A Bowman–Birk Inhibitor from the Seeds of Luetzelburgia auriculata

Article Cite This: J. Nat. Prod. 2018, 81, 1497−1507

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A Bowman−Birk Inhibitor from the Seeds of Luetzelburgia auriculata Inhibits Staphylococcus aureus Growth by Promoting Severe Cell Membrane Damage Thiago F. Martins, Ilka M. Vasconcelos, Rodolpho G. G. Silva, Fredy D. A. Silva, Pedro F. N. Souza, Anna L. N. Varela, Louise M. Albuquerque, and Jose T. A. Oliveira*

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Laboratory of Plant Defense, Department of Biochemistry and Molecular Biology, Federal University of Ceara (UFC), Avenida Mister Hull, 60451-970, Fortaleza, Ceara, Brazil ABSTRACT: Staphylococcus aureus is a multidrug-resistant bacterium responsible for several cases of hospital-acquired infections, which constitute a global public health problem. The introduction of new healthcare strategies and/or the discovery of molecules capable of inhibiting the growth or killing S. aureus would have a huge impact on the treatment of S. aureus-mediated diseases. Herein, a Bowman−Birk protease inhibitor (LzaBBI), with strong in vitro antibacterial activity against S. aureus, was purified to homogeneity from Luetzelburgia auriculata seeds. LzaBBI in its native form is a 14.3 kDa protein and has a pI of 4.54, and its NH2-terminal sequence has high identity with other Bowman−Birk inhibitors. LzaBBI showed a mixed-type inhibitory activity against both trypsin and chymotrypsin, respectively, and it remained stable after both boiling at 98 °C for 120 min and incubation at various pHs. Scanning electron microscopy revealed that LzaBBI disrupted the S. aureus membrane integrity, leading to bacterial death. This study suggests that LzaBBI is a powerful candidate for developing a new antimicrobial to overcome drug resistance toward reducing hospital-acquired infections caused by S. aureus.

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global emergence of antibiotic-resistant bacteria, new strategies of treatments are needed, either by chemical modification of existing drugs or purification of biomolecules with therapeutic potential.5,6 Several proteins with deleterious actions against bacteria have been isolated from plants. Protease inhibitors (PIs) are proteins or peptides that complex with proteolytic enzymes, inhibiting their catalytic activity.7 PIs are ubiquitous in nature, and in plants the highest concentrations are generally found in seeds. PIs perform a variety of functions during plant development, as they control protein degradation during seed dormancy and act as plant defense molecules against insect and pathogen attacks.8−10 Over the last years, PIs have received greater attention due to the possibility of various applications in both human health and crop protection.11,12 Indeed, recent studies reported that some PIs of plant origin like those from Abelmoschus moschatus (AMTI-II)13 and Jatropha curcas (JcTI-I)14 kill human pathogenic bacteria (Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Bacillus subtilis, Streptococcus pneumoniae, Bacillus cereus, and Salmonella enteric) by damaging the plasma cell membrane.8 They also have antifungal, antiprotozoal, and antiviral activities.9 Others, like those from Clitoria fairchildiana (CFPI),15 Ricinus communis (RcTI),16 and Cassia leiandra (ClTI),17 inhibit

wing to increasing medical awareness of the rapid emergence of multidrug-resistant bacteria worldwide, endangering the efficacy of antibiotics and threatening the healthcare systems, two articles were recently published on the antibiotic resistance crisis.1,2 In part 1, in which the causes and threats are discussed, the antibiotic resistance crisis has been attributed to the overuse, inappropriate prescription, extensive agricultural use as growth supplements in livestock, and the availability of only a few novel antibiotics due to economic and regulatory obstacles.1 In part 2, which advanced management strategies and new antibiotics, the author discussed steps to reduce antibiotic resistance by improving diagnosis, tracking, and prescribing correct practices; optimizing therapeutic regimens; eliminating diagnostic uncertainty; and improving tracking methodologies. These procedures should be used by healthcare facilities to electronically report infections, antibiotic use, and resistance, besides preventing infection transmission, which can significantly decrease resistance by eliminating the need for antibiotics.2 Staphylococcus aureus and its methicillin-resistant strains (MRSA) are Gram-positive bacteria that exist in community and hospital settings that often cause bloodstream infections in hospitalized patients. Hospital-acquired MRSA (HA-MRSA) strains are generally multidrug resistant, and although vancomycin has been used to treat MRSA infections, a few cases of vancomycin-resistant S. aureus (VRSA) have recently emerged.3,4 In connection with this recognized reality of rapid © 2018 American Chemical Society and American Society of Pharmacognosy

Received: June 26, 2017 Published: June 21, 2018 1497

DOI: 10.1021/acs.jnatprod.7b00545 J. Nat. Prod. 2018, 81, 1497−1507

Journal of Natural Products

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Table 1. Purification Steps of LzaBBI from L. auriculata Seeds purification steps

total protein (mg)a

total activity (TIU)b

specific activity (TIU/mg protein)

purification (fold)c

yield (%)d

crude extract (CE) heat-treated CE (98 °C, 20 min) sepharose 4B- anhydrotrypsin (A2) μRPC C2/C18 (LzaBBI)

426.24 ± 39.99 271.86 ± 11.21 12.44 ± 1.05 0.88 ± 0.15

31 933.07 ± 184.89 22 416.24 ± 1776.61 15 356.24 ± 1288.60 3199.55 ± 81.07

74.92 ± 2.00 82.45 ± 1.49 1234.42 ± 62.02 3635.85 ± 301.05

1.00 1.10 16.48 48.52

100.00 70.19 48.08 10.01

a

Total amount of proteins recovered from 5 g of dehulled L. auriculata mature seeds. bOne trypsin inhibitory unit (1 TIU) is defined as the decrease in 0.01 unit of absorbance at 410 nm per 15 min assay, at 37 °C. cThe purification index was calculated as the ratio between the specific activity of each purification step and that of the crude extract taken as 1. dCalculated based on the total activity recovered.

