To evaluate the pathogenic potential of Bacillus anthracissecreted proteases distinct from lethal toxin, two neutral zinc metalloproteases were purified to apparent homogeneity from the culture supernatant of a non-virulent delta Ames strain (pXO1 ؊ , pXO2 ؊ ). The first (designated Npr599) is a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species. The second (designated InhA) is a homolog of the Bacillus thuringiensis immune inhibitor A. These proteases belong

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... to the M4 and M6 families, respectively. Both enzymes digested various substrates, including extracellular matrix proteins, endogenous inhibitors, and coagulation proteins, with some differences in specificity. In addition, InhA accelerated urokinase-mediated plasminogen activation, suggesting that InhA acts as a modulator of plasmin in the host inflammatory system. Relevant to epithelial barrier function, Npr599 and InhA significantly enhanced syndecan-1 shedding from cultured normal murine mammary gland cells without affecting their viability through stimulation of the host cell ectodomain shedding mechanism. In addition, Npr599 and InhA directly cleaved recombinant syndecan-1 fused to glutathione S-transferase. Mass spectrometric analysis suggested that the cleavage sites of Npr599 and InhA are the Asp 39 -Asp 40 and Gly 48 -Thr 49 bonds, respectively. We propose that Npr599 and InhA from B. anthracis are multifunctional pathogenic factors that may contribute to anthrax pathology through direct degradation of host tissues, increases in barrier permeability, and/or modulation of host defenses. Downloaded from FIGURE 1. Purification and identification of Npr599 and InhA. Two proteases were purified from a culture of the B. anthracis delta Ames strain by ammonium sulfate saturation and DEAE-cellulose and Sephacryl S-200 column chromatography. Lane M, prestained molecular mass markers of (from top to bottom) 250, 148, 98, 64, 50, 36, 22, and 16 kDa. C-term, C terminus. Downloaded from FIGURE 5. Enhancement of syndecan-1 shedding by Npr599 and InhA. Confluent NMuMG cells in 96-well plates were incubated at 37°C with Npr599 (62.5, 250, and 500 ng/ml) and InhA for 4 h (A) or with protease (250 ng/ml) for 1, 4, and 8 h (B). Shed syndecan-1 ectodomain levels were measured by dotblot analysis as described under "Experimental Procedures." Error bars represent S.D. values determined from triplicate measurements. AU, arbitrary units.