Antibacterial, antifungal and in vivo anticancer activities of chitin-binding lectins from Tomato (Solanum lycopersicum) fruits

1 Introduction

Lectins are a group of multivalent proteins or glycoproteins that bind cell-surface glycoconjugates to get involved in direct defense against pathogens and immune regulation in various biological systems (Barre et al., 2019; Coelho et al., 2017; Hasan et al., 2019; Souza et al., 2013). Beside their physiological roles, functions of lectins in determining the structures of carbohydrates and elucidating mechanisms of biological phenomena are now well-studied. A number of lectins are being used for performing diagnostic and therapeutic perspectives as well.

Chitin is the biopolymer of N-acetylglucosamine units linked by glycosidic bonds, distributed abundantly within arthropods, fungi, crustaceans and marine organisms. A number of plants and animals possess chitin-binding lectins contributing in their immune defense system against pathogens containing chitin through the stimulation of antimicrobial, anticancer and insecticidal activities. Chitin-binding lectins belong to the hevein family possessing one or more hevein domains. These lectins showed varying binding specificity to LacNAc and chito-oligosaccharides (Itakura et al., 2017).These lectins interact mainly with chito-oligosaccharides moieties in different degrees to bring on antimicrobial effects by various mechanisms. They hamper the growth of bacteria through antibiofilm and antiquorum sensing properties whereas pore formation, changes in cell permeability and disintegration of cell wall are the mechanisms involved in their bactericidal and fungicidal activities, apoptosis and autophagy (Gomes et al., 2012; Hasan et al., 2014a,b, Yao et al., 2010).Chitin-binding lectins or CBLs are widely known for their role in plant defense (Chen et al., 2018). Tomato lectins had been used in histochemical analysis, analyzing structural changes during oncogenesis as well as a tool for drug delivery (Oguri et al., 2008).

N-acetylglucosamine specificity of 74-kDa lectin from Tomato (Solanum lycopersicum) fruits was exploited to purify it by affinity adsorption in 1980 (Kilpatrick, 1980). Nachbar et al also reported the purification of a Tomato lectin (71 kDa) by affinity chromatography at the same year (Nachbar et al., 1980). Two isolectins with a molecular weight of 68 kDa were also purified by chromatofocusing (Kilpatrick et al., 1984). Until then, all the purified lectins were similar in size. Later, bigger lectins (130 and 100 KDa) from tomato fruits were isolated using erythroglycan-sepharose column and affinity chromatography on a chitin column, respectively (Zhu and Laine, 1989; Saito et al., 1996).In recent years, molecular structure and properties of Tomato lectins have been elucidated and it became evident that Tomato lectins are typical chitin-binding lectins, chimeric in nature with at least two chitin-binding domains (Peumans et al., 2003; Jain et al., 2021). They possess a range of molecular weights (45–138 kDa) due to the presence of highly glycosylated extension-like domains (Merkle and Cummings, 1987; Naito et al., 2001; Qin et al., 2003; Oguri et al., 2008).

This study focused on biological activities of Tomato lectins rather than the purification as there is insufficient information about their physiological roles, despite of their long history. Previously, antifungal and antimicrobial activities of two lectins from Lycopersicon esculentum and Solanum integrifolium were evaluated (Roh et al., 2010; Chen et al., 2018). This work reports partial purification of a mixture of chitin-binding Tomato lectins (TCLs) through a modified affinity chromatographic procedure, their toxicity as well as antibacterial and antifungal effects in detail. In addition, in vivo and in vitro antiproliferative activities of TCLs against Ehrlich’s ascites carcinoma (EAC) cells were reported for the first time.

2 Experimental

3 Results

3.1

3.1 Purification and determination of molecular mass of TCLs

Chitin-binding proteins with hemagglutinating activity were isolated through a single-step affinity chromatography using acetylated chitin powder (Fig. 1A). Four major bands with molecular masses ranging from 30 to 115 kDa were found in 16% SDS-PAGE (Fig. 1B, Lane 2). This protein was designated as Tomato Chitin-binding lectins or TCLs.