into two protein peaks, A1 and A2 (Figure 1a). A2 showed an approximately 16.5-fold increase in activity compared to the crude extract (Table 1). A2 was pooled and subjected to a reverse-phase chromatography (HPLC step), and the first peak (Figure 1b), eluted with an acetonitrile gradient (with 20% to 25% eluent B gradient, 17−19 min retention time), showed a specific inhibitory activity of 3635.85 trypsin inhibitory unit (TIU)/mg protein and 48.5-fold purification compared to the crude extract (Table 1). This fraction was homogeneous, as confirmed by the presence of a unique protein band after SDSPAGE (Figure 2a). The native molecular mass of LzaBBI, assessed by molecular exclusion chromatography on a Sephacryl S-200 column, is 14.3 kDa (Figure 1c). The apparent molecular mass after SDSPAGE, under nonreduced or reduced conditions, is 17.3 kDa (Figure 2a), which is near to those of the protease inhibitors purified from Populus deltoides (22 kDa),31 Trigonella foenumgraecum (20 kDa),24 Dolichos bif lorus (16 kDa),32 and R. cummunis (14 kDa).16 LzaBBI is not a glycoprotein (Figure 2b), unlike other protease inhibitors that have carbohydrate moieties covalently bound to their structures.23,31 Moreover, LzaBBI has an acidic pI of 4.54 (Figure 2c), which is close to those of other reported Bowman−Birk inhibitors such as the three isoforms found in Vigna unguiculata (PI, PII, and PIII) seeds, with isoelectric points (pIs) of 4.45, 4.68, and 4.95, respectively,33 and the two Bowman−Birk inhibitor isoforms (CLTI-I and -II) of Canavalia lineata seeds, with pIs of 4.57 and 4.50, respectively.34 LzaBBI is a potent inhibitor of the serine proteases trypsin and chymotrypsin (Figure 3). Under optimal conditions LzaBBI could completely inactivate the hydrolytic activities of these enzymes. However, LzaBBI is inactive toward elastase, other serine proteases, and bromelain and papain, which are cysteine proteases (Figure 3). These results are similar to the specificity exhibited by other inhibitors belonging to the Bowman−Birk family.28,35,36 Analysis of the LzaBBI NH 2 -terminal sequence (SIPPQCHCADIRLNSCHSAQCQC) revealed a high similarity (Table 2) to other protease inhibitors that belong to the Bowman−Birk family.37,38 In addition, LzaBBI possesses 52.17% nonpolar and hydrophobic amino acids in its NH2terminal sequence, among which five cysteine residues are likely to be involved in the formation of intramolecular disulfide bonds. This fact is strengthened by the free cysteine (−SH) detection assay. In its native and denaturing forms, LzaBBI showed a single free cysteine. However, under reducing conditions it showed six free cysteine residues (Figure 4), a marked characteristic of the Bowman−Birktype inhibitors,39 which might contribute to their high stability to extremes of pH and temperatures.8,38−40 Indeed, LzaBBI is similar to other inhibitors, as it has high inhibitory activity stability even after exposure to boiling water

insect midgut proteases, leading to larval development delay. In addition, in vitro studies showed that PIs from Peltophorum dubium (PDTI) and Glycine max (SBTI) specifically induce apoptosis in human leukemia Jurkat cells18 and antiangiogenic effects in gastric and colorectal adenocarcinoma.19 Other different human cancers, diseases, and disorders have been treated in vitro with plant PIs.20 Altogether these experimental findings suggest that PIs have potential applications in human health and agriculture. PIs are often classified according to the class of protease they inhibit: serine protease, cysteine protease, aspartic protease, metalloprotease, and threonine protease inhibitors.7 However, the most widely studied is the serine protease inhibitors, which are grouped into 18 families based on their primary and threedimensional structures, and mechanism of inhibition.21 Furthermore, plant serine protease inhibitors are divided into Kunitz, Bowman−Birk, Potato I, Potato II, and Squash families based mainly on their primary structure.22,23 Generally, Bowman−Birk inhibitors are proteins of 8−10 kDa and have seven disulfide bonds and two independent reactive domains, one for trypsin and the other for chymotrypsin.24 Luetzelburgia auriculata (Allemao) Ducke is a plant native to northeastern Brazil and belongs to the Fabaceae family.25 Herein, we report the purification and characterization of a Bowman−Birk serine protease inhibitor from L. auriculata seeds (LzaBBI), which besides having trypsin and chymotrypsin inhibitory activity also inhibits the growth of the human pathogenic bacteria S. aureus by forming pores in the cell membrane and altering its permeability.

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RESULTS AND DISCUSSION Generally protease inhibitors are resistant to heat treatments at high temperatures with insignificant loss of inhibitory activity.26 Therefore, heat treatments have been successfully used as the initial purification step of a protease inhibitor26,27 to get rid of unwanted proteins and other compounds. For example, Klomklao et al.26 found that heat treatment of the mung bean seed extract at 90 °C for 10 min resulted in increased specific trypsin inhibitory activity, leading to 6.46fold purification. In our work, heat treatment of the seed crude extract at 98 °C for 20 min, as the first step of LzaBBI purification, increased 1.1-fold the specific trypsin inhibitory activity (Table 1). Another common step for purification of trypsin protease inhibitors is the use of affinity chromatography on immobilized trypsin,28,29 which remains active and can interact with the trypsin inhibitor, although it can also partially promote hydrolysis of the inhibitor attached to the affinity column.30 To avoid hydrolysis, we used immobilized anhydrotrypsin, a catalytically inactive trypsin derivative, coupled to a Sepharose 4B matrix to further purify LzaBBI. Affinity chromatography of the heat-treated L. auriculata seed protein extract on Sepharose 4B-anhydrotrypsin fractionated it 1498



