (D) show TNS fluorescence of TcdB between pH 5.0 and 6.0. Each spectrum represents the experimental sample with background (TNS and buffer alone) subtracted.</p

Heterologous delivery of the TcdB enzymatic domain.

<p>CHO cells were treated with LFnTcdB_HIST(enz) or LFnTcdB_HV(enz) in the presence of PA for 24 h and cell viability was determined by WST-8 staining. The error bars represent the standard deviation from the mean of three samples.</p

Representative photographs of zebrafish after 24 h exposure to TcdB.

<p>(A) Zebrafish after exposure to 10 nM TcdB_HIST. Cardiac damage is evident by pericardial edema (black arrow) and blood accumulation (white arrow). (B) Exposure to 10 nM TcdB_HV causes tissue necrosis and death of the zebrafish. (C) Zebrafish treated with 1 nM TcdB_HIST appear normal, with little to no edema. (D) Zebrafish after exposure to 1 nM TcdB_HV. Arrow indicates damage to the yolk sac, visualized by tissue discoloration and necrosis. (E) Untreated control.</p

Flow-based analysis of cell binding.

<p>TcdB_HIST and TcdB_HV were tested for their ability to bind CHO (A) and HL-1 cells (B). 40 nM of fluorescently labeled TcdB was incubated with cells on ice, and binding was determined by flow cytometry. TcdB_HIST and TcdB_HV are indicated, and shaded peaks represent cells incubated with unlabeled TcdB. (C) Mean fluorescence intensity (MFI) vs TcdB concentration on HL-1 cells. Please note the difference in axis for TcdB_HIST and TcdB_HV.</p

Tryptophan emission of TcdB_HIST and TcdB_HV at acidic and neutral pH.

<p>The fluorescent spectrum of each sample is shown and labeled; each spectrum represents the experimental sample minus background fluorescence of buffer alone. Panels (A) and (B) show tryptophan emission of TcdB_HIST and TcdB_HV from pH 4.0 to pH 7.0 while panels (C) and (D) highlight the changes in tryptophan fluorescence between pH 5.0 and pH 6.0.</p

Protection against TcdB in vivo and in vitro after immunization with ToxoidB₀₂₇.

<p>(A) Percent viability of CHO cells treated for 24 hrs with TcdB₀₀₃ (black) or TcdB₀₂₇ (gray) alone or after preincubation for 30 minutes with αToxoidB₀₀₃ antiserum or αToxoidB₀₂₇ antiserum. Cell viability was determined by WST-8 staining and the error bars represent the standard deviation from the mean of three samples. ***p<0.001, *p<0.05 (B–C) Kaplan-Meier graphs showing the time to death of C57Bl/6 mice that were injected intravenously with a 2×LD₁₀₀ of TcdB₀₀₃ (A) or TcdB₀₂₇ (B) after immunization with ToxoidB₀₀₃ (red), ToxoidB₀₂₇ (dashed), or control peptide (black) (n = 9). Log-rank analysis performed using GraphPad Prism, *** p<0.001, ** p<0.01.</p