Gene-Trap Mutagenesis Identifies Mammalian Genes Contributing to Intoxication by Clostridium perfringens ε-Toxin

The Clostridium perfringens ε-toxin is an extremely potent toxin associated with lethal toxemias in domesticated ruminants and may be toxic to humans. Intoxication results in fluid accumulation in various tissues, most notably in the brain and kidneys. Previous studies suggest that the toxin is a pore-forming toxin, leading to dysregulated ion homeostasis and ultimately cell death. However, mammalian host factors that likely contribute to ε-toxin-induced cytotoxicity are poorly understood. A library of insertional mutant Madin Darby canine kidney (MDCK) cells, which are highly susceptible to the lethal affects of ε-toxin, was used to select clones of cells resistant to ε-toxin-induced cytotoxicity. The genes mutated in 9 surviving resistant cell clones were identified. We focused additional experiments on one of the identified genes as a means of validating the experimental approach. Gene expression microarray analysis revealed that one of the identified genes, hepatitis A virus cellular receptor 1 (HAVCR1, KIM-1, TIM1), is more abundantly expressed in human kidney cell lines than it is expressed in human cells known to be resistant to ε-toxin. One human kidney cell line, ACHN, was found to be sensitive to the toxin and expresses a larger isoform of the HAVCR1 protein than the HAVCR1 protein expressed by other, toxin-resistant human kidney cell lines. RNA interference studies in MDCK and in ACHN cells confirmed that HAVCR1 contributes to ε-toxin-induced cytotoxicity. Additionally, ε-toxin was shown to bind to HAVCR1 in vitro. The results of this study indicate that HAVCR1 and the other genes identified through the use of gene-trap mutagenesis and RNA interference strategies represent important targets for investigation of the process by which ε-toxin induces cell death and new targets for potential therapeutic intervention

Identification of Amino Acids Important for Binding of <i>Clostridium perfringens</i> Epsilon Toxin to Host Cells and to HAVCR1

<i>Clostridium perfringens</i> epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1

Evaluation of the C.Diff Quik Chek Complete Assay, a New Glutamate Dehydrogenase and A/B Toxin Combination Lateral Flow Assay for Use in Rapid, Simple Diagnosis of Clostridium difficile Disease▿

The diagnosis of Clostridium difficile infection continues to be a challenge for many clinical microbiology laboratories. A new lateral flow assay, the C.Diff Quik Chek Complete assay, which tests for the presence of both glutamate dehydrogenase (GDH) and C. difficile toxins A and B, was evaluated for its ability to diagnose C. difficile disease. The results of this assay were compared to those of both PCR and toxigenic culture. The results showed that this assay allows 88% of specimens to be accurately screened as either positive (both tests positive) or negative (both tests negative) for the presence of toxigenic C. difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant results (one test positive and the other negative) allows the easy, rapid, and highly sensitive (100%; 95% confidence interval [CI], 89.6 to 100%) and specific (99.6%; 95% CI, 97.3 to 99.9%) diagnosis of C. difficile disease. The use of this algorithm would save institutional costs, curtail unnecessary isolation days, reduce the nosocomial transmission of disease, and increase the quality of care for patients

HAVCR1 expression in transfected HEK-293 cells.

<p><b>A</b>. Expression of HAVCR1 in ACHN, HEK-293, and HEK-293 cells transfected with plasmid DNA expressing <i>HAVCR1a</i> from ACHN cells was detected by a cell-surface ELISA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017787#pone.0017787-Feigelstock1" target="_blank">[43]</a>. Representative results are presented as the mean and standard deviation of triplicate samples. The asterisk denotes results that are significantly less than the signal from toxin-sensitive ACHN cells (p<0.05, ANOVA followed by Dunnett's post hoc test). <b>B</b>. Expression of HAVCR1 on ACHN, HEK-293, and HEK-293 cells transfected with plasmid DNA expressing <i>HAVCR1a</i> from ACHN cells was detected by fluorescence microscopy as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017787#s4" target="_blank">Materials and Methods</a>. Green: HAVCR1; red: 7-AAD. <b>C</b>. Binding of ε-toxin to ACHN, HEK-293, and HEK-293 cells transfected with plasmid DNA expressing <i>HAVCR1a</i> from ACHN cells was detected by a cell-surface ELISA. Representative results are presented as the mean and standard deviation of triplicate samples. The asterisks denote results that are significantly less than the signal from toxin-sensitive ACHN cells (p<0.05, ANOVA followed by Dunnett's post hoc test).</p

Amino acid sequence alignment of human and canine HAVCR1 splice variants.

<p>The predicted amino acid sequences of HAVCR1 proteins from human ACHN and 769-P cells and from canine MDCK cells were aligned using ClustalW. These sequences have been deposited in GenBank as accession numbers HQ412639 (HAVCR1a, ACHN cells), HQ412640 (Havcr1a, 769-P cells), HQ412641 (HAVCR1b, 769-P cells), HQ412642 (HAVCR1a, MDCK cells), HQ412643 (HAVCR1b, MDCK cells), and HQ412644 (HAVCR1c, MDCK cells).</p