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100 °C.41,42 The antitrypsin inhibitors from fava beans, peanuts, and cereals43 also exhibited lower thermal stability compared to LzaBBI. With regard to pH, both the antitrypsin and antichymotrypsin activities of LzaBBI were similar in stability and resistance to denaturation at different pH ranges (Figure 5c and d), like the inhibitors purified from E. contortisiliquum, Poecilanthe parvif lora, and Vigna mungo.40,41,44 However, under reducing conditions (1 and 10 mM dithiothreitol, DTT) the antitrypsin inhibitory activity of LzaBBI decreased by 38% and 100%, respectively, after 120 min of treatment (Figure 6a), whereas its antichymotrypsin activity was less affected, with a 33% decrease after incubation with 10 mM DTT (Figure 6b). These results reinforce the presence of disulfide bonds in the LzaBBI molecule and that they are more important for the trypsin inhibitory activity compared to the chymotrypsin inhibitory activity. Several studies have reported on the importance of disulfide bonds for stability of the 3D structure of protease inhibitors39 and resulting inhibitory activity. The inhibition mechanism by which LzaBBI inhibits the trypsin and chymotrypsin activities is of mixed type, as Km and Vmax are both affected (Figure 7a and b). Apparently, besides binding to both enzymes to form the EI (enzyme−inhibitor) complex that next binds to the substrate (S) to form EIS, LzaBBI also interacts with the ES (enzyme−substrate) complex previously formed to subsequently generate EIS.45 However, of biotechnological importance, the effectiveness of inhibitors like LzaBBI, which displays a mixed-type kinetic pattern, is not affected by higher substrate concentrations, contrary to competitive inhibitors that can be displaced from the enzyme active site by an adequately high substrate concentration. Moreover, LzaBBI has dissociation constants (Ki) of 0.86 × 10−3 and 1.20 × 10−3 μM for trypsin and chymotrypsin, respectively (Figure 7c and d), which indicate higher affinity for trypsin in comparison to chymotrypsin. For instance, low Ki values were also reported for trypsin and chymotrypsin inhibitors from Vigna mungo (0.3 and 10.7 μM, respectively) and Dolichus bif lorus (0.04 and 0.48 μM, respectively).32,44 Reports on the antibacterial activity of serine protease inhibitors within the Bowman−Birk family, are scant. Here, relevantly, we report on the purification of a serine protease inhibitor with an MIC and an MBC of 23.1 × 10−4 and 92.5 × 10−4 μM, respectively (Table 3). At sub-MIC concentrations, 2.9 × 10−4 and 5.8 × 10−4 μM, inhibition of S. aureus growth was still observed (Figure 8). S. aureus is a human pathogenic bacteria resistant to commercially available antibiotics that has great ability to acquire antibiotic resistance.46 Regarding the antibacterial mechanistic approach, changes in ROS production, permeabilization, and pore formation in the bacterial cell membrane after exposure to LzaBBI were observed. Scanning electron microscopy analysis revealed that whereas the control cells remained intact and evenly shaped (Figure 9a), LzaBBI-treated S. aureus cells showed several alterations on the cell surface (Figure 9b−f) that appears corrugated (Figure 9c), showing small protruding bubbles (Figure 9c), dents (Figure 9d), holes (Figure 9e,f), and deep craters (Figure 9e,f), in addition to lysed cells (Figure 9e,f), indicative of severe damage (Figure 9). Damage to the S. aureus cell membrane was confirmed by the antibacterial assay in the presence of the fluorescent nuclear marker propidium iodide (PI), which gained access to the S. aureus interior, as fluorescence was observed only in LzaBBI-treated (Figure

Figure 1. (a) Affinity chromatography of the seed crude extract of L. auriculata previously boiled at 98 °C for 20 min and loaded on a Sepharose 4B-anhydrotrypsin column. Peak A1 was eluted with 0.05 M Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl (Tris-7.5 buffer). Peak A2 was eluted with 0.05 M glycine-HCl buffer, pH 2.2, containing 0.5 M NaCl. (b) Reverse-phase chromatography of peak A2 on a μRPC C2/C18 ST 4.6/100 column, equilibrated with 0.1% (v/v) trifluoracetic acid (TFA) in 2% (v/v) acetonitrile (eluent A). LzaBBI was purified after application of a linear gradient (0−100%, v/ v) of B solution composed of 0.1% (v/v) TFA in 80% (v/v) acetonitrile and Milli-Q grade water. (c) Molecular mass of native LzaBBI assessed after molecular exclusion chromatography on a Saphacryl S-200 HR column equilibrated and eluted with 0.05 M Tris-HCl buffer, pH 7.5, containing 0.3 M NaCl. Molecular markers: β-amylase, 200 kDa; alcohol dehydrogenase, 150 kDa; BSA, 66 kDa; carbonic anhydrase, 29 kDa; cytochrome c, 12.4 kDa. Ve and Vo are respectively the elution and void volume (volume of the stationary liquid contained in a column).

