Secreted Neutral Metalloproteases of Bacillus anthracis as Candidate Pathogenic Factors (2006)

To evaluate the pathogenic potential of Bacillus anthracis-secreted 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 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 Asp39–Asp40 and Gly48–Thr49 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. To evaluate the pathogenic potential of Bacillus anthracis-secreted 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 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 Asp39–Asp40 and Gly48–Thr49 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. Bacillus anthracis is a highly pathogenic Gram-positive bacillus that secrets two major virulence factors, lethal toxin and edema toxin, encoded by megaplasmid pXO1. Another plasmid (pXO2) encodes the anti-phagocytic capsule, which substantially contributes to the virulence of the microbe. Lethal toxin is necessary for pathogenicity, as deletion of its gene renders the microbe avirulent, whereas edema toxin-knock-out strains are only partially attenuated (1Pezard C. Berche P. Mock M. Infect. Immun. 1991; 59: 3472-3477Crossref PubMed Google Scholar). Lethal toxin consists of a heptameric protective antigen noncovalently associated with lethal factor. Lethal toxin is a zinc metalloprotease that cleaves and thus inhibits MAPK 2The abbreviations used are: MAPK, mitogen-activated protein kinase; ECM, extracellular matrix; HS, heparan sulfate; MES, 4-morpholineethanesulfonic acid; GST, glutathione S-transferase; pNA, p-nitroanilide; uPA, urokinase-type plasminogen activator; NMuMG, normal murine mammary gland; PMA, phorbol 12-myristate 13-acetate; MCA, 7-methoxycoumarin-4-acetyl; DNP, 2,4-dinitrophenyl; Nva, l-norvaline; DPA, l-diaminopropionyl; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase. 2The abbreviations used are: MAPK, mitogen-activated protein kinase; ECM, extracellular matrix; HS, heparan sulfate; MES, 4-morpholineethanesulfonic acid; GST, glutathione S-transferase; pNA, p-nitroanilide; uPA, urokinase-type plasminogen activator; NMuMG, normal murine mammary gland; PMA, phorbol 12-myristate 13-acetate; MCA, 7-methoxycoumarin-4-acetyl; DNP, 2,4-dinitrophenyl; Nva, l-norvaline; DPA, l-diaminopropionyl; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase. kinase family members in vitro and in vivo, resulting in defective host cell signaling (2Guidi-Rontani C. 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Michael B.F. Bansal A. Cummings R.T. Cunningham B.R. Friedlander A.M. Douglas C.M. Patel S.B. Wisniewski D. Scapin G. Salowe S.P. Zaller D.M. Chapman K.T. Scolnick E.M. Schmatz D.M. Bartizal K. MacCoss M. Hermes J.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7958-7963Crossref PubMed Scopus (145) Google Scholar, 8Turk B.E. Wong T.Y. Schwarzenbacher R. Jarrell E.T. Leppla S.H. Collier R.J. Liddington R.C. Cantley L.C. Nat. Struct. Mol. Biol. 2004; 11: 60-66Crossref PubMed Scopus (176) Google Scholar). Specific lethal toxin blockers are expected to complement the existing antibiotic treatments, which alone are successful only in 55% of inhalation anthrax patients (9Jernigan D.B. Raghunathan P.L. Bell B.P. Brechner R. Bresnitz E.A. Butler J.C. Cetron M. Cohen M. Doyle T. Fischer M. Greene C. Griffith K.S. Guarner J. Hadler J.L. Hayslett J.A. Meyer R. Petersen L.R. Phillips M. Pinner R. Popovic T. Quinn C.P. Reefhuis J. Reissman D. Rosenstein N. Schuchat A. Shieh W.J. Siegal L. Swerdlow D.L. Tenover F.C. Traeger M. Ward J.W. Weisfuse I. Wiersma S. Yeskey K. Zaki S. Ashford D.A. Perkins B.A. Ostroff S. Hughes J. Fleming D. Koplan J.P. Gerberding J.L. Emerg. Infect. Dis. 2002; 8: 1019-1028Crossref PubMed Scopus (537) Google Scholar). It has been reported that the synthetic inhibitor of lethal toxin proteolytic activity in combination with ciprofloxacin is protective in rabbits (7Shoop W.L. Xiong Y. Wiltsie J. Woods A. Guo J. Pivnichny J.V. Felcetto T. Michael B.F. Bansal A. Cummings R.T. Cunningham B.R. Friedlander A.M. Douglas C.M. Patel S.B. Wisniewski D. Scapin G. Salowe S.P. Zaller D.M. Chapman K.T. Scolnick E.M. Schmatz D.M. Bartizal K. MacCoss M. Hermes J.D. