The acidic latex protein from Hevea brasiliensis serves as an anionic antimicrobial peptide

Bacterial strains and media

The bacterial strains utilized in this study, namely Bacillus cereus, Escherichia coli, Staphylococcus aureus and Salmonella typhi, were maintained as glycerol stocks at −80 °C. Prior to use, the bacterial strains were cultured on 1.5% (w/v) Luria-Bertani (LB) agar (Difco™ & BBL™, Sparks, USA) at 37 °C. Phytophthora palmivora, was isolated and cultured on 1.5% (w/v) potato dextrose agar (PDA) (Difco™ & BBL™, Sparks, USA) at 25 °C for the chitinase activity experiment.

The colloidal chitin for the chitinase detection medium was prepared following the method outlined by Roberts & Selitrennikoff (1988) as follows: Chitin flakes sourced from shrimp shells (Himedia™, Maharashtra, India) were hydrolyzed using 37% (w/w) cold HCl and stirred at 4 °C overnight. Subsequently, the mixture was filtered, and extraction was carried out in 95% cold ethanol at 4 °C overnight. The resulting chitin pellet, obtained through centrifugation, was washed with deionized water until the pH reached 7.0 and stored at 4 °C until use. The chitinase detection medium was prepared by combining 0.3 g MgSO₄•7H₂O, 3.0 g (NH₄)₂SO₄, 2.0 g KH₂PO₄, 1.0 g C₆H₈O₇•H₂O, 15 g agar, 200 µL Tween-80, 4.5 g colloidal chitin, and 0.15 g bromocresol purple in one L of distilled water, adjusting the pH to 4.7, and autoclaving the mixture.

Construction of the expression plasmid

The open reading frame (orf) of Hev b5, obtained from GenBank with accession number NM_001405317.1, was synthesized and ligated into the EcoRI and XhoI sites of the pET-28a(+) expression vector (GenScript, Jiangsu, China), which harbors the coding sequence for a hexa-histidine (6xHis) affinity tag. The resulting expression construct, named pET-28a(+) –Hev b5, was introduced into competent DH5α E. coli cells to enhance the plasmid copy number. Plasmids were then isolated from DH5α E. coli cells using the Plasmid Mini Prep Kit (ver. 2.0) (BioFACT, Daejeon, Korea), following the manufacturer’s instructions. Validation of the plasmids was performed through nucleotide sequence analysis.

Sequence analysis of Hev b5

The nucleotide sequence of an orf was translated into the amino acid sequences of Hev b5 and analyzed using Expasy tools. The potential signal peptide was identified by the SignalP 4.1 program. The protein domain characteristics were verified using the Simple Modular Architecture Research Tool (SMART) 7.0. N- and O-glycosylation sites were predicted using the NetNGlyc 1.0 and NetOGlyc 4.0. The tertiary structure of Hev b5 was modeled using the SWISS-MODEL program and visualized with PyMOL.

Expression and extraction of recombinant Hev b5

For the expression of Hev b5, the plasmids containing pET-28a(+)—Hev b5 were introduced into competent BL21 Star™ (DE3) E. coli cells. A 1 ml volume of the overnight-grown culture was inoculated into 100 ml of LB broth supplemented with kanamycin. The culture was then incubated at 37 °C with agitation at 200 rpm until the optical density at 600 nm (O.D.₆₀₀) reached 0.45, at which point protein expression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) (Vivantis, Selangor darul Ehsan, Malaysia) to a final concentration of 0.5 mM. After induction and overnight incubation, the cells were harvested by centrifugation at 5,000× g for 30 min at 4 °C.

The extraction of recombinant Hev b5 (rHev b5) was performed following the protocol described by Peleg & Unger (2011), with minor modifications to suit the specific conditions of this study. Protein extraction was carried out as follows: bacterial cell walls were disrupted by employing 5 ml of lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole, pH 7.4) per milligram of wet weight, supplemented with 2.5 µg/ml of lysozyme. Additionally, sonication with a microtip was performed using a set amplitude of 25A, alternating between 30-second intervals of activation and deactivation, repeated 5 times. To enhance the quality of the extraction, RNase A and DNase I were added to final concentrations of 10 and 5 µg/ml, respectively, and incubated at 37 °C for 15 min. The cell lysate was then centrifuged at 10,000× g for 20 min at 4 °C. The supernatant was collected, while the pellet was resuspended in solubilization buffer (8 M urea, 0.1 M Tris-HCl, pH 8.0, 10 mM DTT). Subsequently, the soluble fraction and inclusion bodies were dialyzed against refolding buffer (0.1 M Tris-HCl, pH 8.0, 5 mM EDTA, 5 mM cysteine) with stirring at 4 °C overnight.