(98 °C) for different periods (Figure 5a and b) and pH changes (Figure 5c and d). Such stability is particularly important for future biotechnology applications. Actually, LzaBBI is a more stable serine protease inhibitor compared to the ECTI inhibitors from Enterolobium contortisiliquum and Acacia conf use, which lost their inhibitory activity against chymotrypsin after 10 and 60 min exposure, respectively, at 1499



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Figure 2. (a) Electrophoresis (SDS-PAGE, 15% v/v) of the protein fractions obtained following the steps used to purify LzaBBI: Lane M, molecular mass markers; lane 1, crude extract (CE) of L. auriculata seeds; lane 2, CE after boiling at 98 °C for 20 min; lane 3, A2 protein fracion obtained after affinity chromatography of the boiled CE on a Sepharose 4B−anidrotripsina affinity column; lanes 4 and 5, purified LzaBBI in the absence and presence, respectively, of dithiothreitol (DTT). Lanes 1−3, lane 4, and lane 5 were loaded with 15, 5, and 10 μg of protein, respectively. (b) Native electrophoresis (PAGE, 15% v/v) staining (Schiff reagent) for glyprotein detection: Lane 1, purified LzaBBI; lane 2, positive control for glycosylated protein (fetuin); lane 3, negative control for glycosylated protein (SBTI). (c) Two-dimentional electrophoresis (SDS-PAGE, 15% v/v) of LzaBBI for pI determination.

Figure 3. Inhibitory activity of LzaBBI toward serine (trypsin, chymotripsin, elastase) and cysteine (bromelain, papain) proteases. Bars indicate the standard deviation of triplicate determinations. Figure 4. Determination of LzaBBI free sulfhydryl content in native, denaturing (10% SDS), and reductive (10 mM DTT) conditions using the DTNB reagent. Bars indicate the standard deviation of five replications.

10b), but not in control cells (Figure 9d). PI is a membraneimpermeable fluorescent dye and interacts only with DNA after penetration into cells with damaged plasma membranes (Figure 9e,f). DCFH-DA was used as an indicator of intracellular ROS for cells treated with LzaBBI. After 30 min of incubation with DCFH-DA, the bacterial cells were in

contact, or not (control), with LzaBBI for 5 h. It was observed that the cells treated with LzaBBI exhibit induction of ROS,

Table 2. NH2-Terminal Amino Acid Sequence of LzaBBI Compared with Sequences of Similar Proteins

a

BLAST search performed on NCBI. bPlant species: Luetzelburgia auriculata; Cicer arietinum; Abarema acreana; Lablab purpureus; Amburana cearensis; Glycine max; Phaseolus vulgaris. (*) Identical amino acid residues. (:) Amino acid residues not identical, but classified within the same group. 1500



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Figure 5. Thermal (a, b) and pH (b, c) stability of LzaBBI inhibitor activity (antitrypsin and antichymotrypsin) after incubation at 98 °C for different time points and different pHs, respectively. Bars indicate the standard deviation of triplicate determinations.

a highly recognized mechanism for antimicrobial activity of several molecules.47 Compromised membranes lead to electrolyte imbalance between the bacterial cytoplasm and environment, resulting in bacterial death. Several studies have reported that other protease inhibitors have a mode of action similar to LzaBBI,46 such as the trypsin/chymotrypsin inhibitor (TP-1) from Solanum tuberosum, which had strong antibacterial activity against Clavibacter michiganense subsp. Michiganinse, a Gram-positive bacteria, and also antifungal activity against both Candida albicans and Rhizoctonia solani. The cell wall structure of Gram-positive bacteria, which is composed of lipoteichoic acid (LTA) and peptidoglycan (PepG)48 units, was porous with dimensions in the range of 40−80 nm.49 Considering the molecular mass of LzaBBI (17.3 kDa) to upload into a tool that estimates protein size (http://www.calctool.org/CALC/ prof/bio/protein_size), LzaBBI is 3.62 nm in diameter. This indicates that LzaBBI could cross the Gram-positive bacterium cell wall and reach the cell plasma membrane, where it might interact and disturb its surface structure (Figure 9f), because LzaBBI possesses 52.17% hydrophobic amino acid residues in its NH2-terminal sequence (Table 3). Therefore, it is plausible to suggest that upon contact with the S. aureus cell membrane surface, LzaBBI interacts with the membrane phospholipid bilayer and forms transbilayer pores or channels, according to the Carpet model, in which the protein acts in a detergent-like manner.50 For example, two AMPs (Cn-AMP2 and CnAMP3) purified from coconut water exhibited growth inhibitory activity against the Gram-positive bacteria B. subtilis and S. aureus by interacting with their respective plasma membranes.51 This is the first report on the purification and characterization of a serine protease inhibitor belonging to the Bowman−Birk family from the mature seeds of L. auriculata (LzaBBI), a leguminous plant. The results show a potent in

Figure 6. Residual inhibitory activity (antitrypsin and antichymotrypsin) of LzaBBI after incubation with 0.5, 1, and 10 mM DTT. Bars indicate the standard deviation of triplicate determination.

indicated by high green fluorescence (Figure 10f), when compared to the control cells (Figure 10h). Oxidative stress is 1501



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Figure 7. Kinetics of the inhibition reaction of trypsin (a) and chymotrypsin (b) by LzaBBI, assesed using the Lineweaver−Burk plot. The inhibition constants (Ki) of LzaBBI for trypsin (c) and chymotrypsin (d) in the presence of two different BApNA (1.2 and 2.5 mM) and BTpNA (1.2 and 1.5 mM) substrate concentrations, respectively, assessed using the Dixon plot.

Table 3. MICa and MBCb of LzaBBI against S. aureus

a

LzaBBI concentration (mM)

MIC

MBC

2.9 × 10−7 5.8 × 10−7 11.5 × 10−7 23.1 × 10−7 46.2 × 10−7 92.5 × 10−7

Yes Yes Yes No No No

Yes Yes Yes Yes Yes No

Minimal inhibitory concentration. tration.

b

antibiotic-resistant bacteria S. aureus. Moreover, it is suggested that the antibacterial effect of LzaBBI is primarily due to oxidative stress by ROS production and cell membrane damage that leads to cell lysis. Therefore, LzaBBI is a powerful candidate for the development of a new drug that may help reduce hospital-acquired infections caused by S. aureus.