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7958-7963Crossref PubMed Scopus (145) Google Scholar). However, a number of anthrax pathological features such as massive hemorrhages and intensive organ and tissue damage cannot be explained by the sole activity of lethal toxin and edema toxin, indicating the involvement of other virulence factors. Our animal experiments using culture supernatants of the non-toxigenic B. anthracis delta Ames strain demonstrated their hemorrhagic effect on skin and high toxicity upon intratracheal administration (10Popov S.G. Popova T.G. Hopkins S. Weinstein R.S. MacAfee R. Fryxell K.J. Chandhoke V. Bailey C. Alibek K. BMC Infect. Dis. 2005; 5: 25Crossref PubMed Scopus (39) Google Scholar). Consistent with this, several broad-spectrum protease inhibitors, as well as immune sera against anthrax M4 and M9 metalloproteases, display high efficacy in the post-exposure treatment of murine systemic anthrax (10Popov S.G. Popova T.G. Hopkins S. Weinstein R.S. MacAfee R. Fryxell K.J. Chandhoke V. Bailey C. Alibek K. BMC Infect. Dis. 2005; 5: 25Crossref PubMed Scopus (39) Google Scholar). These observations prompted us evaluate the pathogenic potential of the secreted proteolytic enzymes, which might serve "accessory" functions to lethal toxin and therefore might be required for the full virulence of B. anthracis. To the best of our knowledge, B. anthracis extracellular proteases have not been previously characterized with respect to their enzymatic properties and activity toward pathologically relevant substrates. Bacterial proteases may exert tissue damage directly by cleaving the extracellular matrix (ECM) components, including collagen, laminin, fibronectin, and elastin (11Miyoshi S. Shinoda S. Microbes Infect. 2000; 2: 91-98Crossref PubMed Scopus (282) Google Scholar, 12Harrington D.J. Infect. Immun. 1996; 64: 1885-1891Crossref PubMed Google Scholar). Another general mechanism involves microbial interference with the homeostatic balance between endogenous proteases and their inhibitors, which determines tissue integrity. One of the examples is the effect of Pseudomonas aeruginosa elastase on the balance between neutrophil elastase and the inhibitors α1-protease inhibitor and α2-macroglobulin (13Dinwiddie R. Respiration. 2000; 67: 3-8Crossref PubMed Scopus (56) Google Scholar). The mammalian plasminogen system can also serve as a target of bacterial proteases, which can accelerate the urokinase-catalyzed activation of plasminogen and degrade endogenous plasmin inhibitors such as α2-antiplasmin and α2-macroglobulin (14Lahteenmaki K. Edelman S. Korhonen T.K. Trends Microbiol. 2005; 13: 79-85Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). As a result, the activated plasmin can directly digest laminin, a major glycoprotein of basement membranes, and indirectly further damage tissue barriers by activating latent matrix metalloproteases (15Lahteenmaki K. Virkola R. Pouttu R. Kuusela P. Kukkonen M. Korhonen T.K. Infect. Immun. 1995; 63: 3659-3664Crossref PubMed Google Scholar). In addition to ECM degradation, bacterial proteases are also involved in the pathogenic cleavage of host cell-surface molecules in a process of ectodomain shedding (16Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (208) Google Scholar, 17Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3064Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Shed ectodomains play pivotal roles in diverse pathophysiological events, including septic shock, host defense, and wound healing (18Haynes III, A. Ruda F. Oliver J. Hamood A.N. Griswold J.A. Park P.W. Rumbaugh K.P. Infect. Immun. 2005; 73: 7914-7921Crossref PubMed Scopus (55) Google Scholar, 19Marshall L.J. Ramdin L.S.P. Brooks T. Charlton P.A. Shute J.K. J. Immunol. 