ARTICLE Enzyme-Driven Bacillus Spore Coat Degradation Leading to Spore Killing Ruchir V. Mundra,1,2 Krunal K. Mehta,1,2 Xia Wu,1,2 Elena E. Paskaleva,2 Ravi S. Kane,1,2 Jonathan S. Dordick1–5 1

Howard P. Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, CBIS 4005E, 110 8th Street, Troy, New York 12180; telephone: +518-276-2536; e-mail: [email protected]; telephone: +518-276-2899; fax: +518-276-2207; e-mail: [email protected] 2 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 3 Department of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, New York 4 Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 5 Department of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, New York

ABSTRACT: The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14-fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus the enzymatic route reduces the Correspondence to: J.S. Dordick and R.S. Kane Contract grant sponsor: Defense Threat Reduction Agency Received 6 August 2013; Revision received 30 September 2013; Accepted 7 October 2013 Accepted manuscript online 12 October 2013; Article first published online 6 November 2013 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25132/abstract). DOI 10.1002/bit.25132

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environmental burden of chemically mediated spore killing, and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable. Biotechnol. Bioeng. 2014;111: 654–663. ß 2013 Wiley Periodicals, Inc. KEYWORDS: spore coat; bacillus; proteases; cortex lytic enzymes; spore killing; decontamination

Introduction Bacillus spores are causative agents of a wide range of diseases ranging from food-borne illnesses (Bottone, 2010) to inhalation anthrax (Little and Ivins, 1999). The myriad bacillus spores are highly resistant to heat (Nicholson et al., 2000; Setlow, 2006), organic solvents, UV and gamma irradiation (Plomp et al., 2007; Riesenman and Nicholson, 2000; Setlow, 2006), mechanical disruption (Riesenman and Nicholson, 2000), and toxic chemicals such as hydrogen peroxide (Plomp et al., 2007; Riesenman and Nicholson, 2000; Setlow, 2006) and chloroform. Moreover, bacillus spores are resistant to bactericidal enzymes including lysozyme (Driks, 2002; Gould and Hitchins, 1963; Riesenman and Nicholson, 2000), which further complicates decontamination. Understanding the basis of this remarkable resistance has been of significant interest to the microbiology community (Driks, 2002; Henriques and Moran, 2000, 2007; Lai et al., 2003; McKenney et al., 2012). While resistance to heat and organic solvents has been attributed to the structure and chemical composition of the spore cortex and core (Nicholson et al., 2000; Riesenman and Nicholson, 2000; Setlow, 2006), the spore coat, essentially a proteinaceous ß 2013 Wiley Periodicals, Inc.

multilayered structure composed of intricate crosslinks of over 30 different polypeptides (Henriques and Moran, 2000, 2007; Lai et al., 2003), is primarily responsible for conferring resistance against harsh chemicals and lytic enzymes (Atrih et al., 1996; Driks, 2002; Henriques and Moran, 2007; Leggett et al., 2012). Targeting the spore coat, therefore, represents an opportunity to overcome one of the primary protective mechanisms of the bacillus spore. Despite the critical protective function of the spore coat, it remains the least studied structure among the various spore components (Moir, 2006). Nevertheless, some information is available. For example, the spore coat of Bacillus subtilis is composed of three main layers—an electron dense outer coat, a lamellar lightly staining inner coat and an electron diffuse undercoat (McKenney et al., 2012; Riesenman and Nicholson, 2000). This complex coat architecture is chemically comprised of keratin-like disulfide crosslinks, oo0 -dityrosine linkages, and protease-resistant transglutaminase-mediated peptide bonds, which act together to form a highly rugged molecular sieve that permits only small molecules to pass through (Scherrer et al., 1971). More specifically, cortex lytic enzymes, such as lysozyme or SleB (Heffron et al., 2010), which can potentially hydrolyze the cortex and lead to spore germination, cannot penetrate the spore coat. This further prevents cell lytic enzymes to gain access to the bacterial cell wall to kill germinated spores. Thus, the bacterial spore coat is capable of serving as an impenetrable barrier protecting the integrity of the spore (Fernando and Othman, 2006). In the absence of a spore coat, for example, following chemical decoating (Henriques and Moran, 2000; Pandey and Aronson, 1979) or through genetic mutations (Driks, 1999; Riesenman and Nicholson, 2000), bacillus spores become vulnerable to lysozyme (Riesenman and Nicholson, 2000). Gould and Hitchins (1963) successfully demonstrated sensitization of Bacillus cereus spores to lysozyme after treatment with b-mercaptoethanol and 8 M urea at 70 C. More recently, Riesenman and Nicholson (2000) used a combination of 1% (v/v) SDS, 50 mM dithiothreitol (DTT), and 8 M urea at pH 9.8 and 60 C to decoat B. subtilis spores. While such approaches are fundamentally intriguing, their application in a real-life environment (particularly the use of detergents and denaturants, and use of high temperature) remains a key challenge. Herein, we provide the first demonstration of an enzymatic decoating technique, which uses mild protease-mediated partial degradation of the spore coat of B. cereus and B. anthracis. Our approach results in sufficient permeabilization of the spore coat, thereby rendering the underlying cortex susceptible to lysozyme and the cortex lytic enzyme, SleB (Heffron et al., 2010), to initiate germination. The germinated spores were further vulnerable to lytic enzymes resulting in spore killing. This approach may enable an environmentally friendly route to the rapid germination of recalcitrant bacillus spores, leading to effective decontamination.