Purification of rHev b5

The purification process of rHev b5 involved fast protein liquid chromatography (FPLC) (ÄKTA start, Uppsala, Sweden) utilizing a Ni-NTA HisTrap™ FF 1 ml column packed with Ni Sepharose™ 6 Fast Flow, which selectively binds to the 6xHis tag of rHev b5. Binding buffer (20 mM NaH₂PO₄, 0.5 M NaCl, pH 7.4) and rHev b5 were injected through the sample loop at a constant flow rate of 0.5 ml/min. Elution was achieved by applying a gradient of elution buffer (20 mM NaH₂PO₄, 0.5 M NaCl, 500 mM imidazole, pH 7.4), allowing the separation of fractions, each collected at 1 ml/tube. Subsequently, fractions containing rHev b5 were analyzed via 12% SDS-PAGE, and their concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Bio Basic, Markham, Ontario, Canada). The purified rHev b5 band was confirmed by Western blot analysis. Briefly, the protein was separated by 12% SDS-PAGE and electrophoretically transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 10% skim milk in TBS (25 mM Tris-HCl, pH 7.5, 0.5 M NaCl) and incubated with an anti-rHevb5 antibody (or anti-His antibody), produced by GenScript (1:2,500 dilution). After washing the membrane, peroxidase-conjugated goat anti-rabbit IgG (1:20,000 dilution) was added. The target protein band was visualized by a colorimetric reaction using 1-Step™ TMB-Blotting Substrate Solution (Thermo Fisher Scientific, Waltham, MA, USA), and the reaction was stopped by washing with water for 5 min.

Chitinase activity assay

The chitinase activity of rHev b5 was initially assessed using the gel diffusion method on chitinase-selective media described by Agrawal & Kotasthane (2012). The appearance of red-purple diffusion zones on the media, indicated by the color change of bromocresol purple in response to pH variations, served as an indicator of chitinase activity. The chitinase-selective media were spread with 450 µg of rHev b5. Subsequently, P. palmivora, previously isolated and cultivated on 1.5% (w/v) PDA at 25 °C for 72 h, was transferred onto the selective media and further incubated at the same temperature for an additional 72 h. The extent of diffusion zones was documented with photographs, and the data were presented in terms of the annular radius value of the diffusion zone (mm), reflecting the red-purple coloration on the selective media. As controls, the experiment was repeated using 50 mM Tris-HCl, pH 8.0, and 1x Anti-Anti as negative controls, while 0.25 mg BSA was used as the internal control group.

The sample, standard, control, and blank tubes were prepared according to the chitinase activity assay protocol provided by Abbexa, ensuring that each tube contained a reaction volume of 80 µl. A volume of 80 µl of substrate was added to the sample and control tubes, and the contents were mixed thoroughly. The tubes were then incubated at 37 °C for 1 h and centrifuged at 5,000× g for 10 min at 4 °C. After centrifugation, 80 µl of the supernatant was transferred to new tubes and mixed with 40 µl of reaction buffer. The mixtures were heated in boiling water for 7 min, and the tubes were subsequently centrifuged at 5,000× g for 2 min. A volume of 80 µl of the resulting supernatant was transferred into each well of a microplate and mixed with 120 µl of dye reagent solution. The plate was incubated at 37 °C for 1 h. The absorbance was then measured and recorded at 585 nm. Chitinase activity was calculated, where one unit of chitinase activity is defined as the amount of enzyme required to produce one µg of N-acetylglucosamine per hour at 37 °C.

