Protease inhibitors from plants: Biotechnological insights with emphasis on their effects on microbial pathogens

Patrícia M.G. Paiva, Emmanuel V. Pontual, Luana C.B.B. Coelho and Thiago H. Napoleão Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, 50670-420, Recife,

Pernambuco, Brazil.

The chapter reports structural characteristics and biotechnological applications of proteinaceous protease inhibitors with emphasis on their antifungal and antibacterial activities. Protease inhibition occurs through the formation of a complex between an enzyme and the inhibitory molecule which can interfere in several biological processes such as inflammation, apoptosis, blood clotting and hormone processing pathways. These molecules have been reported as part of defense mechanism of plants against fungal and insect attack. In general, protease inhibitors are able to affect fungi by inhibiting extracellular and/or intracellular proteases that display important roles in nutrition and infection processes since the invasion of host tissue and fungal development depends on the degradation of membrane and/or cell wall proteins. Antifungal trypsin inhibitors may also act directly at level of fungal cell membrane. Protease inhibitors have also been reported as antibacterial agents. This property has been attributed to inhibition of bacterial proteases involved in several physiological processes as well as to interaction between the inhibitor and the cell wall or proteins from plasma membrane leading to changes in cell permeability and inducing the death of bacteria. This chapter also presents methodologies used for evaluation of antifungal and antibacterial activities of several samples, including protease inhibitors. The remarkable ability to affect fungi and bacteria growth stimulates the evaluation of using protease inhibitors in strategies to control microorganisms pathogenic for human and plants.

Keywords protease inhibitor; antifungal activity; antibacterial activity.

1. Protease inhibitors

Protease inhibition occurs through the formation of a complex between an enzyme and an inhibitory molecule, which can be proteinaceous or not. This enzyme-inhibitor complex (EI) has a decreased catalytic potential or is not able to hydrolyze the substrate. Protease inhibitors can interfere in several biological processes – such as inflammation, apoptosis, blood clotting and hormone processing pathways – by modulating the activity of proteases [1]. Protease inhibitors have already shown biotechnological potential as antitumor, insecticidal and antimicrobial agents [2-7]. Proteinaceous inhibitors bind to enzyme active site through a structural portion named reactive or inhibitory site; in a competitive inhibition the enzyme is blocked and cannot bind the substrate [8,9]. The contact between inhibitor and proteinase is complementary and several interactions occur between the active site of enzyme and the segment in inhibitor polypeptide chain containing the reactive site [10]. It is common that the EI formation produces conformational alterations in inhibitor molecule, including rotation of side chains, and little movement in principal enzyme chain [11-13]. EI is established fast and usually its dissociation occurs slowly in free enzyme and unmodified or modified inhibitor [10]. A specific nomenclature for amino acid residue positions in the reactive and active sites is employed to describe the interaction between inhibitor and protease. This system uses S and P letters for residues in enzyme active site and inhibitor reactive site, respectively. According to inhibitor amino acid position, in relation to amino and carboxy-terminal sides of the scissile bond, the nomenclature P and P’ is used, respectively (Figure 1).

Fig. 1 Interaction between the side-chains of amino acid residues (P) of a peptide substrate (or an inhibitor) and the subsites (S) of the enzyme active site. P1 residue usually determines the specificity of the enzyme. The inhibitor specificity is in general defined by the residue at P1 position, which also confers resistance to hydrolysis.

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The inhibitor specificity is in general defined by the residue at P1 position; however, it has been observed that amino acid substitutions in another position interfere in inhibitory property. For example, variation at P2’ position gave marked differences in trypsin inhibitory potency of several Bowman-Birk inhibitors [14]. Different residues at P5’ position in Torresea acreana and Torresea cearensis inhibitors conferred distinct kinetic patterns of human plasmin inhibition; comparison of their primary sequences revealed the same amino acid residue at P1 position but differences in the carboxy-terminal charge of the molecules. The T. cearensis and Dioclea glabra… factor XII [15, 16].

