CASD1: A novel player in anthrax toxin entry into cells

Anthrax is a deadly disease caused by the large rod-shaped Gram-positive bacterium Bacillus anthracis. Two factors are fundamental to anthrax pathogenesis; the bacterium's capsule and its secreted three-component toxin, termed anthrax toxin. The toxin actions on cells are deadly for the host. Anthrax toxin enters cells via two related but separate cell surface proteins, TEM8 and CMG2. For entry into cells, the protective antigen (PA; 83 kDa) of anthrax toxin binds the receptors and is then immediately cleaved by furin. The larger of the two polypeptides, PA63, then oligomerizes, and the oligomers bind the two catalytic components of the toxin, edema factor (EF) and lethal factor (LF). The PA oligomers then deliver EF and LF to cytosol by receptor-mediated endocytosis. EF is a highly efficient adenylate cyclase. It intoxicates cells by raising cAMP to deadly levels. LF is a Zn++-dependent protease that cuts MAP kinase kinases, causing a myriad of adverse effects that prove deadly for the infected host. Other proteins that influence anthrax toxin entry into cells are LRP6 and ARAP3. These were identified as players in toxin entry by retroviral vector mediated antisense expression of expressed sequence tag libraries.

This work reports identification of CASD1 as a novel player in anthrax toxin entry. It has been reported that this gene encodes an o-acetyltransferase that largely localizes to Golgi apparatus membranes. Identification of the gene involved random genome-wide insertional mutagenesis of cells with a lentiviral vector, selection of toxin-resistant clones, and then determination of the vector insert sites by genome walking. The toxin-resistant clone RC132 harbors the vector insert in CASD1. The clone showed significantly reduced PA binding, which accounts for its resistance to the toxin. To determine whether CASD1 has any causal relationship with toxin resistance, the gene was down-regulated with siRNA in two naïve cell lines, one of them the toxin-sensitive parent of RC132. Both cell lines acquired a degree of toxin resistance upon down-regulation of CASD1, thus linking CASD1 disruption to toxin resistance. Further, upon CASD1 down-regulation, both cell lines bound less PA, a parallel with RC132 phenotype. Together these results show that CASD1 is a player in anthrax toxin entry, and that its disruption hinders toxin entry at the receptor binding level. In vitro furin-cleaved PA63 toxicity assays gave similar results, suggesting the block is only at the initial receptor binding level.

To determine whether RC132 showed resistance due to a lesion in elongation factor 2 (EF2), we tested RC132 for resistance to Pseudomonas aeruginosa exotoxin A (ETA). These results showed that RC132 is as sensitive to ETA as is its parent. Thus, RC132 has no defect in EF2, the target for ETA and FP59. We also tested RC132 sensitivity to diphtheria toxin, another toxin that targets EF2. Surprisingly, RC132 showed more than 20-fold resistance to this toxin.

Overall, the results reported in this thesis show that CASD1is a player in PA and DT entry into cells, but not ETA. However, much more work is needed to elucidate the molecular mechanisms by which CASD1 plays its role in anthrax toxin, as well as DT entry into cells.