The pag Gene of pXO1 Is Involved in Capsule Biosynthesis of Bacillus anthracis Pasteur II Strain

The protective antigen (PA) of the anthrax toxin binds to a cell surface receptor and thereby allows lethal factor (LF) to be taken up and exert its toxic effect in the cytoplasm. Here, we report that clustering of the anthrax toxin receptor (ATR) with heptameric PA or with an antibody sandwich causes its association to specialized cholesterol and glycosphingolipid-rich microdomains of the plasma membrane (lipid rafts). We find that although endocytosis of ATR is slow, clustering it into rafts either via PA heptamerization or using an antibody sandwich is necessary and sufficient to trigger efficient internalization and allow delivery of LF to the cytoplasm. Importantly, altering raft integrity using drugs prevented LF delivery and cleavage of cytosolic MAPK kinases, suggesting that lipid rafts could be therapeutic targets for drugs against anthrax. Moreover, we show that internalization of PA is dynamin and Eps15 dependent, indicating that the clathrin-dependent pathway is the major route of anthrax toxin entry into the cell. The present work illustrates that although the physiological role of the ATR is unknown, its trafficking properties, i.e., slow endocytosis as a monomer and rapid clathrin-mediated uptake on clustering, make it an ideal anthrax toxin receptor.

By using genetic complementation tests with various in vitro-constructed mutants with mutations in the cap region (which is essential for encapsulation in Bacillus anthracis), we identified three cistrons, capB, capC, and capA, in this order of arrangement. Minicell analysis revealed that these cistrons produce proteins of 44, 16, and 46 kilodaltons, respectively. The complete nucleotide sequence of 3,244 base pairs covering the whole cap region was determined and revealed the existence of the three open reading frames of capB (397 amino acid residues; molecular weight, 44,872), capC (149 amino acid residues; molecular weight, 16,522), and capA (411 amino acid residues; molecular weight, 46,420) arranged in the order predicted by complementation tests. These three cistrons were all transcribed in the same direction from promoters unique to each cistron. Judging from the predicted amino acid sequence of the three proteins and from their localization and their sensitivity to various physicochemical treatments, they appeared to be membrane-associated enzymes mediating the polymerization of D-glutamic acid via the membrane. Capsular peptides immunologically identical to that of B. anthracis were found in B. subtilis, B. megaterium, and B. licheniformis, but no sequence homologous to the cap region was found in any of these bacilli other than B. anthracis. Using strains of B. anthracis with or without insertional inactivation of the cap region, we found that the capsule of B. anthracis conferred strong resistance to phagocytosis upon the bacterial host.

Before intoxication can occur, anthrax toxin protective antigen (PA), Pseudomonas exotoxin A (PE), and diphtheria toxin (DT) must be activated by proteolytic cleavage at specific amino acid sequences. Previously, it was shown that PA and DT can be activated by furin. In Chinese hamster ovary (CHO) cells, wild-type (RKKR) and cleavage site mutants of PA, each administered with a modified form of anthrax toxin lethal factor (the N terminus of lethal factor fused to PE domain III), had the following potencies: RKKR (wild type) (concentration causing 50% cell death [EC50] = 12 ng/ml) > or = RAAR (EC50 = 18 ng/ml) > FTKR (EC50 = 24 ng/ml) > STRR (EC50 = 49 ng/ml). In vitro cleavage of PA and cleavage site mutants of PA by furin demonstrated that native PA (RKKR) and PA with the cleavage sequence RAAR are substrates for furin. To characterize eukaryotic proteases that play a role in activating bacterial toxins, furin-deficient CHO cells were selected after chemical mutagenesis. Furin-deficient cells were resistant to PE, whose cleavage site, RQPR, constitutes a furin recognition site and to all PA cleavage site mutants, but were sensitive to DT (EC50 = 2.9 ng/ml) and PA (EC50 = 23 ng/ml), whose respective cleavage sites, RKKR and RVRR, contain additional basic residues. Furin-deficient cells that were transfected with the furin gene regained sensitivity to PE and PA cleavage site mutants. These studies provide evidence that furin can activate the three toxins and that one or more additional proteases contribute to the activation of DT and PA.