Fungal growth inhibition assay

To evaluate the inhibitory potential of rHev b5 on fungal growth, P. palmivora, a common pathogenic fungus, was used in this study following a method adapted from Sowanpreecha & Rerngsamran (2018). rHev b5 was serially diluted two-fold to final concentrations of 70, 35, 17.5, 8.75, and 0 µM in 50 mM Tris-HCl buffer, pH 8.0 with a total volume of one ml, before being combined with 4 ml of PDB. P. palmivora, pre-cultured on 1.5% (w/v) PDA at 25 °C for 120 h, was then introduced into each tube. Fungal growth was assessed by measuring the optical density at 600 nm following a 120-hour incubation at 25 °C. A parallel experiment was conducted using 50 mM Tris-HCl buffer, pH 8.0 without rHev b5, serving as the negative control.

To assess the inhibitory effect of rHev b5 on fungal growth, P. palmivora, a common pathogenic fungus, was used in this study. The procedure was adapted from Hendricks, Christman & Roberts (2017). Briefly, rHev b5 was prepared at concentrations of 70 and 35 µM in 50 mM Tris-HCl (pH 8.0) and spread (100 µL) onto 1.5% (w/v) potato dextrose agar (PDA). P. palmivora, pre-cultured on 1.5% PDA at 25 °C for 7 days, was inoculated at the center of the plate using a No. 5 cork borer. The plates were then incubated at 25 °C for 2 and 5 days, and fungal growth inhibition was assessed by measuring radial inhibition (Ra) and capturing images. A parallel experiment was conducted using 50 mM Tris-HCl buffer (pH 8.0) without rHev b5 as the negative control. The percentage of growth inhibition was calculated using the following formula:  ${\displaystyle \text{\%}\mathrm{I}=\left[\left({\mathrm{Ra}}_{\mathrm{c}}-{\mathrm{Ra}}_{\mathrm{t}}\right)/{\mathrm{Ra}}_{\mathrm{c}}\right]\times 100.}$ where Ra_c is the mean colony diameter in the control group (assuming circular growth), and Ra_t is the mean colony diameter in the test group (Astiti & Suprapta, 2012).

Liquid growth inhibition assay

To assess the basic inhibitory capability of rHev b5 against bacteria (Zheng et al., 2023), both Gram-positive bacteria such as B. cereus and S. aureus, and Gram-negative bacteria such as E. coli and S. typhi were diluted 1:100 in LB broth during the mid-logarithmic phase for the experiment. The rHev b5 solution, at a concentration of 5.00 mg/ml, was serially diluted two-fold in a 96-well plate containing 50 mM Tris-HCl, pH 8.0, with a final volume of 100 µl in each well. Subsequently, 100 µl of the previously prepared bacterial suspension was added to each well. The plate underwent incubation at 37 °C with continuous agitation at 200 rpm, and bacterial growth was assessed every hour over a period of 16 h by measuring absorbance at 600 nanometers using the SPECTROstar Nano absorbance reader (BMG LABTECH, Ortenberg, Germany). LB broth served as the control group and underwent the same experimental conditions described above.

Colony counting method

The colony counting method, adapted from Scilletta et al. (2021), was employed to evaluate the antibacterial efficacy of rHev b5 at a specific time point. Mid-logarithmic phase cultures of both Gram-positive bacteria (B. cereus, S. aureus) and Gram-negative bacteria (E. coli, S. typhi) grown in LB broth were diluted at a ratio of 1:100 and then incubated with 70 nmol of rHev b5 at 37 °C for 12 h, with agitation at 200 rpm. Subsequently, the culture media were collected after specified durations and subjected to 10-fold serial dilution with 50 mM Tris-HCl, pH 8.0 until an appropriate dilution was achieved for subsequent measurements. Eventually, the bacterial suspensions were spread plated on 1.5% (w/v) LB agar and incubated at 37 °C for 16 h. The bacterial colonies within the range of 25 to 250 (Tomasiewicz et al., 1980) were counted and expressed as colony forming units per milliliter (CFU/ml). LB broth and ampicillin were employed as representatives of the negative and positive control groups, respectively.