2003; 171: 2057-2065Crossref PubMed Scopus (117) Google Scholar, 20Elenius V. Go¨tte M. Reizes O. Elenius K. Bernfield M. J. Biol. Chem. 2004; 279: 41928-41935Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). During infection, secreted pathogenic factors enhance host ectodomain shedding, which contributes to epithelial barrier disruption, endothelial damage, and tissue penetration by bacilli (16Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (208) Google Scholar, 17Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3064Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). For instance, LasA, a secreted virulence factor of P. aeruginosa, enhances shedding of syndecan-1, which belongs to a family of cell-surface heparan sulfate (HS) proteoglycans. The resulting soluble syndecan-1 ectodomains enhance bacterial virulence in newborn mice (16Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (208) Google Scholar, 17Park P.W. Pier G.B. Preston M.J. Goldberger O. Fitzgerald M.L. Bernfield M. J. Biol. Chem. 2000; 275: 3057-3064Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Quite notably, inhibition of syndecan-1 shedding or neutralization of the HS of the shed ectodomain prevents P. aeruginosa lung infection (16Park P.W. Pier G.B. Hinkes M.T. Bernfield M. Nature. 2001; 411: 98-102Crossref PubMed Scopus (208) Google Scholar). These facts indicate that proteolysis of the ECM and shedding of the cell-surface ectodomain can play roles not only in signaling, but also in establishment of infection by acting as mediators of lethality, perturbing different mechanisms of the host defense response. We report here the purification, biochemical properties, and substrate specificity, with regard to ECM molecules, plasma proteins, and the cell-surface protein syndecan-1, of two neutral zinc metalloproteases. The first (designated Npr599 for neutral protease) is a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species (21Wetmore D.R. Wong S.-L. Roche R.S. Mol. Microbiol. 1992; 6: 1593-1604Crossref PubMed Scopus (68) Google Scholar, 22Narasaki R. Kuribayashi H. Shimizu K. Imamura D. Sato T. Hasumi K. J. Biol. Chem. 2005; 280: 14278-14287Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The second (designated InhA for immune inhibitor A metalloprotease) is a homolog of the Bacillus thuringiensis immune inhibitor A. These proteases belong to the M4 and M6 families, respectively. Both of these enzymes can serve as possible pathogenic factors, enhancing tissue destruction, bacterial invasion, and perturbation of host defense responses. Inhibition of Npr599 and InhA activities in vitro correlates with the protective effects of the anti-protease treatments reported previously (10Popov S.G. Popova T.G. Hopkins S. Weinstein R.S. MacAfee R. Fryxell K.J. Chandhoke V. Bailey C. Alibek K. BMC Infect. Dis. 2005; 5: 25Crossref PubMed Scopus (39) Google Scholar), indicating that they can be considered as potential therapeutic targets. Microbial Strain, Cultivation, and Supernatant Preparation—The non-encapsulated and non-toxigenic B. anthracis strain delta Ames (pXO1–, pXO2–) was kindly provided by Dr. J. Shiloach (National Institutes of Health, Bethesda, MD). To obtain a culture supernatant, 1 liter of LB medium was inoculated with an overnight seed culture (50 ml), incubated at 37 °C with vigorous agitation until the cells had reached stationary phase, and centrifuged at 17,000 × g for 10 min. The supernatant was removed and passed through a 0.22-μm cellulose acetate filter. Purification—All operations during enzyme purifications were performed at 4 °C unless indicated otherwise. Solid ammonium sulfate was added to 940 ml of culture supernatant to 75% saturation. The precipitated proteins were then collected by centrifugation at 17,000 × g for 20 min, dissolved, and dialyzed against 50 mm Tris-HCl (pH 7.6) containing 3 mm sodium azide. The resulting proteins were loaded onto a DEAE-cellulose anion-exchange column (bed volume of 60 ml) equilibrated with 50 mm Tris-HCl (pH 7.6) containing 3 mm sodium azide. Elution was achieved stepwise with 10, 50, 100, 200, 500, and 1000 mm NaCl in the same buffer. The substances of two peaks were collected: a flow-through fraction (P1) and a 200 mm NaCl eluate (P2). The P1 and P2 protease fractions were loaded onto a Sephacryl S-200 gel filtration column equilibrated with 20 mm Tris-HCl (pH7.6) and 150 mm NaCl and eluted at flow rate of 1.3 ml/min. Fractions (5 ml) were collected, and protease activity was assayed. Protease Assay—Protease activity was