Materials and Methods Spore Preparation and Purification B. cereus Frankland and Frankland 1887 AL strain was a kind gift from Prof. P. Setlow, University of Connecticut. B. anthracis delta Sterne strain was obtained from Naval Surface Warfare Center, Dahlgren. Spores of B. anthracis were prepared using a previously described protocol (Banerjee et al., 2012). In brief, Difco sporulation medium (DSM) was prepared in distilled water by dissolving Bacto nutrient broth (Difco) (8 g/L) and adding 0.10% (w/v) KCl, and 0.012% (w/v) MgSO4·7H2O. NaOH was added to bring the pH to 7.6. The medium was autoclaved and cooled sufficiently. Prior to use, 1-mL sterile solutions of 1 M Ca(NO3)2, 0.01 M MnCl2, and 10 mM FeSO4 were added to 1 L of the autoclaved medium (FeSO4 was prepared fresh every time). B. anthracis was first propagated in 25 mL DSM at 30 C and 220 rpm until mid-log phase (A600  0.5) was reached (usually 3 h). The culture was then transferred to 225 mL of prewarmed (37 C) DSM in a 1-L flask (Banerjee et al., 2012). The mixture was further incubated for 5 days at 30 C in a shaker at 220 rpm. The spore-containing 250 mL culture was washed in sterile distilled water (SDW) and then centrifuged at 4,000g for 20 min until most of the germinating spores, cell debris, and vegetative cells were eliminated as assessed by phase contrast microscopy. The spore suspension was then stored at 4 C in sterile water and protected from light. Spores of B. cereus were prepared as described previously in a defined sporulation medium (Ghosh and Setlow, 2009; Ghosh et al., 2008; Stewart et al., 1981) at 30 C with good aeration for 48 h. Specifically 125 mL of spores were cultured in a 1-L flask. Spores were harvested by centrifugation (4,000g, 15 min) and washed with SDW until they were free of any germinating cells, vegetative cells, and cell debris. Spores were then stored at 4 C protected from light. Chemical Decoating of Spores Clean spores (109 CFU/mL), which were 95% phase bright, were resuspended in a freshly prepared decoating solution (Pandey and Aronson, 1979) containing 5 mM N-cyclohexyl2-aminoethanesulfonic acid (CHES) (Acros Organics, NJ), 50 mM DTT (MP Biomedicals, Santa Ana, CA), 1% sodium dodecyl sulfate (SDS, Sigma Aldrich, St. Louis, MO), and 8 M urea (Sigma Life Science, St. Louis, MO) at pH 9.8 for 3 h at 37 C. The treated spores were centrifuged three times at 12,000g for 2 min and washed three times with sterile water. The supernatant from the first wash, which consisted of solubilized coat fractions, was stored at 4 C for further testing for protease-catalyzed degradation, while the pellet was used as a positive control for germination experiments. Trinitrobenzene Sulfonic Acid (TNBS) Assay for Screening Proteases on Solubilized Coat Fraction To remove SDS, urea, and DTT, the solubilized coat was dialyzed against 0.1 M sodium bicarbonate buffer (pH 8.5)