Bacterial binding activity of rHev b5

To verify that rHev b5 functions as an antimicrobial peptide through binding with microorganisms, the bacteria-binding assay described by Huo et al. (2024) was modified and conducted to suit the requirements of this study. S. aureus and S. typhi were selected to represent Gram-positive and Gram-negative bacteria, respectively, and were cultured. Then, 10 micrograms of rHev b5 was incubated with microorganisms (1 × 10⁶ cells) in TBS buffer containing 50 mM CaCl₂ or ZnCl₂ at room temperature for 20 min with rotation. The cells were pelleted by centrifugation at 10,000× g for 1 min, and the supernatant was collected as the S fraction. The resulting pellet was washed three times with TBS buffer, and the supernatant from these washes was designated the W fraction. The bound protein was eluted with 7% SDS (E fraction). All samples were mixed with SDS sample loading buffer and heated at 100 °C for 2 min. Finally, the binding ability of rHev b5 to microorganisms was analyzed by 12% SDS-PAGE and western blotting with an anti-rHevb5 antibody. To confirm the contribution of divalent cations to the antibacterial activity of rHev b5, TBS without 50 mM CaCl₂ or ZnCl₂ was also tested.

Statistical analysis

The chitinase activity and colony counting assays were conducted in triplicate, while liquid growth inhibition was performed in duplicate. Results were reported as the mean ± standard deviation. Statistical analysis of the colony counting assay was carried out using one-way ANOVA with IBM SPSS Statistics version 29.0.2.0. Duncan’s multiple range tests were used to assess significant differences between mean values, with statistical significance set at p < 0.05.

Sequence characterization of Hev b5

The orf sequence, including a stop codon, of the acidic protein Hev b5 from H. brasiliensis under accession numbers NM_001405317.1, DQ306732.1, U51631.1, and U42640.1, showed 100% identity over 456 bp. This sequence encoded a peptide of 151 amino acids. SignalP 4.1 prediction indicated the absence of a signal peptide. The protein had a predicted isoelectric point (pI) of 4.06 and a molecular mass of 16.09 kDa.

SMART analysis revealed that the protein comprises two low complexity regions: the first region starts at position 17 and ends at position 82, and the second region starts at position 90 and ends at position 151. These regions may form surfaces for interaction with phospholipid bilayers. Both low complexity regions are rich in glutamic acid (E). Additionally, SCOPe analysis identified the d2ae2a_ domain, which starts at position 61 and ends at position 102, as being involved in classes comprising alpha and beta proteins.

The tertiary structure of Hev b5 was constructed using the SWISS-MODEL algorithm and viewed with the PyMOL program (Fig. 1). The protein was aligned with the major latex allergen Hev b5 under accession number Q39967.1, which was built using the AlphaFold v2 method. The model quality was assessed using the GMQE score, which was 0.57. The tertiary structure comprised long random coils separated by a very short strand of α-helix.

Expression, extraction, and purification of rHev b5

The recombinant Hev b5 orf protein, fused with a 6xHis tag at the N-terminus, was successfully produced in E. coli BL21 Star (DE3) following IPTG induction, as evidenced by the prominent blue band at approximately 40 kDa in lane 2 of Fig. 2. After protein extraction, the rHev b5 bands were shown to be expressed in both the soluble and insoluble fractions (Fig. 2, lanes 3 and 4). However, only the soluble fraction was then purified using a Ni-NTA column and dialyzed to remove imidazole, resulting in purified rHev b5 with a molecular mass of 36 kDa (Fig. 2A, lane 5). Thus, the putative rHev b5 with His-tag was calculated to be 19.91 kDa, which does not match the estimated molecular mass based on its deduced amino acid sequence.

The anti-Hev b5 antibody, produced using a specific peptide of Hev b5, confirmed that the expressed bands in lanes 2 and 3, as well as the purified rHev b5 in lane 5, correspond to rHev b5 (Fig. 2B, lane 5). However, a discrepancy was noted in the molecular weight of the band: the SDS-PAGE gel showed a band at 36 kDa, while the western blot result displayed a band at 54 kDa. This difference was further validated using an anti-His antibody (Fig. 2C, lane 5). Both the anti-Hev b5 and anti-His antibodies detected a single protein band of the same size, confirming the identity and integrity of the rHev b5 protein.

Chitinase activity assay

The qualitative assay of chitinase activity demonstrated that rHev b5 exhibited low chitinolytic activity when screened on solid medium supplemented with 0.45% colloidal chitin as the sole carbon source, resulting in a purple color (Fig. 3A). Weak activity was achieved by 450 µg of the protein which shown as light purple. This observation was made in comparison with a positive control, which also displayed a very dark purple color, affirming the presence of moderate chitinolytic activity. In contrast, the negative control exhibited a yellow color, signifying the absence of chitinolytic activity. Additionally, the quantitative chitinase activity was evaluated using a chitinase assay kit. The results showed that the purified rHev b5 exhibited high chitinase activity at 1,723 U/mg, whereas the cell lysate of rHev b5 displayed only 36.73 U/mg. These findings suggest that rHev b5 is capable of effective enzymatic degradation of chitin, highlighting its chitinase activity in the experimental setup.