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EXPERIMENTAL SECTION

General Experimental Procedures. LzaBBI was purified from the heat-treated L. auriculata seed extract by affinity chromatography on Sepharose 4B-anhydrotrypsin, followed by reverse-phase chromatography on a C2/C18 matrix. LzaBBI purity, molecular mass, subunit composition, and the presence or absence of disulfide bridges were assessed by polyacrylamide gel electrophoresis under denaturing (SDS-PAGE) and reducing conditions (DTT). The isoelectric point, native molecular mass, and NH2-terminal amino acid sequence of LzaBBI were determined by electrofocusing, exclusion chromatography on Saphacryl S-200 HR, and Edman degradation on a Shimadzu PPSQ-10 automated protein sequencer, respectively. Kinetic analysis, thermal and pH stability, antibacterial activity, and mode of action of LzaBBI by using scanning electron microscopy (SEM) and fluorescence microscopy were also carried out. Materials. Mature seeds of L. auriculata (Allemao) Ducke were collected from the ground under trees growing in the semiarid region of the State of Ceara, Brazil. Acrylamide, azocasein, bovine serum albumin (BSA), bis-acrylamide, chymotrypsin (type I-S, 58 units mg−1 protein), Nα-benzoyl-L-arginine p-nitroanilide (BApNA), N-benzoylL-tyrosine p-nitroanilide (BTpNA), 2,7-dichlorofluorescein diacetate (DCFH-DA), propidium iodide, 5,5-dithiobis(2-nitrobenzoic acid) (DTNB), bovine pancreatic trypsin (type I, 10,000 BAEE units mg−1 protein), sodium dodecyl sulfate (SDS), and other reagents for polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Sigma-Aldrich Ltd. Hexamethyldisilazane (HMDS) was purchased from Electron Microscopy Science, and the other

Minimal bactericidal concen-

Figure 8. S. aureus growth curve in the absence (control) and presence of different LzaBBI concentrations.

vitro antibacterial activity of LzaBBI at low concentrations and strongly indicate its potential use to control the multiple 1502



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Figure 9. Scanning electron micrographs of S. aureus cells not treated (a) or treated with LzaBBI (5.8 × 10−4 μM) observed after 1 (b), 2 (c), 3 (d), 4 (e), and 5 (f) h contact. chemicals were of analytical grade and commercially acquired. S. aureus (ATCC 25923), a Gram-positive bacterium culture, was obtained from the Laboratory of Plant Toxins at the Biochemistry and Molecular Biology Department, Federal University of Ceara, Fortaleza, Brazil. Protein Extraction and Inhibitor Purification Procedures. L. auriculata seeds were peeled, ground to a fine powder in an electric coffee grinder, and defatted with n-hexane in the proportion of 1:3 (m/v) at room temperature (22 ± 2 °C), with three solvent changes. The soluble proteins were extracted from the defatted powder (5 g) with 0.15 M NaCl at 1:40 (m/v) ratio for 30 min, under stirring, at 4 °C. The homogenate was filtered through one layer of muslin cloth and centrifuged at 18000g, 20 min, 4 °C (high-speed refrigerated centrifuge, Hitachi, Tokyo, Japan,), and the supernatant, designated crude extract (CE), was recovered. The CE was boiled at 98 °C for 20 min, cooled in an ice bath, and centrifuged at 12000g for 20 min at 4 °C (high-speed refrigerated centrifuge, Hitachi, Tokyo, Japan). The supernatant was saved and dialyzed against 0.05 M Tris-HCl buffer, pH 7.5, containing 0.5 M NaCl (Tris-7.5 buffer) (1:10 volume supernatant: buffer) under gentle stirring, with four changes of the buffer every 3 h within a 12 h period. Aliquots were used to test for the inhibitory activity against trypsin and chymotrypsin and for chromatography on an affinity matrix (Sepharose 4B-anhydrotrypsin, 6.5 × 2.1 cm) previously equilibrated with the Tris-7.5 buffer. The nonretained proteins (A1) were eluted with the equilibration buffer, and the retained proteins (A2) were eluted with 0.05 M glycine-HCl buffer, pH 2.2, containing 0.5 M NaCl, at a 60 mL h−1 flow rate. Fraction A2 was pooled, dialyzed against Milli-Q grade water, lyophilized, and applied on a C2/C18 column (μRPC C2/C18 ST 4.6/ 100, GE Healthcare, Uppsala, Sweden) coupled to an HPLC system

(PU-2089s, Jasco). The C2/C18 column was equilibrated with 0.1% (v/v) trifluoracetic acid (TFA) in 2% (v/v) MeCN (eluent A) and Milli-Q grade H2O. A2 was dissolved (1:100, m/v) in eluent A, centrifuged at 12000g for 10 min at 4 °C, and applied (50 μL) on the C2/C18 column. Chromatography was done using a linear gradient with the eluent B (0.1% [v/v] TFA in 80% [v/v] MeCN and Milli-Q grade H2O) from 0 to 100% (v/v), at 30 mL/h−1 flow rate. The protein peaks eluted were monitored at 230 nm, frozen (−80 °C) after collection, lyophilized, and stored at −20 °C for later analysis. Protein quantification was done as previously described,52 using BSA (Sigma) as the standard protein. Determination of Protease Inhibitor Activity. Trypsin and chymotrypsin inhibitor activities were determined by measuring the residual enzyme activity toward BApNA (pH 7.5) and BTpNA (pH 8.0) substrates, respectively. Trypsin and chymotrypsin (10 μL, at 0.3 mg mL−1), solubilized in 0.05 M Tris-HCl buffer, pH 7.5 and 8.0, respectively, containing 0.02 M CaCl2 (Tris-CaCl2 buffer), were preincubated for 10 min at 37 °C with 100 μL of test samples and 690 μL of the respective Tris-CaCl2 buffer to allow enzyme and inhibitor interaction. Enzyme reactions were initiated by addition of 500 μL of the trypsin substrate BApNA (1.25 × 10−3 M) prepared in 1% (v/v) DMSO and 500 μL of the chymotrypsin substrate BTpNA (1.00 × 10−3 M) prepared in 20% (v/v) DMSO, both completed to 1300 μL with the respective Tris-CaCl2 buffer. After 15 min at 37 °C, reactions were stopped by adding 120 μL of 30% (v/v) HOAc. In the control samples, the inhibitor source was added after HOAc. The enzymatic hydrolysis of BApNA and BTpNA was evaluated by recording the absorbance readings at 410 nm.53 Results are shown as the percent of residual enzymatic activity with respect to control activity. The ability of the purified inhibitor (LzaBBI) to inhibit other serine (porcine 1503