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using ultracentrifuge filters with a 3 kDa cut-off. The bufferexchanged coat fractions were then subjected to protease digestion. Ten proteases—trypsin, a-chymotrypsin, pronase, proteinase K, elastase, thermolysin, papain, pepsin, subtilisin Carlsberg, and collagenase, all obtained from Sigma, were used at a concentration of 10 mg/mL to digest the isolated coat in 96-well plates for 3 h at room temperature. The free amino groups released upon coat digestion were then quantified with the TNBS reagent (0.01% (v/v) in 0.1 M sodium bicarbonate buffer pH 8.5) at 37 C for 2 h (Goodwin and Choi, 1970). Absorbance was measured at 335 nm on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA) to quantify the released amino groups, which correlated with the extent of coat degradation. Protease Treatment of B. cereus Spores B. cereus spores (2  109 CFU/mL) were incubated with 50 mM DTT in Tris buffer (pH ranging from 8.0 to 9.5) for 3 h at 37 C. Following the addition of DTT, the pH was adjusted using NaOH. The spores were then washed three times with SDW by centrifuging at 12,000g for 2 min at 4 C to remove the DTT. Spores were then treated with proteases at a working concentration of 50 mg/mL for 3–5 h at 37 C. B. anthracis spores (2  109 CFU/mL) were treated with 50 mM DTT in Na-carbonate/bicarbonate buffer at varying pH at 37 C. Similar to that with B. cereus, DTTwas washed off and the spores were subsequently incubated with proteases at a working concentration of 500 mg/mL at 37 C for varying incubation times.

concentration of 5  107 CFU/mL and were incubated with lysozyme/SleB (0.1 mg/mL) for a period of 1 h. Spores were counted using optical microscopy and then diluted to 105 CFU/mL in 100 mM PBS with 0.1% Tween80 (Sigma Life Science) added to prevent aggregation. Part of the spore suspension was then heated at 70 C for 20 min. The suspensions, both with and without heating, were plated onto nutrient agar plates. The plates were incubated overnight at 37 C. Because germinated spores cannot survive at 70 C, the viable colonies that grew on these plates were used to quantify germination (Shah et al., 2008). Appropriate controls were used as described in previous section. Imaging Spore Surfaces Using Scanning Electron Microscopy (SEM) A drop of clean spore suspension was added to an SEM silicon wafer and allowed to air dry (Murphy and Campbell, 1969). The wafer was then attached to a metallic stub via doublesided carbon tape. A thin metallic coating was then evaporated onto the stub and the samples were analyzed via SEM using a JEOL-JSM 840 Scanning Electron Microscope (Evex Analytical, Inc.) with a field emission gun operating at 10.0 kV. Samples subjected to various treatments were characterized in the same way. Images were taken at different magnifications and from different areas in the specimen to obtain a broader and statistically relevant analysis of the sample. Intact spores and spores treated with reductant alone were used as negative controls, whereas chemically decoated spores were used as a positive control to analyze the surface characteristics.

Spore Germination Using Lysozyme and SleB Protease-treated spores were centrifuged (12,000g, 2 min at  4 C) and washed three times with SDW to eliminate proteolytic interference in successive steps. All spore samples were diluted to A600 ¼ 2 (approximate cell density of 2  108 CFU/mL). Both lysozyme and SleB, the latter expressed and purified as previously described (Heffron et al., 2010) were used at a working concentration of 0.05 mg/ mL. Germination was tested in black 96-well plates, where 50 mL of spore suspension was mixed with 100 mL of cortex lytic enzymes, and 50 mL of terbium-containing buffer (50 mM TbCl3 in 25 mM Tris–HCl buffer, pH 8.8). Dipicolinic acid (Ca2þ-DPA) release was monitored by real-time fluorescence (lex ¼ 270 nm, lem ¼ 545 nm) and a cut-off of 530 nm (Hindle and Hall, 1999; Yi and Setlow, 2010) Untreated spores and spores incubated with reductant only with no protease treatment were used as negative controls. Chemically decoated spores (obtained as described above) were used as a positive control to analyze effectiveness of proteolytic decoating. Extent of Germination Using a Plating Assay Following proteolytic treatment, spores were washed as described above. Washed spores were used at a working