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Fig. 1

(A) The acetylated chitin powder was packed in a chromatographic column and the column was equilibrated with 10 mM Tris-HCl buffer, pH 8.0.The crude Tomato protein was applied and the column was washed by the same buffer. The proteins were eluted by 0.5 M acetic acid (3 ml/tube) and hemagglutination activity of each tube was determined after dialysis. B. The isolated protein fraction showing hemagglutination activity showed four major bands (115, 76, 40 and 30 kDa) in SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

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3.2

3.2 Toxicity of TCLs against Artemia nauplii

TCLs dose-dependently killed the shrimp nauplii whereas 40% mortality rate was found at a concentration of 200 µg/ml. Mild toxicity of the protein mixture was indicated by an LC₅₀ value of 261.0 μg/ml (Fig. 2).

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Fig. 2

Toxicity of TCLs. Percentages of mortality brine shrimp nauplii treated with different concentrations of TCLs after a 24-hour exposure. Data are expressed in mean ± S. D.; p < 0.05.

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3.3

3.3 Antibacterial activity of TCLs against pathogenic bacteria

After the incubation of Shigella boydii for 8, 16 and 24 h with TCLs, percentages of growth inhibition were calculated to be 2.58%, 6.57% and 4.03%, respectively, comparing to the growth of control bacteria (Fig. 3A). After treating with TCLs for 8, 16 and 24 h, time-dependent growth inhibition was found for Shigella dysenteriae as 5.17%, 3.67% and 2.85%, respectively (Fig. 3B). This trend continued for Staphylpcoccus aureus as well. TCLs inhibited their growth at the rate of 6.84% and 0.51% when treated for 8 and 16 h, respectively, but lost the activity during a 24 h treatment (Fig. 3C). In case of Escherichia coli, TCLs could not inhibit their growth and mitogenic growth (7–9%) was observed instead (not shown in the figure).

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Fig. 3

Growth inhibition of various bacterial species treated with TCLs comparing to their corresponding controls. (A) Shigella boydii (B) Shigella dysenteriae (C) Staphylococcus aureus. ‘A’, ‘T’ and ‘C’ represent antibiotic (15 µg/disc of Ampicillin), TCLs-treated (500 µg/disc) and Control discs, respectively.

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3.5

3.5 Antibiofilm activity of TCLs

Though TCLs could not inhibit the growth of Escherichia coli, it showed antibiofilm activity against the bacteria. The lectin could inhibit the formation of biofilm by 53.32%, 62.62% and 77.29% at concentrations of 250, 500 and 1000 μg/ml (Fig. 4A). In another experiment, formation of biofilm was observed in the test tube containing E. coli bacterial suspension whereas no biofilm was found in the TCLs-treated bacterial suspension (Fig. 4B).

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Fig. 4

(A) Antibiofilm activity of TCLs against Escherichia coli. Data are expressed in mean ± S.D; p < 0.05. (B) Inhibition of biofilm formation in TCLs-treated E. coli suspension.

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3.6

3.6 Antifungal activity of TCLs by disc diffusion method and agglutination of fungal spores

TCLs exerted moderate antifungal activity against the fungi Aspergillus niger by inhibiting fungal growth at a concentration of 600 µg/ml (Fig. 5A). Fungal spores from Aspergillus niger became agglutinated at a concentration of 200 µg/ml of TCLs whereas no agglutination was observed without TCLs (Fig. 5B).

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Fig. 5

Antifungal activity of TCLs against Aspergillus niger. (A) A disc (‘T’) soaked with 600 µg/ml of TCLs inhibited the fungal growth, ‘A’ represents standard antifungal agent (1% Clioquinol + 0.02% Flumetasone Pivalate and ‘C’ is the control disc. (B) Agglutination of fungal spores in the absence (a) and presence (b) of 200 µg/ml of TCLs. Scale bar indicates 50 µm.