assayed during purification using an EnzChek Ultra protease kit for casein hydrolytic activity, an EnzChek gelatinase/collagenase kit for gelatin hydrolytic activity, and an EnzChek elastase kit for elastin hydrolytic activity, respectively, according to the manufacturer's recommendation (Molecular Probes). Briefly, 5 μl of supernatant or fractions in 45 μl of digestion buffer (100 mm Tris-HCl (pH 8.0), 0.1% Triton X-100, 5 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride) were mixed with 50 μl of fluorescein-labeled substrate, and then fluorescence intensity was measured after a 1-h incubation at 37 °C at 485-nm excitation and 510-nm emission wavelengths. One unit of protease activity was defined as the amount of protease required to liberate 1 mmol of the fluorescent dye from substrate-dye conjugates in 1 min. Characterization of the Proteases—To study the effect of pH on protease activity, the proteases were assayed at 37 °C in the following 0.1 m NaCl-containing buffers at various pH values: 50 mm sodium acetate/acetic acid buffer (pH 4–5.5), MES/NaOH buffer (pH 6–7), and 50 mm Tris-HCl (pH 7.5–10). The optimum temperature was determined by measuring the caseinolytic activity of the protease at 21, 37, 50, and 70 °C for 1 h (without the temperature-pH correction). To test the effect of inhibitors on protease activity, the proteins were preincubated with inhibitors, divalent ions, or other reaction components in 10 mm Tris-HCl (pH 7.8) for 30 min at room temperature. An equal volume of 2× casein substrate was then added, followed by further incubation at 37 °C for 1 h. SDS-PAGE and Determination of Protein Concentration—Proteins were separated by Tris/glycine/SDS-PAGE using 10 or 14% gels under reduced and denaturing conditions. The gels were stained using Coomassie Brilliant Blue R-250 and then destained. Protein concentration was determined colorimetrically using the Bio-Rad protein assay (Bradford) dye reagent and the bovine serum albumin as standard. N-terminal Amino Acid Sequencing—Partial N-terminal amino acid sequencing of the purified proteases was performed on polyvinylidene difluoride-electroblotted proteins at the Midwest Analytical, Inc. (St. Louis, MO), using an automated Edman degradation sequencer (Applied Biosystems, Foster City, CA). Substrate Digestion by Proteases—Approximately 0.2 μg of proteases was incubated for 4 h at 37 °C with various proteins in 20 mm Tris-HCl (pH 7.4) containing 1 mm CaCl2 and 1 mm MgSO4. Denaturation of human collagen types I, III, and IV was performed at 95 °C for 2 min. A plasmid expressing recombinant rat syndecan-1 with a glutathione S-transferase (GST) tag at the N terminus was kindly provided by Dr. Eok-Soo Oh (Ewha Womans University, Seoul, Korea). Recombinant syndecan-1 protein was partially prepared from host Escherichia coli BL21(DE3) cells using a glutathione-Sepharose affinity column. Digested substrates were separated by SDS-PAGE. Determination of Kinetic Parameters—Synthetic collagenase substrates (0.4–25 μm) were prepared in assay buffer (50 mm Tris-HCl (pH 7.5), 1 mm CaCl2, and 0.01% Tween 20). A collagenase assay was carried out in the substrate solution by incubation with 4 nm enzymes at 37 °C. Fluorescence was measured at λex = 320 nm and λem = 390 nm. Initial velocities were obtained from plots of fluorescence versus time using the data points corresponding to <40% full hydrolysis. The slopes of these plots were divided by the fluorescence change corresponding to complete hydrolysis and then multiplied by the substrate concentrations to obtain initial velocity in units of μm s–1. N-(3-(2-furyl)acryloyl)-Leu-Gly-Pro-Ala-hydrolyzing activity was measured by continuously monitoring the decrease in absorbance of the substrate at 324 nm after the addition of enzyme. Assay for Plasmin and Plasminogen Activation—Plasmin activity was assayed by monitoring Val-Leu-Lys-p-nitroanilide (pNA) hydrolysis at a of in 50 mm Tris-HCl (pH and 1 mm was preincubated with proteases at 37 °C for 4 h. Plasminogen activation in the of plasma was assayed by monitoring hydrolysis. plasminogen was incubated at 37 °C with 2 μg of protease or in 50 μl of 50 mm Tris-HCl (pH and 1 mm The resulting were and added to μm (50 in the of plasminogen plasminogen activation was carried out with 200 0.1 μm and or 10 purified proteases in a reaction The of from the substrate was at nm. of in shedding from normal murine mammary gland cells was assayed as previously T.G. B. Bradburne C. S. Bailey C. Chandhoke V. S.G. BMC Microbiol. 