Clostridium difficile infection is a major cause of nosocomial diarrhoea, and can culminate in the life-threatening conditions pseudomembranous colitis and toxic megacolon. Disease is produced in C. difficile infection through the release of toxin A and toxin B. Current diagnostics rely on a combination of immunoassays, histopathological findings, and PCR. Immunoassays, though fast, lack sensitivity compared to more expensive and time-consuming histopathology methods. PCR, though sensitive, only indicates the presence of toxin genes and not whether they are being expressed. Matrix Assisted Laser Desorption/Ionization Time-of-Flight mass spectrometry (MALDI-TOF) has been gaining popularity in medical laboratories as a means of identifying bacterial isolates due to high sensitivity and fast turnaround time. The purpose of this study is to investigate MALDI-TOF as a means of detecting the presence of C. difficile toxins A and B. The method developed was based on the proposed cellular processing mechanism of toxin A and B – both are large toxins (approximately 300 kDa) that are endocytosed and undergo a pH-induced conformation change in the early endosome. This causes the active domain and protease domain of the toxin to be translocated into the cytoplasm. Inositol hexakisphosphate, an abundant cystosolic signaling molecule, then binds to the protease domain of the toxin and induces the release of the 63 kDa active toxin. The active component of both toxin A and B glucosylates cellular Rho GTPases, which results in cytoskeleton derangement and cell death. The method developed required fecal material to be prepared as a 1:10 dilution in deionized water followed by filter sterilization. The filtrate volume was then divided into two equal volumes, and each volume was subjected to an acetonitrile precipitation. One pellet was resuspended in deionized water, and the other in sodium acetate buffer (100mM, pH 4.7) to mimic the conditions of the early endosome. The spectra produced from each condition were overlaid, which demonstrated the presence of a high-intensity peak at approximately 60 kDa in known toxin positive specimens. If this peak can be shown to be correlated to the presence of toxin in a larger sample set, this method may hold promise as a means of detecting C. difficile toxins A and B. This protocol avoids the extensive time and labour associated with a mass-fingerprinting approach, while maintaining higher degree of specificity than simple intact protein detection due to the comparison of spectra between acid and neutral conditions.