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3.7

3.7 Growth inhibition assay of Ehrlich ascites carcinoma (EAC) cells in vivo and in vitro

TCLs could inhibit the growth of EAC cells when treated for five days. Comparing to the untreated or control cells, 19.63% and 59.91% of in vivo cell growth inhibition was observed at low (2 mg/kg/day) and high (4 mg/kg/day) doses of TCLs (Fig. 6A). Proliferation of EAC cells was measured in vitro by MTT assay after treating those with various doses of TCLs. At concentrations 37.5, 75 and 150 μg/ml, percentage of growth inhibition slowly increased from 27.61% to 38.74% and 49.23%, respectively (Fig. 6B).

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Fig. 6

Growth inhibition of EAC cells from mice treated with TCLs. Data are expressed in mean ± S.D (n = 6); p < 0.05. Dose-dependent growth inhibition of TCLs-treated EAC cells cultured in DMEM media for 48 h. Data are expressed in mean ± S.D. (n = 3); p < 0.05.

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3.8

3.8 Observation of morphological changes and nuclear damages of EAC cells through fluorescence microscope

Comparing to control mice, number of EAC cells got reduced in the lectin-treated mice (Fig. 7A). EAC cells from Swiss albino mice treated with TCLs were stained with Hoechst-33342 to observe the morphological alterations. EAC nuclei in cells from the control group were found to be in regular round shape in fluorescence microscope (Fig. 7B) and homogeneously stained with Hoechst-33342. On the other hand, cells from TCLs-treated mice were found to be irregular in shape and exhibited apoptotic morphological changes (e.g., membrane blebbing, cell shrinkage, chromatin condensation, and nuclear fragmentation) when observed in fluorescence microscope (Fig. 7C & D).

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Fig. 7

Observation of morphological features of EAC cells (A) Reduced number of EAC cells in lectin-treated mice (left) comparing to untreated mice (right) (B) EAC cells (stained) from untreated or control group of mice, EAC cells (stained) from mice treated with (B) 1 mg/kg/day and (C) 2 mg/kg/day of TCLs. Scale bar indicates 50 µm.

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3.9

3.9 Isolation of RNA from EAC cells and expression of apoptosis-related genes

Considerable appearance of apoptosis-related genes like Fas, Caspase 8 and Caspase 3 was found. Expression of these genes from EAC cells of TCLs-treated mice were upregulated than those cells from the control mice. Expression of caspase 3 was much stronger than that of caspase 8. Quality of mRNA from lectin-treated and control EAC cells was confirmed through the ample expression of 18S gene (Fig. 8).

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Fig. 8

Expression of apoptosis-related genes. PCR reactions were carried out with primers specific for Fas, Caspase 8, Caspase 3 and 18S whereas products were separated on 1.5% agarose gel and stained with ethidium bromide. L represents DNA ladder; T and C represents RNA from TCLs-treated EAC cells and untreated control EAC cells, respectively.

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4 Discussion

Tomato lectins were actually isolated long ago following various isolation procedures. Physicochemical properties of Tomato lectins were revealed to be parallel to other solanaceae lectins from Solanum tuberosum (Potato) and Datura stramonium (Datura) (Marinkovich, 1964; Allen et al., 1978; Kilpatrick and Yeoman, 1978). These three lectins were similar in structure though both potato and tomato lectins were fusion proteins consisting of Cys-rich and Hyp-rich domains (Kieliszewski et al., 1994; Allen et al., 1996). TCLs with apparent molecular weights of 30, 40, 76 and 115 KDa migrated in the SDS-PAGE as four major bands and were eluted as a mixture of proteins. Previously, mixtures of Potato lectins had been reported to possess antimicrobial and anticancer activities (Hasan et al., 2014a,b). Antifungal property of another mixture of lectins from cherry tomato was also observed (Roh et al., 2010). All these results encouraged us to investigate the antibacterial, antifungal and in vivo anticancer activities of this mixture of Tomato lectins, instead of a purified lectin.

TCLs were found to be mildly toxic with an LC₅₀ value of 261.0 µg/ml against brine shrimp nauplii. Two other solanaceae lectins, STL-S and STL-D, showed more toxic nature against the nauplii with LC₅₀ values 75 and 90 µg/ml, respectively (Hasan et al., 2014a,b). Plant lectins from Momordica charantia, Nymphaea nouchali, Moringa oleifera and Sebastiania jacobinensis possessed varying toxicity (49.7, 120 ± 29, 131 and 715.89 ± 1 µg/ml) (Kabir and Reza, 2014); Kabir et al., 2011; Asaduzzaman et al., 2018; Vaz et al., 2010) whereas a lectin from snake gourd shared very similar LC₅₀ value (261 ± 29 μg/ml) with TCLs (Kabir et al., 2012).