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Spore Killing Using PlyPH (Bacteriophage Lytic Enzyme) PlyPH was expressed and purified as described by Yoong et al. (2006). Spores treated with proteases and germinated using SleB, as described above, were then diluted to an approximate concentration of 107 CFU/mL in 100 mM PBS (0.1% (v/v) Tween80). Spore suspension (100 mL) at a working concentration of 106 CFU/mL was incubated with 500 mg/mL of PlyPH for 6 h. Subsequently, the spores were diluted to 105 CFU/mL and aliquots were plated on nutrient agar. Similar to the aforementioned plating assays, part of the spore suspension was heated at 70 C for 20 min to evaluate germination. Appropriate controls were used at each step.

Results and Discussion Identifying Proteases That Digest Isolated Spore Coat To evaluate whether proteases could catalyze the degradation of bacillus spore coats, we isolated the spore coats of B. cereus and B. anthracis using a previously published methodology (Pandey and Aronson, 1979). Ten commercially available proteases were screened for their ability to digest the coat. The increase in amino groups due to protease action was quantified using a standard TNBS assay (Goodwin and Choi,

Table I. Screen of commercial proteases for their abilities to proteolyze isolated spore coats of B. cereus and B. anthracis.

Proteasesa Trypsin a-Chymotrypsin Elastase Proteinase K Pronase Collagenase Subtilisin Carlsberg Pepsin Papain Thermolysin

B. cereus (net absorbance of digested coat)

B. anthracis (net absorbance of digested coat)

0.00  0.00 0.00  0.00 0.02  0.00  0.26  0.03  0.31  0.06 0.02  0.01  0.08  0.02 0.00  0.00 0.00  0.00 0.00  0.00

0.06  0.02 0.02  0.00 0.00  0.01  0.28  0.05  0.25  0.02 0.01  0.00  0.38  0.04 0.00  0.00 0.00  0.00 0.00  0.00



All proteases were used at a concentration of 10 mg/mL. Statistically significant within 99% confidence level.

a 

presence of extensive disulfide crosslinks in the coat, which may act as a barrier to proteases. To test the latter hypothesis, we used DTT (a strong reducing agent) to reduce the disulfide links prior to addition of the proteases, as depicted in Scheme 1 (Step 1).

1970) (Table I). Background proteolytic autolysis activity was subtracted from the measured amino formation due to coat digestion. Table I summarizes the proteolytic degradation of isolated spore coats from both species. B. cereus spore coat degradation was most effectively catalyzed by proteinase K and pronase, while for B. anthracis, in addition to proteinase K and pronase, subtilisin Carlsberg was also an effective degradation catalyst (Table I). The slight difference in protease selectivity on the two bacilli spore coats is not surprising, as it is known that the B. cereus and B. anthracis have somewhat dissimilar polypeptide compositions (Lai et al., 2003). Proteolytic Degradation of Intact Bacillus Spore Coat The isolation of the bacillus spore coats required the use of harsh denaturants and reductants. Thus, it is likely that the coat proteins were significantly denatured. To ascertain whether protease activity was an artifact of this denaturation, or whether their intrinsic activities were sufficient to act on intact spore coats, we treated intact spores with the aforementioned proteases and evaluated the resulting permeabilization by lysozyme. This approach takes advantage of the known ability of enzymes such as lysozyme and SleB to trigger the germination of chemically decoated spores, by catalyzing spore cortex lysis and subsequent Ca2þ-DPA release (Li et al., 2013). The released Ca2þ-DPA can be detected through the formation of a fluorescent complex with Tb3þ allowing facile measurement of germination (Hindle and Hall, 1999). The effectiveness of protease-catalyzed coat degradation that would lead to coat permeabilization, therefore, can be assessed by the release of Ca2þ-DPA through subsequent lysozyme incubation. None of the proteases tested, however, catalyzed release of measurable Ca2þ-DPA indicating the inability to effect sufficient coat permeabilization of lysozyme (data not shown). This inability may be due to the purported presence of protease-resistant transglutaminase-mediated peptide bonds on the spore surface (Henriques and Moran, 2000, 2007), and/or to the