2. Families of plant protease inhibitors

Plant protease inhibitors have been classified into the families Arrowhead, Bowman-Birk, Kunitz, Potato I, Potato II, Cereal, Rapeseed and Squash [10]. Bowman-Birk and Kunitz families are the most well studied and these inhibitors have been mainly isolated from leguminous plants. Bowman Birk inhibitors (BBI) are highly stable proteins containing disulfide bridges, which contribute to their stability, although the removing of one bridge does not necessarily results in structural modifications. BBI contains two reactive sites that bind two different proteases or two molecules of the same enzyme; the first reactive site generally inhibits trypsin while the second site inhibits proteinases of different specificities [8,17,18]. BBI from dicotyledonous have shown high amino acid sequence homologies [19]; this fact probably is related to their in vivo functional importance. For example, in interactions between BBI and insect trypsins the conserved lysine at the inhibitor P1 position confers resistance to hydrolysis and inhibitory effect. On the other hand, some trypsins appear to have been adapted to resist BBIs such as lepidopteran trypsins, which are able to induce BBI hydrolysis [20]. Usually, leguminous seeds contain BBI multiple molecular forms that may arise due to protein post-translational modification. For example, isoinhibitors formed due to BBI self-association properties were found in Medicago scutellata [21] and pea seeds [22] by formation of electrostatic intermolecular interactions and hydrogen bonds plus hydrophobic interactions, respectively. The Kunitz inhibitors are proteins with molecular mass from 18 to 22 kDa containing little residues of cysteins and only one binding site for trypsin [23]. Major and Constabel [24] worked with five genes for production of Kunitz trypsin inhibitors from plants belonging to Populus genus. The proteins produced were active toward trypsin, chymotrypsin and elastase (all serineproteases).

3. Purification and biotechnological potential of protease inhibitors A variety of conventional chromatographic methods have been described to obtain purified protein inhibitors and generally the isolation protocols involve a combination of them, resulting in procedures that demand long time. Alternatively, to fast purification, affinity chromatography on columns containing an immobilized enzyme have been used to start the inhibitor purification procedure since this method usually provides very high yields and purification folds. Trypsin inhibitors have been isolated by binding to columns of immobilized trypsin (Figure 2); the inhibitor-trypsin complex is dissociated by loading a buffer of low pH that promotes alterations in amino acid ionizations at the enzyme binding site.

Fig. 2 Schematic representation of isolation of a trypsin inhibitor by affinity chromatography. (1) The column containing a matrix composed by trypsin immobilized on an inert support is usually equilibrated with Tris buffer at pH between 7.5 and 8.5. (2) Sample loading; the sample should be in the equilibration buffer. (3) In washing step using the equilibration buffer, molecules without affinity for trypsin are eliminated and the trypsin inhibitor adsorbs on the matrix. (4) In elution step, trypsin inhibitor molecules are usually desorbed and recovered using a buffer solution at a low pH value (usually between 2.0 and 3.0).

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Columns containing immobilized inhibitor can be used for enzyme purification. Two trypsin-like proteinases from the lepidopteran Sesamia nonagrioides were isolated through affinity chromatography on benzamidine-Sepharose 6B column [25]. Proteinaceous inhibitors can be used for enzyme characterization. For example, two hydrolytic activities on N-benzoyl-DL-arginine-p-nitroanilide (trypsin-like activity) of crude extracts from digestive tracts of four tropical fishes (Caranx hippos, Pseudupeneus maculatus, Sparisoma sp. and Hoplias malabaricus) were differently inhibited by two trypsin inhibitors suggesting distinct enzyme structures [26]. Also, the trypsin-like activity of alkaline proteinase isolated from intestine of Nile tilapia (Oreochromis niloticus) was identified using different trypsin inhibitors [27]. Isolated inhibitors have several biotechnological applications. Drug delivery systems can be obtained by covalent attachment of EDTA and proteinaceous enzyme inhibitors on polymers aiming to protect embedded therapeutic peptides from degradation by metallo and serine proteinase enzymes. An insulin delivery system constituted of tablets containing hormone and chitosan-EDTA-BBI conjugate was evaluated in vitro using a fluid containing trypsin, chymotrypsin and elastase; the formulation avoided the total insulin degradation by pancreatic serine proteases [28, 29]. Plant natural defense to insect attack involves the expression of protein inhibitors against insect gut proteinases and the search by biopesticides has stimulated the study of the interaction between plant protease inhibitors and insect proteases. When ingested by insects, protease inhibitors can persist in the insect body and cause severe deleterious effects such as digestion and absorption impairment and decrease in the availability of essential amino acids which usually results in death by starvation. The use of trypsin inhibitors to control populations of several pest insects such as Callosobruchus maculatus, Spodoptera litura, Achaea janata, Anthonomus grandis, Helicoverpa armigera and Ephestia kuehniella has been investigated [30]. Koblinski et al. [31] reported that protease inhibitors have shown antitumor activity being able to block the ability of tumor cells in forming metastasis. Some inhibitors are also used in the treatment of acquired immunodeficiency syndrome (AIDS) and arterial hypertension being administrated as monotherapy or associated with other agents, such as inhibitors of reverse transcriptase. Other studies have demonstrated the efficacy of these molecules as antiprotozoal drugs and agents for treatment of diabetes mellitus and Alzheimer’s disease [4].