It is already recognized that gram-positive bacteria are surrounded by much thicker layers of peptidoglycan comparing to gram-negative bacteria (Silhavy et al., 2010). Glycan strands in bacterial peptidoglycan are composed of repeating units of N-acetylglucosamine and N-acetylmuramic acids with different degrees of polymerization. As a result, chitin (polymer of N-acetylglucosamine units)-binding lectins can bind and interact with cell surface glycans present in bacterial peptidoglycan and this is probably the reason behind the susceptibility of gram-positive bacteria (S. aureus) towards TCLs comparing to gram-negative bacteria (E. coli and S. boydii). StL-20, a potato lectin, also showed strong antibacterial activity against gram-positive bacteria than gram-negative bacteria (Hasan et al., 2014a,b). Moreover, antibacterial activity of Eugenia uniflora lectin (EuniSL) against gram-positive and gram-negative bacteria was very much in line with the finding of this study (Oliveira et al., 2008). Minimum inhibitory concentration (MIC) values of TCLs against S. aureus and E. coli were 140 and 200 µg/ml, respectively which indicated their potential as antibiotic. Two seed lectins from Archidendron jiringa and Eugenia uniflora showed bacteriostatic activity against S. aureus (with MIC values 56.7 and 1.5 µg/ml). In case of gram negative E. coli bacteria, strong growth inhibition (MIC 16.5 µg/ml) was observed by E. uniflora seed lectin (EuniSL) though A. jiringa seed lectin could not affect their growth (Charungchitrak et al., 2011; Oliveira et al., 2008). Minimum bactericidal concentrations (MBC) of EuniSL against the same bacterial species were 16.5 µg/ml and 180 µg/ml, respectively (Oliveira et al., 2008) whereas for TCLs, MBC values were 600 and 840 µg/ml, respectively. TCLs moderately (53.32% at 250 μg/ml) suppressed the formation of biofilm by E. coli. StL-20 exerted stronger anti-biofilm activity (20% at 80 μg/ml) against P. aeruginosa whereas that activity of AGL (Amaranthus gangeticus lectin) was weaker (37.14% at 250 μg/ml) than TCLs (Hasan et al., 2014a,b; Hasan et al., 2021).

TCLs showed strong antifungal activity against the fungi, Aspergillus niger, at a concentration of 600 µg/ml and agglutinated fungal spores at a concentration of 200 µg/ml. Potent fungal growth inhibition by the lectin from cherry tomato against Cladosporium cucumerinum and Monosporascus cannonballus was also reported (Roh, 2010). Other antifungal lectins from solanaceae family (Solanum tuberosum, Capsicum annuum and Solanum integrifolium) had also been reported (Hasan et al., 2014a,b; Gozia et al., 1993; Kuku et al., 2009; Chen et al., 2018). Another 48 kDa chitin-binding lectin from Setcreasea purpurea rhizome exerted fungicidal activity against a number of fungi but was inactive against Aspergillus niger even at much higher concentration (1.51 mg/ml) (Yao et al., 2010). Strong fungal growth inhibition was also documented by lectins from Archidendron jiringa (Charungchitrak et al., 2011), Phaseoulus vulgaries and Glycine max (Mohsen et al., 2018).