6: PubMed Scopus Google Scholar). Briefly, cells were in medium in cultured to and with the indicated proteins using culture supernatants (100 were collected and with μl of buffer mm 50 mm and 0.1% Tween 20 (pH were to using a The was with in and incubated with rat syndecan-1 followed by The were using an kit and The were by the and the intensity of using the are as the amount of syndecan-1 shed to the using a by of culture supernatants from epithelial cells with as a for shedding as previously T.G. B. Bradburne C. S. Bailey C. Chandhoke V. S.G. BMC Microbiol. 6: PubMed Scopus Google Scholar). For of the and were using of medium from cells for 4 h with purified proteases or phorbol 12-myristate 1 μm) was collected, and acetate and 3 of were overnight at the were in digestion and volume of were digested overnight with 20 and 20 at 37 °C. These digested and were by SDS-PAGE using gels and to The was with rat syndecan-1 and then and by the Mass proteins were separated by SDS-PAGE. Protein were from the gel and digested with according to a A. F. M. O. P. A. H. M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). were by using an and a fused column (100 μm × 10 with a and with (5 the column was for 5 min with A and were eluted using a of acid and to 30 min at and then to an 5 min. The was in a in which full spectrometric was followed by spectrometric the were for using a of were against the rat with III, J.K. D. Chem. 1995; 67: PubMed Scopus Google Scholar). For a to be considered had to of for for and for and a of of Npr599 and InhA in the Supernatant of the B. anthracis Ames anthrax the extracellular secreted proteins distinct from lethal toxin virulence factors that and other tissue damage J. PubMed Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). We previously determined the properties of anthrax culture supernatants and the of the neutral protease to the M4 family to in mice (10Popov S.G. Popova T.G. Hopkins S. Weinstein R.S. MacAfee R. Fryxell K.J. Chandhoke V. Bailey C. Alibek K. BMC Infect. Dis. 2005; 5: 25Crossref PubMed Scopus (39) Google Scholar). To the mechanisms of anthrax infection and to new therapeutic the purification and of proteases secreted by B. anthracis. The delta Ames strain (pXO1–, pXO2–) was cultured in a LB medium at 37 °C with vigorous agitation until stationary from the culture supernatant were precipitated with ammonium sulfate and used for DEAE-cellulose anion-exchange major peaks with enzymatic activities against casein and elastin were eluted from the column in the flow-through fraction (P1) and in the 200 mm NaCl eluate (P2). The P1 and P2 protease fractions were and further purified to apparent homogeneity on a Sephacryl S-200 gel filtration column. reduced and denaturing the purified enzymes a protein for P1 with a of and two protein for P2 with of and The proteases are highly T. O. Y. N. A. J. PubMed Scopus Google and therefore a purification rate of only the culture To the proteases and to the proteins to the of the N-terminal amino by automated Edman We determined that the P1 protease as a major and as a These the as the cleaved N-terminal of the of the M4 thermolysin-like neutral protease with a of is The full P1 gene in the B. anthracis Ames encodes the which is to to Bacillus neutral protease to and to of which belong to the neutral protease and which has to P. aeruginosa N. H. J.A. E. J. M. M.J. R.J. M. R.T. R. J.D. M. D. J.L. Y. L. K.J. A.M. A. R. C. O. B. Friedlander A.M. C.M. Nature. 2003; PubMed Scopus Google Scholar). We P1 as The N-terminal of P2 protease were determined as for the protein and for the Both of the proteins from the M6 family which is to the from B. and B. thuringiensis S. L. A. J. Microbiol. 1999; PubMed Scopus Google and which is encoded by the Based on