4. Antifungal activity of protease inhibitors

Protease inhibitors have been reported as part of defense mechanism of plants against fungal attack. In general, protease inhibitors are able to affect fungi by inhibiting extracellular and/or intracellular proteases that display important roles in nutrition and infection processes since the invasion of host tissue and fungal development depends on the degradation of membrane and/or cell wall proteins. Antifungal proteins with trypsin inhibitor activity may also act directly at fungal membrane level [2, 32-35]. Figure 3 schematizes some effects of protease inhibitors on fungi.

Fig. 3 (A) Schematic representation of fungal mycelia emphasizing the roles of extracellular and intracellular proteases (B) Antifungal activity of protease inhibitors may be linked to the inhibition of extracellular or intracellular proteases involved in mycelia growth. Insets show spore germination (1) and inhibition of spore germination (2).

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Evidences of the involvement of protease inhibitors on plant resistance to fungi have been documented. Analysis of cDNA from Nicotiana tabacum revealed a dramatic expression of a Kunitz protease inhibitor (NtKTI1) through the whole plant but preferentially in roots and stems. The NtKTI1 showed strong antifungal activity against Rhizoctonia solani and moderate effect on growth of Rhizopus nigricans and Phytophthora parasitica var. nicotianae [36]. A 14-kDa trypsin inhibitor from maize is reported to be associated with host resistance to aflatoxin production by Aspergillus flavus; this inhibitor was also able to reduce the activity and extracellular production of a glucosidase (α-amylase) from A. flavus [37]. Trypsin inhibitor from maize, expressed in Escherichia coli, inhibited the conidial germination and the growth of Fusarium moniliforme hyphae suggesting its participation in the plant defense mechanism [38]. A cystatin (proteinaceous inhibitor of cysteine protease) from sugarcane, also expressed in E. coli, was able to inhibit the growth of the filamentous fungus Trichoderma reesei and microscopy analysis suggested that this inhibitor acted directly on the proteases of fungi, decreasing germination and impairing the normal development of hyphae [34]. Trypsin inhibitor from Pseudostellaria heterophylla demonstrated antifungal activity toward Fusarium oxysporum similar to aprotinin, a bovine peptide with inhibitory activity on several proteases [39]. Three isoforms of a serineprotease inhibitor from Acacia plumosa seeds blocked the growth of Fusarium moniliforme, Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp. [35]. The trypsin inhibitor from Psoralea corylifolia seeds also impaired the growth of Fusarium oxysporum, Alternaria brassicae, Aspergillus niger and Rhizoctonia cerealis [3]. The germination of the phytopathogenic fungi Sclerotinia sclerotiorum was inhibited after exposure to the trypsin inhibitor from Helianthus annuus flowers [2]. Trypsin-chymotrypsin inhibitor from Solanum tuberosum strongly inhibited the pathogenic fungi Candida albicans and Rhizoctonia solani; this inhibitor, which showed 62% homology with protease inhibitors from Kunitz family, was not able to induce lysis of human erythrocytes constituting an initial evidence of safety for use as antimicrobial agent [40]. Different methods have been developed in order to identify antifungal agents. For example, the method described by Wang and Ng [39] is a simple assay which enables easy visualization of fungal growth inhibition. The method (schematized in Figure 4) consists in transferring a mycelia disk (around of 0.6 cm diameter) to the center of a petri dish containing potato-dextrose-agar medium. Next, sterile filter papers (around of 0.6 cm diameter) should be soaked with 20 μL of the test sample or the control solution. Four disks are placed in each dish, one corresponding to the control and three containing different concentrations of the test sample. The disks are placed approximately 1 cm distant from the mycelia disk and equidistant from each other. After assembly the experiment, the plates should be incubated at 28 °C for at least 72 h and then the formation or not of inhibition rings around the paper disks containing the test sample should be recorded.