Cancer is a group of diseases identified according to their preliminary affected cell types. Various plant lectins have been shown to have antiproliferative properties against various cancer cell types (Kabir et al., 2016; Kabir and Reza, 2014). Given the lack of H2 histocompatibility antigen, EAC cells are very suitable to check the potentiality of newly synthesized or purified chemopreventive agents because of their proliferative capacity (Chen and Watkins, 1970). In this work, TCLs dose dependently inhibited the growth of EAC cells and its antiproliferative activity (49.23% inhibition of cell growth at a concentration of 150 µg/ml) was higher than potato lectins (36% at 160 µg/ml) (Kabir et al., 2016). Other plant lectins from Keampferia rotunda and Geodorum densiflorum repressed 43.7% and 60% of EAC cell growth in similar (160 µg/ml) concentrations (Ahmed et al., 2017; Kabir et al., 2019). At lower concentrations of 120 µg/ml and 128 µg/ml, anti-proliferative activity of lectins from Pisum sativum and Amaranthus gangeticus varied a lot, giving values like 84% and 33.7%, respectively (Kabir et al., 2013; Hasan et al., 2021). In the in vivo experiment, growth inhibition of EAC cells by TCLs was 59.91% at a dose of 2 mg/kg/day. KRL-2 and Pea lectins were weaker in action 41% and 63% at concentrations 3 and 2.8 mg/kg/day whereas Potato lectins showed stronger effects (83% at 1.38 mg/kg/day) (Ahmed et al., 2017; Kabir et al., 2013; Hasan et al., 2014a,b). So, it can be recommended that TCLs showed moderate anticancer activity comparing to the lectins mentioned here.

Apoptosis is characterized by shrinking of cells, blebbing of plasma membrane, fragmented DNA and nuclear condensation (Procopio et al., 2017). Signs of apoptosis in TCLs-treated EAC cells became evident due to the presence of apoptotic symptoms when compared to control EAC cells . Similar signs of apoptosis in EAC cells have also been induced by other lectins from plants and invertebrates (Kabir et al., 2013; Ahmed et al., 2017; Asaduzzaman et al., 2018; Kabir et al., 2019; Swarna et al., 2019). Fas-mediated pathway is well-reported among several extrinsic pathways of apoptosis. Fas ligands bind with cell-surface death receptors (Fas receptors) to send the ‘First Apoptotic Signal’. Fas associated death domains (FADD) become recruited causing the formation of death-inducing signaling complex or DISC including caspase 8, the initiator caspase. Caspase 8 directly or indirectly cleaves downstream caspases and cell death finally occurs due to the action of effector caspases like caspase 3. Expression of Fas, caspase 8 and caspase 3 genes in the TCLs-treated EAC cells suggested a possible Fas-mediated pathway of their apoptotic death. Involvement of caspase 8 and 3 in apoptosis was previously observed in CNE-1 cancer cells treated by another chitin-binding lectin from Setcreasea purpurea (Yao et al., 2010).

5 Conclusion

From this present study, it became obvious that TCLs were mildly toxic with low chitinase activity. It is demonstrated that TCLs has strong antibacterial and antifungal activities. To dispel biofilm from medical instruments, TCLs may contribute effective roles in clinical microbiology because of their antibiofilm activity. In vitro and in vivo studies indicated that TCLs has also moderate anti-proliferative activities against Ehrlich ascites carcinoma cells. These lectins have the ability to change the morphology of EAC cells by inducing apoptosis. These observations are commencing that TCLs has potent biomedical properties and further investigations are required to explore their chemopreventive properties in more details.

Ethics approval and consent to participate

Ethical clearance of the experiments using Swiss albino mice was provided by the Institutional Animal, Medical Ethics, Bio-safety and Bio-security Committee (IAMEBBC) for Experimentations on Animals, Human, Microbes and Living Natural Sources (Memo No. 102(6)/320/IAMEBBC/IBSc), Institute of Biological Sciences (IBSc), University of Rajshahi, Bangladesh.

Authorship contribution statement

Nawshin Arfin: Methodology, Writing – original draft. Munna Kumar Podder: Methodology, Validation. Syed Rashel Kabir: Methodology, Validation, Writing – review & editing. A.K.M. Asaduzzaman: Methodology, Data curation, Validation. Imtiaj Hasan: Conceptualization, Supervision, Validation, Writing – original draft, review & editing.

Consent for publication

Authors declare consent for publication.

Funding

This study was partly supported by the research grant from Ministry of Science and Technology (Grant No. 39.00.0000.009.14.011.20-BS-231/1567), Government of Bangladesh.