Establishing Optimum Pretreatment Parameters Pretreatment of B. cereus and B. anthracis with DTT for 3 h at pH 8.0–9.7, followed by removal of DTT (via washing) and then incubation with proteinase K, pronase, or subtilisin Carlsberg resulted in significant lysozyme permeability at pH 9.3 (for B. cereus) and pH 9.7 (for B. anthracis) (data not shown). The enhanced nucleophilicity of DTT at higher pH, therefore, was sufficient to effect its reaction with coat protein disulfide linkages. Indeed, for B. cereus spores, at pH 9.3, each of the proteases tested resulted in measurable Ca2þ-DPA release following lysozyme incubation. Based on a standard curve of measured fluorescence as a function of Ca2þ-DPA concentration (data not shown), the level of Ca2þ-DPA released and the steady state rates of release were expressed in terms of nmol Ca2þ-DPA/min. The time course for protease action on B. cereus is depicted in Figure 1a, with Ca2þ-DPA release plotted as a percentage of maximum Ca2þ-DPA released (obtained after boiling spores in water for 30 min). As expected from above, pretreatment with DTT alone led to minimal Ca2þ-DPA release following lysozyme incubation. Although all three proteases catalyzed some Ca2þ-DPA release, proteinase K was identified to be the most effective (for B. cereus), with an optimal pretreatment condition of pH 9.3 for 3 h, leading to rapid Ca2þ-DPA release upon lysozyme incubation. There was a noticeable lag phase with pronase, and a far more significant lag phase with subtilisin. Ca2þ-DPA release with 100 mg/mL proteinase K or pronase resulted in at least 75% of maximum Ca2þ-DPA release within 20 min of lysozyme incubation, whereas subtilisin catalyzed the release of 30% maximum Ca2þDPA. The initial rate of Ca2þ-DPA release as a result of proteinase K pretreatment was ca. 14-fold faster than with DTT alone. After 10 h, minimal additional activity was observed with the protease-DTT pretreatments, including upon addition of fresh proteinase K. Interestingly, B. anthracis required pH 9.7 and 7 h to effect protease-mediated Ca2þ-DPA release. Subtilisin was the most effective protease under these conditions reaching ca. 85% of maximal Ca2þ-DPA release within 20 min of lysozyme treatment (Fig. 1b). Similarly, DTT pretreatment alone without protease catalysis resulted in less than 20% of maximum Ca2þ-DPA release, once again highlighting the importance of the protease-mediated breakdown. The observed differences in optimal pretreatment conditions and protease effectiveness suggest that there are key differences in the structural features of the spore coats for the two bacillus species. Nevertheless, these results establish that commonly available subtilisin-type proteases partially degrade, or at least permeabilize, the largely impregnable spore coat to enable lysozyme to degrade the newly accessible

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Scheme 1.

Three-step DTT/enzymatic decontamination strategy for B. anthracis.

cortex and effect release of Ca2þ-DPA. Although the proteases required DTT to break disulfide linkages in the spore coat, traditional denaturants such as SDS and urea were not required in the decoating process. Influence of Protease Concentration on Ca2þ-DPA Release Rates and Lag Times As shown in Figure 1, proteinase K was the most effective protease for B. cereus, resulting in the shortest lag times and fastest Ca2þ-DPA release rates when compared to pronase and subtilisin Carlsberg (at a similar concentration of 100 mg/mL). Similarly, subtilisin Carlsberg was the optimal enzyme for B. anthracis. Hence, in the case of B. cereus, we studied the influence of proteinase K concentration on Ca2þDPA release rates and lag times, while for B. anthracis we used subtilisin Carlsberg. The two-step enzymatic process is complex and leads to an indirect measurement of protease activity through the secondary action of lysozyme. The concentrations of proteinase K and subtilisin Carlsberg (against B. cereus and B. anthracis, respectively) were varied at a fixed lysozyme concentration of 3.3 mM (50 mg/mL) and the rate of Ca2þDPA release and lag time prior to steady-state Ca2þ-DPA release were measured. At this lysozyme concentration, the rate of proteinase K-catalyzed Ca2þ-DPA release was unaffected by higher concentrations of lysozyme (data not shown) and intrinsic proteolytic activities on the spore coats