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4000g, using molecular mass cutoff membranes of 3 kDa. After inhibitor recovery, 10 μL of samples was mixed with 190 μL of DTNB working solution and incubated for 5 min, at 37 °C, in 96-well microplates. Absorbance readings at 412 nm were recorded, and the number of free sulfhydryl groups was determined based on the molar extinction coefficient value57 of 13 600 M−1 cm−1. The DTNB working solution was prepared by mixing 20 μL of 1 M Tris-HCl, pH 8.0, 160 μL of distilled H2O, and 10 μL of DTNB stock solution (50 mM NaOAc in distilled H2O containing 2 mM DTNB). Polyacrylamide Gel Electrophoresis and Molecular Mass Determination. The purity of the fractions that showed Bowman− Birk inhibitor activity was checked by SDS-PAGE58 carried out in a Hoefer miniVE Vertical electrophoresis system (Amersham Pharmacia Biotech, San Franciso, CA, USA). Samples were prepared by dilution (1:3, v/v) in the sample buffer (0.5 M Tris-HCl, pH 6.8, containing 1% [m/v] SDS and 0.1% [m/v] bromophenol blue) in the presence or absence of 3.8% (m/v) DTT and boiled at 98 °C, for 5 min. The samples were loaded on the 3.5% (m/v) stacking gel and fractionated in the 15% (m/v) separating gel. Electrophoresis was performed at 20 mA per gel. Protein bands were revealed with Coomassie Brilliant Blue G-250,59 and the apparent molecular mass of LzaBBI was determined (GelAnalyzer 2010a software) by comparison with standard molecular markers (phosphorylase b, 97 kDa; bovine serum albumin, 67 kDa; ovalbumin, 45 kDa; carbonic anhydrase, 29 kDa; soybean trypsin inhibitor, 20.1 kDa; and α-lactalbumin, 14.2 kDa). The molecular mass of native LzaBBI was assessed by molecular exclusion chromatography on a Saphacryl S-200 HR column (HiPrep 16/60, GE Healthcare Life Sciences) coupled to an Ä KTAprime Plus system (GE Healthcare Life Sciences). The column was equilibrated and eluted with 0.05 M Tris-HCl buffer, pH 7.5, containing 0.3 M NaCl, at a 0.8 mL/min flow rate. To determine the void volume (Vo), blue dextran (2000 kDa) was applied and then the standard proteins (ß-amylase, 200 kDa; alcohol dehydrogenase, 150 kDa; BSA, 66 kDa; carbonic anhydrase, 29 kDa; cytochrome c, 12.4 kDa) at the concentrations recommended by the supplier (Sigma-Aldrich, Inc., USA). Samples were run individually on the column, eluted with the equilibration buffer, 1.6 mL fractions were collected, and absorbance readings were taken at 280 nm to build the calibration curve by plotting the logarithm of the molecular mass of the standard proteins against the ratio of the respective elution volume (Ve) and Vo (Ve/Vo). LzaBBI was applied on the column as above, its Ve measured, and the molecular mass calculated using the linear equation obtained. Evidence of the glycoprotein nature of LzaBBI was estimated after electrophoresis by specific Schiff’s reagent staining.60 Fetuin and soybean trypsin inhibitor (SBTI) (both at 10 μg of gel) were used as positive and negative control, respectively. LzaBBI was loaded (10 μg) concomitantly on the gel, and the electrophoresis run as described above. After electrophoresis, the gel was fixed in 12% (v/v) TCA for 30 min, washed with Milli-Q grade water, and transferred and kept incubated with the Schiff’s reagent for 50 min in the dark (4 °C). The gel was washed (three times) with 0.5% (m/v) potassium metabisulfite and visualized. Isoelectric Point Determination. To determine the isoelectric point, 15 μg of LzaBBI were solubilized in 250 μL of the rehydration buffer (8 × 103 mM urea, 1.0 × 103 mM thiourea, 10% [v/v] glycerol, 2% [m/v] CHAPS, 2% [v/v] IPG buffer pH 3−10, and 0.001% [m/v] bromophenol blue). An immobilized pH gradient polyacrylamide gel strip (IPG, 13 cm), pH 3−10 (GE Healthcare), was incubated with LzaBBI for 14 h, and the isoelectric focusing (IEF) was performed in an Ettan IPGphor II system (Amersham Bioscience) using the following schedule: 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, and a gradient from 4000 V up to 18 000 V/h total, exposed to 50 μA continuous electric current, at 20 °C.16 After IEF, the strip was incubated in the reducing solution (50 mM Tris-HCl, pH 8.8, containing 30% [v/v] glycerol, 6 × 103 mM urea, 2% SDS [m/v], 2% [m/v] DTT, 0.1% [m/v] bromophenol blue) for 15 min under gentle stirring. After reduction, the strip was contacted with alkylating solution (50 mM Tris-HCl, pH 8.8, containing 30% [v/v] glycerol, 6 × 103 mM urea, 2% SDS [m/v], 2.5% [m/v] iodoacetoamide, 0.1%