Fig. 4 Antifungal assay. (A) A mycelial disk is placed on the center of a petri dish containing culture medium. After sterile disks of filter paper are soaked with control solution or test sample at different concentrations and disposed approximately 1 cm distant from the mycelia disk and equidistant from each other. (B) After incubation a positive result (antifungal activity) is evidenced by formation of a zone of growth inhibition around the paper disk. If the sample does not have antifungal activity the mycelia grows bypassing the filter paper, similar to what happens for control.

Pontual et al. [41] detected the Moringa oleifera flower trypsin inhibitor (MoFTI) in extract obtained by homogenization of flowers with distilled water and showed its ability to block the activity of bovine trypsin from commercial origin (inhibition constant, Ki, of 0.38 nM) and the trypsin-like activity (Ki of 0.6 nM) found in an extract from gut of fourth-stage larvae of Aedes aegypti. The authors also showed that MoFTI was involved in the larvicidal activity of the flower extract. Antifungal activity of MoFTI was evaluated against Fusarium species using the assay schematized in Figure 4. Fusarium are phytopathogens responsible for damaging economically important plants, such as tomato, corn, potato, banana, bean and cocoa; these fungi produce toxins, affect the permeability of cell membranes and may disrupt plant metabolism [42]. MoFTI at concentrations of 10, 20 and 40 μg was not an antifungal agent on F.

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solani since it was not observed the formation of inhibition rings around the paper disks containing the inhibitor. MoFTI also did not affect the growth of F. moniliforme, F. lateritium, F. oxysporum f.sp. lycopersici, F. oxysporum f.sp. cubensis and F. poae. The absence of growth inhibitory effect of MoFTI on Fusarium does not exclude the possibility of antifungal effect on other fungal species. Trypsin inhibitor isolated from Clausena lansium seeds showed antifungal activity toward Physalospora piricola but not affected Mycosphaerella arachidicola, Botrytis cinerea, Fusarium oxysporum or Coprinus comatus [43].

5. Antibacterial activity of protease inhibitors

Protease inhibitors have been reported as antibacterial agents and this biological activity has been attributed to inhibition of bacterial proteases involved in several physiological processes as well as interactions between the inhibitor and the cell wall or proteins from plasma membrane leading to changes in cell permeability and inducing the death of bacteria [44-46]. Figure 5 shows possible effects of trypsin inhibitors on bacteria.

Fig. 5 (A) Schematic representation of bacteria cell. (B) Antibacterial activity of protease inhibitors may be linked to inhibition of bacterial proteases, interactions between the inhibitor and the cell wall or membrane proteins leading to leakage of cellular contents. Figure 6 shows the scheme of disk diffusion assay that is used as a screening method for determination of antibacterial activity. Fistulin, a protease inhibitor from Cassia fistula leaves, was active by this method against Bacillus subtilis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli; the assay revealed the formation of zones of growth inhibition (clear area which was devoid of bacterial growth) ranging from 12 to 18 mm around paper disks containing the purified inhibitor [47]. Rakashanda et al. [48] reported that the protease inhibitor from seeds of Lavatera cashmeriana was an antibacterial agent on K. pneumoniae and P. aeruginosa (zones of growth inhibition of 10 and 12 mm, respectively) but did not affect significantly the E. coli growth (inhibition zone of 3 mm).

