Caspase Activation as a Versatile Assay Platform for Detection of Cytotoxic Bacterial Toxins

Abstract

Pathogenic bacteria produce several virulence factors that help them establish infection in permissive hosts. Bacterial toxins are a major class of virulence factors and hence are attractive therapeutic targets for vaccine development. Here, we describe the development of a rapid, sensitive, and high-throughput assay that can be used as a versatile platform to measure the activities of bacterial toxins. We have exploited the ability of these toxins to cause cell death via apoptosis of sensitive cultured cell lines as a readout for measuring toxin activity. Caspases (cysteine-aspartic proteases) are induced early in the apoptotic pathway, and so we used their induction to measure the activities of Clostridium difficile toxins A (TcdA) and B (TcdB) and binary toxin (CDTaCDTb), Corynebacterium diphtheriae toxin (DT), and Pseudomonas aeruginosa exotoxin A (PEA). Caspase induction in the cell lines, upon exposure to toxins, was optimized by toxin concentration and intoxication time, and the specificity of caspase activity was established using a genetically mutated toxin and a pan-caspase inhibitor. In addition, we demonstrate the utility of the caspase assay for measuring toxin potency, as well as neutralizing antibody (NAb) activity against C. difficile toxins. Furthermore, the caspase assay showed excellent correlation with the filamentous actin (F-actin) polymerization assay for measuring TcdA and TcdB neutralization titers upon vaccination of hamsters. These results demonstrate that the detection of caspase induction due to toxin exposure using a chemiluminescence readout can support potency and clinical immunogenicity testing for bacterial toxin vaccine candidates in development.

Bithionol blocks pathogenicity of bacterial toxins, ricin and Zika virus

“…It has previously been shown that PE exploits several host proteins for its binding to and entry into host cells 5 and initiates programmed cell death by inducing activities of host caspase-3, -6, and -7 6 . We investigated whether known mutations in host proteins exploited by PE associate with altered cytotoxicity of the toxin in cells from tissues that are naturally attacked by this toxin.…”

Cell-based in vitro models in environmental toxicology: a review

“…Caspase-3/7 enzyme activity assays (apopain and CPP32 assays) are fluorescence-based test systems designed for quantification of apoptosis [57,58]. Different probes have been developed to quantify caspase activity, including Förster resonance energy transfer (FRET)-based systems [59] and rhodamine-110 labeled substrates [60].…”

Multiplex Drug Screenings to Discover Host Oriented Therapies Against Infectious Diseases

“…2b) is likely a result of a polygenic inheritance model, consistent with evidence that multiple host proteins mediate PE lethality 21,76 . To learn whether genetic variations in the genes encoding for these proteins account for any of the variation in sensitivity of B-cells to PE seen in Fig.…”

“…It has previously been shown that PE exploits several host proteins for its binding to and entry into host cells 76 and initiates programmed cell death by inducing activities of host caspase-3, -6, and -7 21 . We investigated whether known (Fig.…”

“…These results show that the activity of caspases affects host cell sensitivity to toxins, and that these proteins are potential therapeutic intervention points and targets for the following drug screens. In addition to PE, ricin and toxins of anthrax, diphtheria, Botulinum, and cholera induce programmed cellular death by activating host caspases 21,22,45,[84][85][86][87][88] (Fig. 3a).…”