Figure 10. Fluorescence microscopy of S. aureus cells not treated (c, d and g, h) and treated (a, b and e, f) with LzaBBI (5.8 × 10−4 μM) for 5 h and subsequently incubated with propidium iodide (a−d) and DCFH-DA (e−h). elastase) and cysteine (bromelain) proteases was assessed using azocasein as substrate.53 For analysis of papain (cysteine protease) inhibition, BANA was used as substrate.54 Automated NH2-Terminal Sequence Analysis. The NH2terminal sequence of LzaBBI was performed on a Shimadzu Co PPSQ-10 automated protein sequencer (Kyoto, Japan) by Edman degradation. Phenylthiohydantoin amino acid derivatives were detected at 269 nm after separation on an RP-HPLC C18 column (4.6 × 2.5 mm) under isocratic conditions according to the supplier’s instructions. The query sequence was used in the BLASTP search tool against the NCBI database to identify similar sequences with other trypsin inhibitors.55 Determination of Free Sulfhydryl Groups. This was done for native, denatured, and reduced LzaBBI as previously reported,56 using the Ellman’s reagent 5,5-dithiobis(2-nitrobenzoic acid) (DTNB). The assay was performed with 200 μM LzaBBI dissolved in 0.15 M NaCl. For reduced LzaBBI, the native inhibitor was treated with 10 mM DTT for 2 h and absorbance readings were recorded. Next, SDS (10%, v/v) was added, and absorbance readings were taken again. DTT removal was achieved by molecular exclusion chromatography on a Saphacryl S-200 HR column (HiPrep 16/60, GE Healthcare Life Sciences, Uppsala, Sweden) coupled to an Ä KTAprime Plus system (GE Healthcare Life Sciences). The column was equilibrated with 150 mM NaCl and eluted with the same equilibration solution. Pooled column eluates were concentrated by ultrafiltration (Vivaspin protein concentrator spin columns, GE Healthcare Bio-Sciences, USA) at 1504



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that prevents the visible growth of S. aureus incubated in 96-well polypropylene plates. The MBC was determined after broth microdilution by subculturing samples (5 μL) from the wells in Petri dishes containing the nutrient agar medium. MBC corresponded to the lowest LzaBBI concentration yielding negative subcultures after incubation at 37 °C for 24 h.64 Antibacterial Assay. This was performed in microplates against the human pathogenic bacterium S. aureus as previously described.65 One hundred microliters of the culture medium (MHB) containing the bacterial cells (0.1 absorbance unit, at 600 nm) was incubated with 100 μL of LzaBBI sub-MIC (1.4 × 10−4, 2.9 × 10−4, 5.8 × 10−4 μM protein) at 37 °C. Absorbance readings at 630 nm were taken at 1 h intervals, for up to 5 h, using an automated microplate reader (Epoch microplate spectrophotometer, Bio Tek Instruments, USA). The negative control (100% growth) consisted of the bacterial cells incubated in the presence of 100 μL of Tris-7.5 buffer, and the positive control (100% inhibition) in the presence of 100 μL 10% (v/ v) formaldehyde. SEM Analysis. To assess the morphological effect of LazBBI on S. aureus, SEM was carried out using a modified method based on Kozlowska et al.66 The samples analyzed were the same used as those for the antibacterial assays. After 5 h treatment of the bacterial cells with 5.8 × 10−4 μM LzaBBI in Tris-7.5 buffer, the suspension was centrifuged for 5 min at 3.000g, 25 °C, and the supernatant discarded. The pellet was fixed with 200 μL of 1% (v/v) glutaraldehyde/4% (v/ v) formaldehyde in 0.15 M Na3PO4 buffer, pH 7.2, for 5 h at room temperature (22 ± 2 °C). The bacterial cells were recovered by centrifugation as above, washed three times with the Na3PO4 buffer, and finally fixed with 0.2% (m/v) OsO4. The samples were dehydrated in a graded EtOH series (30%, 1×; 70%, 1×; 100%, 3×) for 10 min per change. Next, samples were dried with EtOH/ HMDS (50:50, v/v) for 10 min, followed by 100% HMDS for a further 10 min. The solution was poured off, and the samples were transferred to coverslips previously treated with 0.1% (m/v) gelatin prepared in sterile H2O. The samples were air-dried in a cabinet, and the slides assembled on stubs and metalized with gold particles. The images were obtained with an Inspect-50 FEI scanning electronic microscope (FEI Company, Hillsboro, OR, USA). Cell Membrane Permeabilization Assay. S. aureus cell suspensions (Absorbance at 620 nm = 0.400) were obtained as previously described,65 and 100 μL was incubated with 5.8 × 10−4 μM LzaBBI or Tris-7.5 buffer (negative control) at 37 °C for 5 h. After incubation, 0.4 μL of 0.001 M propidium iodide was added, and the suspension gently stirred for 1 h at 37 °C. Cell suspension drops were deposited on microscope glass slides, covered with coverslips, and observed under a fluorescence microscope (Eclipse 80i, Nikon, Japan).67 Reactive Oxygen Species (ROS) Detection. DCFH-DA staining was used as an indicator of intracellular ROS generation47 induced by LzaBBI. S. aureus cells were incubated or not (control) with LzaBBI (5.8 × 10−4 μM) dissolved in Tris-7.5 buffer for 5 h and recovered after three washings with the above buffer under low-speed centrifugation (2000g, 5 min, 25 °C). Next, they were incubated with 10 μM DCFH-DA at 37 °C for 30 min and recovered as previously mentioned. Cell suspension drops were then deposited on microscope glass slides, covered with coverslips, and observed under a fluorescence microscope (Eclipse 80i, Nikon, Japan). Statistical Analysis. The results were compared by one-way ANOVA, and significance between means was determined by the Tukey test (p ≤ 0.05) using the Assistat software (version 7.7 beta).68