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were obtained. The dependence of spore degradation rates on protease concentrations was observed to be roughly linear for both spores at lower protease concentrations (Fig. 2). The absence of perfect linearity could be attributed to the heterogeneous nature of spore coat. Moreover, at a higher enzyme concentration, there was a significant leveling off of the rate of Ca2þ-DPA release (data not shown). This is understandable, as the enzymes have likely exhausted the available reactive sites in the spore coat on which they act. The time course of lysozyme-catalyzed Ca2þ-DPA release from both spores also indicated a lag-time in lysozyme action; shorter lag times were observed at higher protease concentrations (Fig. 2). This decrease in lag times was not unexpected, as an enhanced protease mediated coat degradation would imply faster access towards lysozyme, and thus, more rapid release of Ca2þ-DPA. Single Step Coat Degradation and Comparison With Traditional Decoating Methods Owing to the lack of disulfide bonds within subtilisin Carlsberg, we tested whether it was feasible to combine DTT and protease pretreatment into a single step to degrade the B. anthracis spore coat. As shown in Figure 3, 500 mg/mL subtilisin combined with 50 mM DTT permeabilized the spore coat towards 1 mM lysozyme. The effectiveness of the combined protease-DTT pretreatment was similar to that performed in two sequential steps (Fig. 1b). Importantly, the

hand is a muramidase (Rau et al., 2001), and thus able to hydrolyze the accessible cortex causing subsequent Ca2þDPA release. B. anthracis Germination Using SleB and Comparison With Lysozyme SleB, a cortex lytic transglycosylase, hydrolyses the B. anthracis spore cortex and initiates spore germination (Heffron et al., 2010; Li et al., 2012). We expressed SleB as a 67 kDa MBP fusion protein, which was considerably larger than the 14.4 kDa lysozyme and posed a vital test in evaluating spore coat porosity. As chemically decoated spores have been shown to be vulnerable to SleB (Li et al., 2013), we tested the ability of SleB to permeate through protease treated spores. Remarkably, we found that not only did 1 mM SleB gain access to the underlying cortex, but the rate of cortex hydrolysis and subsequent steady state Ca2þ-DPA release was approximately fourfold faster than that caused by 1 mM lysozyme (Fig. 4). SleB is essentially an endogenous cortex lytic enzyme responsible for B. anthracis germination, and therefore, the spore cortex acts as a natural substrate. This may explain the enhanced rate of germination over the less selective lysozyme. In addition, and consistent with the higher catalytic activity of SleB, there was no measurable lag observed as compared to a lag time of approximately 75 s for lysozyme hydrolysis. Collectively these results imply that proteases generate large pores in the spore coat architecture, thereby allowing facile permeability even towards larger enzymes. Figure 1. Protease-catalyzed degradation of bacillus spore coats in vivo for (a) B. cereus and (b) B. anthracis. Spores at a concentration of 2  109 CFU/mL were subjected to a solution containing 50 mM DTT at a pH of 9.3 for B. cereus and 9.7 for B. anthracis. Proteases were used at 0.1 mg/mL for B. cereus and 0.5 mg/mL for B. anthracis. Spores treated with DTT (^), DTT þ Proteinase K (&), DTT þ Pronase (*), DTT þ Subtilisin Carlsberg (~). The extent of coat degradation achieved was quantified by measuring fluorometrically Ca2þ-DPA released upon germination using 1 mM lysozyme.