Fig. 6 Schematic representation of disk diffusion assay for evaluation of antibacterial activity. (A) A bacterial culture is sowed in petri dish containing culture medium with agar. (B) Next, disks of filter paper soaked with control solution or different concentrations of test sample are placed over the culture medium. (C) After incubation bacterial colonies will grown throughout the dish and formation of zones of growth inhibition will be observed if the sample exerted antibacterial effect.

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Another methodology widely employed to detect antibacterial agents allows the determination of minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations of the test sample (Figure 7). This method consists in incubating (37 ºC, 24 h) different concentrations of a test sample (180 μl in liquid culture medium) in the wells of a microtiter plate containing a bacterial inoculum (20 μl). After incubation time, the optical density at 490 nm (OD490) should be measured and the MIC value is determined as the lowest concentration at which is observed a ≥50% reduction in optical density relative to the control well OD490 [49]. MBC can be determined from the MIC assay. For this, aliquots (10 μl) from each well of the treatments that were found to inhibit bacterial growth should be transferred to a petri dish containing a medium with agar and incubated for 48 h at 37 °C. MBC will correspond to the lowest concentration showing no bacterial growth, which could be different from MIC.

Fig. 7 Schematic representation of assay for determination of minimal inhibitory (MIC) and minimal bactericidal (MBC) concentrations. The assay was schematized in quadruplicate (A, B, C and D plate lines). Plate columns 1, 2 and 3-12 correspond to blank (only culture medium), control (bacteria and culture medium) and tested sample concentrations, respectively. Satheesh and Murugan [50] reported that the protease inhibitor from Coccinia grandis leaves showed antibacterial activity by killing or inhibiting the growth of S. aureus (MBC of 1.2 mg⁄mL; MIC of 1 mg⁄mL), B. subtillis (MBC of 1.25 mg⁄mL; MIC of 1 mg⁄mL), K. pneumoniae (MBC of 0.5 mg⁄mL; MIC of 0.01 mg⁄mL), E. coli (MBC of 1 mg⁄mL; MIC of 0.63 mg⁄mL) and Proteus vulgaris (MBC of 0.5 mg⁄mL; MIC of 0.2 mg⁄mL). The authors suggested that the inhibitor may have formed a channel on cell membrane and thus the cell death resulted of the out flowing of cellular contents. Moura et al. [51] showed that a protein preparation from M. oleifera flowers containing the inhibitor MoFTI was active against Gram-negative (Escherichia coli, Proteus mirabilis and Salmonella enteritidis) and Gram-positive

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(Bacillus subtilis, Enterococcus faecalis and Staphylococcus aureus) bacteria. The preparation was most active on E. coli (MIC of 0.018 mg⁄mL; MBC of 0.297 mg⁄mL) which is the best indicator of water fecal contamination as well as inhibited the growth of bacteria from natural environment water. These findings led authors to conclude that the flower preparation has potential for use in disinfection of contaminated water. Ngai and Ng [52] showed that a napin-like polypeptide with trypsin inhibitor activity isolated from Brassica chinensis seeds inhibited the growth of Pseudomonas fluorescens with a concentration that inhibits bacterial growth by 50% (IC50) of 66 µM. This polypeptide also inhibited the growth of Mycobacterium phlei (IC50 of 146 µM), Bacillus subtilis (IC50 of 236 µM), Bacillus cereus (IC50 of 222 µM), and Bacillus megaterium (IC50 of 215 µM). Protease inhibitors from other organisms have also shown antibacterial effect. Soares et al. [53] reported the antibacterial activity of an elastase inhibitor from hemocytes of Lasiodora sp. (EILaH), which was active against Enterococcus faecalis (MIC of 227.5 µg/ml) but did not inhibit the growth of B. subtilis, S. aureus, E. coli and K. pneumoniae. According to the authors, EILaH probably act in the protection of Lasiodora against infection by E. faecalis, a pathogen of arthropods.

Acknowledgments The authors express their gratitude to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for research grants and fellowships (L.C.B.B. Coelho and P.M.G. Paiva), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) and Ministério da Ciência, Tecnologia e Inovação (MCTI) for research grants. E.V. Pontual would like to thank FACEPE for graduate and post-doctoral scholarships.

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