bromophenol blue [m/v]) for 15 min under gentle stirring. The second dimension (SDS-PAGE) was performed on a vertical electrophoresis system (Hoefer SE 600 Ruby, Amersham Bioscience). The strip was placed on the top of a 15% gel that was initially submitted to 15 mA for 30 min and next to 25 mA up to the end of the run. The gel was stained with Coomassie Brilliant Blue G-250.59 The image was acquired using ImageScanner (Armesham Bioscience), and pIs were determined using the ImageMaster 2D Platinum 6.0 software (GE Healthcare). Kinetic Analysis of LzaBBI Inhibition Interaction. To determine both the inhibition mechanism and inhibition constants (Ki) of LzaBBI, the Lineweaver−Burk double-reciprocal (1/V vs 1/ [S], where V is the reaction velocity and [S] the substrate concentration) and Dixon plots (1/V vs [I], where [I] is the inhibitor concentration) were used, respectively. Assays were performed using increasing LzaBBI (2.3 × 10−3, 3.4 × 10−3, 4.6 × 10−3, and 5.8 × 10−3 μM) and BApNA (0.5, 0.7, 1.0, 1.2, and 2.0 mM) concentrations for the kinetics of trypsin inhibition and increasing BTpNA (0.5, 0.7, 1.0, 1.2, and 1.5 mM) concentrations for the kinetics of chymotrypsin inhibition. Both bovine trypsin and chymotrypsin were used at 0.15 mg mL−1 fixed concentration. Reactions were performed as previously described (Protease Inhibitor Activity subsection). The reciprocal of the enzyme reaction rate was expressed as 1/v (OD410 nm, h−1 mL−1 of the reaction medium), and Ki values were calculated from the intersection of the two lines plotted for two different BApNA (1.2 and 2.5 mM) and BTpNA (1.2 and 1.5 mM) concentrations in the Dixon plot.61 The Lineweaver−Burk plots were obtained by the reciprocal of the rate of the enzymes reactions (1/v) vs the reciprocal of the substrate concentrations (1/[S]), in the absence and presence of LzaBBI. Analysis of the Thermal and pH Stability of LzaBBI. Aliquots (0.6 mL for each temperature) of LzaBBI were incubated at 98 °C (boiling temperature) for 10, 20, 30, 40, 50, 60, 90, and 120 min in a water bath. After each incubation period, the samples were cooled to 4 °C. After centrifugation (12000g, 10 min, 25 °C), 100 μL of each heat-treated sample (0.1 and 0.8 μg of LzaBBI for trypsin and chymotrypsin, respectively) was used for the inhibitor assay53 performed in triplicate for control and sample tests, as previously described (Protease Inhibitor Activity subsection). The effect of pH on the LzaBBI stability was performed according to Gomes et al.,62 with modifications. LzaBBI aliquots (1.0 mL) were dialyzed (1:100 volume sample:buffer) under gentle stirring with four changes of the buffer every 3 h within a 12 h period, at 4 °C, against the following buffers, all at 0.05 M concentration: glycine-HCl, pH 2 and 3; NaOAc, pH 4 and 5; K3PO4, pH 6; Tris-HCl, pH 7 and 8; and glycine-NaOH pH 9, 10, and 11. Then, 0.1 and 0.8 μg of LzaBBI per 100 μL of each buffer were used for trypsin and chymotrypsin inhibitor assay,53 respectively, carried out in triplicate, as described above (Protease Inhibitor Activity subsection). Analysis of the LzaBBI Stability to DTT. The methodology described by Garcia et al.40 was employed to evaluate the LzaBBI activity stability in the presence of the reducing agent DTT. Samples were incubated with different DTT concentrations (1, 10, and 100 mM) at 37 °C. Aliquots were taken after 15, 30, 60, and 120 min of treatment, and iodoacetamide (2, 20, and 200 mM, respectively) was immediately added. For every time interval, a control sample without DTT was performed. After treatments, the samples were tested for inhibition of trypsin and chymotrypsin as previously described (Protease Inhibitor Activity subsection). Preparation of the Bacterium Inoculum. It was prepared in accordance with the Cockerill et al. recommendation.63 The absorbance at 600 nm of the S. aureus cell suspension, cultured for 16 h in the Mueller-Hinton broth (MHB) medium (Himedia, India), was adjusted to 0.08−0.1 absorbance unit (105−106 CFU mL−1) and used in the assays. Analysis of the MIC and MBC of LzaBBI. The MIC was determined following the broth microdilution method63 on 96-well polypropylene plates. Different LzaBBI concentrations, from 2.9 × 10−4 to 92.5 × 10−4 μM, were incubated with the bacterial cells for 20 h at 37 °C. The MIC was taken as the lowest LzaBBI concentration

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*Tel: +55 (85) 33669823. Fax: +55 (85) 33669789. E-mail: [email protected] (Jose. T. A. Oliveira). ORCID

Jose T. A. Oliveira: 0000-0003-1207-1140 1505



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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support by the National Council for Scientific and Technological Development (CNPq, grant nos.v308107/2013-6 and 483097/2013-6) and the Council for Advanced Professional Training (CAPES) and the technical support in the scanning ́ from UFC electron microscopy analysis from Central-Analitica (CAPES, CT-INFRA, MCTI-SISNANO, Pro-Equipamentos), Brazil. T.F.M. acknowledges a Master grant from CAPES.

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A Bowman–Birk Inhibitor from the Seeds of Luetzelburgia auriculata

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