Antifungal activity of rHev b5

The antifungal activity of rHev b5 was assessed by monitoring the growth of P. palmivora in PDB. After 5 days, fungal growth was measured by OD600. The results indicated that at 70 µM of rHev b5 (Fig. 3B), the OD was the lowest compared to the control group. However, no significant difference in growth was observed between the 70 and 35 µM of rHev b5 treatments. Additionally, at lower concentrations, P. palmivora growth was comparable to the control, suggesting that these concentrations of rHev b5 did not inhibit fungal growth after 5 days.

Since P. palmivora growing in PDB does not exhibit a homogeneous distribution, measuring OD600 may not clearly reflect differences in growth. Therefore, antifungal activity was further evaluated by observing radial mold growth inhibition. Figure 3C illustrates the radial growth of P. palmivora in the presence of 35 and 70 µM rHev b5 over 2 and 5 days. The results demonstrated that fungal inhibition was dose-dependent. At 2 days, agar plates containing rHev b5 exhibited a low inhibition percentage, ranging from 5.48% to 9.59%. However, by day 5, the inhibition had decreased to 0. 90%–1.35%.

Antibacterial activity using liquid growth inhibition assay

The antibacterial activity of rHev b5 was assessed by measuring bacterial growth in broth media incubated with varying concentrations of rHev b5 over different time intervals. Optical density at 600 nanometers was monitored for 16 h to generate growth curves, as depicted in Fig. 4. The presence of rHev b5 slowed down the growth of both Gram-positive bacteria (B. cereus, S. aureus) and Gram-negative bacteria (E. coli, S. typhi) compared to the control group without rHev b5. The inhibitory effect was most pronounced at a concentration of 70 µM, followed by 35, 17.5, and 8.75 µM, indicating a dose-dependent relationship between rHev b5 concentration and bacterial growth inhibition. Specifically, incubation with 70 µM rHev b5 resulted in sustained inhibition of S. aureus and S. typhi growth over the 16-hour period, similar to the effect observed with 35 µM rHev b5 on S. typhi. However, the inhibitory activity of rHev b5 against B. cereus and E. coli was lower. No apparent antibacterial activity was observed in the control groups.

Antibacterial activity using colony counting method

The bacterial inhibition ability of rHev b5 was confirmed by using the colony counting method (Tasci, 2011). The remaining bacteria after culturing in medium containing rHev b5 were expressed as CFU/ml (as shown in Fig. 5). Comparing the groups with rHev b5 to the negative control group (without rHev b5), a significant decrease in both Gram-positive (B. cereus, S. aureus) and Gram-negative (E. coli, S. typhi) bacteria was observed. Particularly, S. aureus showed the most pronounced reduction with a residual bacterial count of 1.77 × 10³ ± 1.15 × 10² CFU/ml, followed by B. cereus, E. coli, and S. typhi with residual bacterial counts of 3.50 × 10⁵ ± 2.31 × 10⁴, 1.01 × 10⁶ ± 5.51 × 10⁴, and 1.80 × 10⁶ ± 4.16 × 10⁴ CFU/ml, respectively. The positive control group, treated with ampicillin, exhibited the lowest residual bacterial count compared to the other groups. These results indicate that rHev b5 possesses significant bacteria inhibition ability.

Bacterial binding activity of rHev b5

Since rHev b5 possesses antimicrobial properties, its interaction with microorganisms was analyzed by assessing its binding to S. aureus (representing Gram-positive bacteria) and S. typhi (representing Gram-negative bacteria). The results showed that rHev b5 had a binding affinity for both types of bacteria. In the absence of divalent cations, rHev b5 exhibited minimal binding to the microbes. However, in the presence of calcium, its binding to both bacteria was enhanced compared to the ion-free condition (Figs. 6 and 7). Notably, the strongest binding was observed in the presence of zinc. Furthermore, rHev b5 demonstrated a significantly higher binding affinity to Gram-negative bacteria than to Gram-positive bacteria when zinc was present (Figs. 6 and 7).