combined pretreatment nearly reached the level of decoating/ permeabilizing achieved using traditional chemical decoating methods (e.g., 1% (v/v) SDS, 8 M urea, and 50 mM DTT (pH 9.8)). Specifically, upon saturation, traditional decoating methods permeabilized the coat to release approximately 82% of the total Ca2þ-DPA content, whereas protease mediated permeabilization released 75% of total Ca2þ-DPA (Fig. 3). Consistent with the previous experiments, spores treated with DTT alone released less than 10% of the total Ca2þ-DPA. Irrespective of whether decoating was achieved through proteolysis or traditional chemical methods, no germination was observed in the absence of lysozyme (Fig. 3). This result was expected because the subtilisin used for proteolytic decoating is a serine endopeptidase and would only digest the peptide linkages in the spore coat and not hydrolyze the underlying cortex peptidoglycan. Lysozyme on the other

Probing and Analyzing Spore Coat Surface Using Scanning Electron Microscopy (SEM) To assess the enhanced porosity of protease-mediated coat degradation, we analyzed the spore coat surface in response to various pretreatments using SEM at 45,000. Images were taken from different areas in the specimen to obtain a statistically relevant analysis of the sample. Images obtained for intact spores (Fig. 5a) were consistent with the literature (Murphy and Campbell, 1969), with parallel ridges along the spore surface. These ridges are part of the spore coat, which folds into a wrinkled pattern during sporulation to accommodate changes in spore volume without compromising the coat’s structural integrity. Thus, these ridges confer a dynamic nature to the spore coat allowing it to unfold and swell during germination (Chada et al., 2003). While these ridges were present following DTT pretreatment (Fig. 5b), they were either absent or greatly reduced after proteolytic pretreatment (Fig. 5c). Interestingly, coat structures observed with chemically decoated spores (Fig. 5d) resembled closely to that observed in spores treated with protease (Fig. 5c). Recent studies have suggested critical roles for a number of coat proteins in ridge formation. Of particular importance are CotA and CotB (present in the outer coat) and CotE (responsible for outer coat assembly) (Chada et al., 2003). The absence of ridges following protease treatment could

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Figure 2. Steady state rates of Ca2þ-DPA release (^) and lag times (&) as a function of (a) proteinase K concentration on B. cereus spores and (b) subtilisin Carlsberg concentration on B. anthracis spores. Lysozyme was used at 3.3 mM to trigger subsequent Ca2þ-DPA release.

Figure 3. Subtilisin Carlsberg used simultaneously with DTT resulting in a single step degradation approach. B. anthracis spores (2  109 CFU/mL) were treated with a combination of 50 mM DTT and 500 mg/mL subtilisin. Spores treated with DTT þ lysozyme (~), DTT þ Subtilisin (*), DTT þ Subtilisin þ lysozyme (&), chemically decoated spores (^), chemically decoated spores þ lysozyme ( ). Chemically decoated spores were used as a positive control.

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Figure 4.

Germination of B. anthracis spores after proteolytic breakdown and the action of lysozyme (1 mM) or SleB (1 mM). Spores treated with DTT þ Lysozyme (^), DTT þ Subtilisin Carlseberg þ Lysozyme (&), DTT þ SleB (~), DTT þ Subtilsin Carlsberg þ SleB ().

Figure 5.

Scanning electron microscope images of (a) intact B. anthracis spores, (b) DTT-treated spores, (c) DTT þ subtilisin Carlsberg treated spores, and (d) chemically decoated spores (positive control). Images (a) and (b) clearly show parallel ribbing/ridges along the spore surface. The ridges are absent in (c) and these spores very closely resemble chemically decoated spores (d). Images were obtained at a magnification of 45,000.

