Clostridium difficile Toxin B causes epithelial cell necrosis through an autoprocessing-independent mechanism

Clostridium difficile is the most common cause of antibiotic-associated nosocomial infection in the United States. C. difficile secretes two homologous toxins, TcdA and TcdB, which are responsible for the symptoms of C. difficile associated disease. The mechanism of toxin action includes an autoprocessing event where a cysteine protease domain (CPD) releases a glucosyltransferase domain (GTD) into the cytosol. The GTD acts to modify and inactivate Rho-family GTPases. The presumed importance of autoprocessing in toxicity, and the apparent specificity of the CPD active site make it, potentially, an attractive target for small molecule drug discovery. In the course of exploring this potential, we have discovered that both wild-type TcdB and TcdB mutants with impaired autoprocessing or glucosyltransferase activities are able to induce rapid, necrotic cell death in HeLa and Caco-2 epithelial cell lines. The concentrations required to induce this phenotype correlate with pathology in a porcine colonic explant model of epithelial damage. We conclude that autoprocessing and GTD release is not required for epithelial cell necrosis and that targeting the autoprocessing activity of TcdB for the development of novel therapeutics will not prevent the colonic tissue damage that occurs in C. difficile - associated disease

Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities

Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.Wellcome Trust, United States a Trevor D Lawley bajo el número 098051Consejo Nacional de Rectores/[803-B1-654]/CONARE/Costa RicaConsejo Nacional de Rectores/[803-B4-652]/CONARE/Costa RicaUniversidad de Costa Rica/[803-B5-107]/UCR/Costa RicaUniversidad de Costa Rica/[803-B5-108]/UCR/Costa RicaConsejo Nacional para Investigaciones Científicas y Tecnológicas/[FV-0004-13. HHS]/CONICIT/Costa RicaNational Institutes of Health/[R01AI095755]/NIH/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Rapid Single-Shot Synthesis of the 217 Amino Acid-Long N-Terminal Domain of Pyocin S2

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remains challenging. To address this limitation, we employed automated fast-flow peptide synthesis (AFPS) for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 hours and purified to yield milligrams quantities of the desired product. To our knowledge, the 217 amino acid sequence of PyS2NTD is among the longest peptides produced from a “single-shot” synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor (FpvA) and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching (FRAP). These modified, synthetic S-type pyocins domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane

Rapid Single-Shot Synthesis of the 214 Amino Acid-Long N-Terminal Domain of Pyocin S2

The impermeable outer membrane of Pseudomonas aeruginosa is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against Pseudomonas infections. Their production and modification, however, remain challenging. To address this limitation, we employed automated fast-flow peptide synthesis for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2NTD) was synthesized in under 10 h and purified to yield milligram quantities of the desired product. To our knowledge, the 214 amino acid sequence of PyS2NTD is among the longest peptides produced from a “single-shot” synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2NTD with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2NTD binds to P. aeruginosa expressing the cognate ferripyoverdine receptor and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching. These modified, synthetic S-type pyocin domains can be used to probe import mechanisms of P. aeruginosa and leveraged to develop selective antimicrobial agents that bypass the outer membrane

TcdB induces necrosis in epithelial cells.

<p><i>A</i>, TcdB does not induce caspase-3/7 activation in HeLa cells, as detected by a fluorescent indicator, Apo-One. <i>B</i>, TcdB induces rapid death in HeLa cells, as detected by a luminescent indicator, CellTiterGlo. Caspase-3/7 activation and viability values represent the average of three experiments in which each condition was tested in triplicate. The error bars indicate the standard deviation between three experiments. <i>C</i>, HeLa cells were synchronized and incubated with or without TcdB for 2.5 hours at 37°C. A representative image obtained by light microscopy indicates rounding in cells treated with 10 pM TcdB and a loss of membrane integrity in cells treated with 10 nM TcdB. <i>D</i>, Extracellular LDH was detected in TcdB-treated HeLa cells after 2.5 hours using a luminescence-based indicator, Cytotox-Glo. Increased levels of LDH release were apparent after 8 hours. LDH release values represent the average of three experiments in which three replicates were averaged. Error bars indicate the standard deviation between the values obtained from the three experiments. <i>E</i>, HeLa cells were treated with a buffer control or 10 nM TcdB for 1 h and then fixed with 4% formaldehyde. Cells were stained with an antibody specific for HMGB1 and an Alexa Fluor 488 anti-mouse antibody. The cells were visualized with a LSM510 Confocal microscope. The representative images show that HMGBI is released from the nucleus of HeLa cells when treated with 10 nM TcdB and remains nuclear in the untreated cells.</p

TcdB and TcdB autoprocessing mutants cause cell rounding with concentration dependent kinetics.

<p>HeLa cells were treated with multiple concentrations of wild-type and mutant TcdB proteins and imaged every 10 minutes over a 2 hour time course. The percentage of round cells was quantified over six fields for each concentration and time point. The kinetics of rounding induced by TcdB and the TcdB autoprocessing mutants is shown at concentrations of <i>A</i>, 10 pM. <i>B</i>, 100 fM and <i>C</i>, 1 fM. <i>D</i>, Representative images of cells treated with 1 fM TcdB and TcdB autoprocessing mutants for 50 minutes. Green cells are alive; red cells are dead. Images were collected with an Opera High-Throughput Confocal Screening Microscope in an environment-controlled chamber at 37°C, 5% CO₂. Round cells were defined as having an area less than 500 um² and a width-to-length ratio of less than 0.4. Analysis was performed using Columbus Analysis software.</p

TcdB glucosyltransferase mutants cause epithelial cell death.

<p><i>A</i>, TcdB and TcdB glucosyltransferase domain mutants (100 nM) were tested for their capacity to glucosylate purified Rac1 (2 uM) in the presence of 20 mM UDP-[¹⁴C]-glucose over the course of 1 h. The proteins were resolved by SDS-PAGE, and the gels were analyzed by phosphorimaging. <i>B</i>, TcdB D270N was tested with higher concentrations of both toxin and UDP-[¹⁴C]-glucose. Only at the highest concentrations of both toxin and UDP-[¹⁴C]-glucose is residual activity apparent. <i>C</i>, The TcdB glucosyltransferase mutants are impaired in their glucosyltransferase activities in HeLa cells, as determined by Western and an antibody specific for unglucosylated Rac1. <i>D</i>, Wild-type TcdB and the TcdB glucosyltransferase mutants induced comparable levels of HeLa cell death, as determined by CellTiterGlo, after 2.5 h of treatment. Percent viability was determined by normalizing the signal from treated cells to the signal from untreated cells. Values reflect the average signal from three experiments in which each condition was tested in triplicate. Error bars correspond to the standard deviation in the percent viability from the three experiments.</p