imply the degradation of these critical spore coat components. We speculate that proteases could either selectively target one or more of these coat proteins responsible for ridge formation and/or breakdown the overall structural arrangement by degrading several morphogenic coat proteins thereby hindering proper ridge formation. Moreover, spores treated solely with DTT had vastly different spore surface (Fig. 5b) than those treated with DTT and protease (Fig. 5c), once again emphasizing the importance of protease catalyzed coat degradation. SEM imaging on B. cereus yielded similar results (data not shown). Enzymatic Decontamination of B. anthracis Using Bactericidal PlyPH Having established the proteolytic degradation of the B. anthracis spore coat, we next tested whether we could combine this approach with cell lytic enzymes to result in spore killing/decontamination. Along these lines, we adopted a three-step tri-enzymic decontamination strategy (Scheme 1), which includes spore coat degradation using DTT and subtilisin Carlsberg (Step 1), cortex hydrolysis using SleB to trigger germination (Step 2), and bacterial cell wall lysis using the cell lytic enzyme PlyPH (Yoong et al., 2006) (Step 3). Untreated (control) spores (Fig. 6a) showed no germination in the presence of SleB or PlyPH alone or in combination. Subtilisin pretreated spores (500 mg/mL subtilisin and 50 mM DTT) (Fig. 6b) resulted in a 50% reduction in the number of viable colonies using 1 mM SleB even without heating. Heating at 70 C further reduces the number of viable colonies by 65%. As germinated spores cannot survive 70 C, this reduction in viability was equivalent to the extent of

Figure 6.

Influence of heat treatment to assess germination and killing of B. anthracis due to various treatment methods; (a) intact spores, (b) protease-treated spores, and (c) chemically decoated spores were all treated with 1 mM SleB for 3 h and then incubated with 500 mg/mL PlyPH for 7 h. Half of the spores are suitably diluted and plated (gray). Viable colonies (gray) are used to estimate sporicidal effect of various treatments. Half of spores were heated (black) at 70 C to kill germinated spores. Viable colonies, which represented the non-germinated spores, were used to estimate percentage germination.

germination (Fig. 6b). Even in the absence of heat, a combination of SleB and PlyPH led to a roughly 75% reduction in viability. This result suggested that SleB germinated protease-treated spores and rendered them vulnerable to PlyPH. Chemically decoated spores (Fig. 6c) behaved similarly. These results are consistent with our results with Ca2þ-DPA release and SEM analysis. An unexpected outcome of this study was that for protease treated and chemically decoated spores, SleB and PlyPH individually had observable sporicidal activity (Fig. 6b and c).

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In the case of PlyPH, this result might be due to its ability to degrade the cortex, albeit to a small extent, yet sufficient to gain access to the cell wall and kill the cell. However, the sporicidal activity of SleB was surprising, since there is no direct evidence pointing towards this enzyme having bactericidal activity. Nevertheless, the key inference from this set of experiments is the ability of SleB to germinate the protease-treated spores making them susceptible to PlyPH lysis.

Conclusions We have developed a relatively mild and environmentally benign enzymatic route for decontaminating B. cereus and B. anthracis spores, which has both fundamental and practical implications. Fundamentally, we identified simple commercial proteases capable of catalyzing the degradation of bacillus spore coats. This degradation led to a morphological change in the coat, leading to increased permeability of cortex and cell lytic enzymes, and ultimately spore killing. Practically, protease mediated coat degradation leads to an “outside in” enzymatic decontamination strategy, from spore coat degradation to bacterial cell wall lysis. The extent of coat degradation achieved using proteases is similar to that achieved by traditional chemical decoating methods. Although DTT is required to enhance proteolytic degradation, the elimination of detergents and strong denaturants reduces the environmental burden of chemically mediated spore killing. Other biological routes are being explored that may replace the nucleophilic thiol function of DTT. Moreover, more rapid spore coat degradation may be achieved through molecular evolution techniques coupled with high throughput screening. Finally, nature is self-regulated, and endogenous proteases exist in the spore coat that must play a role in coat permeabilization, leading to germination. These enzymes may also serve as suitable candidates for coat degradation. Our work clearly demonstrates that biocatalytic spore killing is achievable, and this opens up a broad array of novel approaches towards decontamination of bacillus spores. We acknowledge the financial support from the Defense Threat Reduction Agency via cooperative research agreement with the US Army Corps of Engineers Engineer Research and Development Center. Discussions with Dr. K. Solanki are gratefully acknowledged.

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Enzyme-driven Bacillus spore coat degradation leading to spore killing.

The bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based c...
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