To Extract Lectin from Selected Fungal and Plant Sources and To Evaluate of Their Hemagglutination and antibacterial activities.

Introduction

Proteins are macromolecules composed of one or more chains of amino acids linked together through peptide bonds (21). Among all the diverse classes of proteins “lectin” possess unique characteristic to bind with specific carbohydrate moiety (10).

Lectins occur widely across different forms of life including humans, plants, animals, algae, cyanobacteria, yeasts, mushrooms, and microfungi. (16). In plants, lectins occusrs in various tissues such as such as seeds, leaves, barks, roots, tubers, flower & fruits (8,6). Among fungal organisms, lectins have been reported from several genera including Penicillium sp., Fusarium sp., and Aspergillus sp. (31).

The term “lectin” was first introduced in 1954 by William c. Boyd and was derived from the latin word “legere” meaning “to select” (5). Proteins that possess the ability to agglutinate red blood cells (RBCs) based on definrd sugar specificity are referred to as lectins. with its known specificity of sugar referred to as lectins In cases where the sugar specificity is not yet known, these proteins are often described as hemagglutinins (16). Lectins are generally defined as carbohydrate-binding proteins or glycoproteins that interact specifically with carbohydrate structures through reversible and non-covalent interactions (10,8). Plant lectins that demonstrate hemagglutinating activity are commonly referred to as phytohemagglutinins (9).

Structurally, lectins typically contain at least one non-catalytic domain known as the Carbohydrate Recognition Domain (CRDs) (26). This domain determines the type of carbohydrate molecule that the lectin can recognize and bind. The interaction between the CRD and its target carbohydrate resembles a reversible “lock-and-key” mechanism in which the lectin selectively binds to specific sugar residues. Many lectins are multivalent molecules and may possess multiple CRDs (16). As a result, a single lectin molecule can bind simultaneously to several carbohydrate structures present on different cells or molecules. This multivalent binding results in cross-linking of cells and ultimately leads to visible agglutination or precipitation (25, 24).

Lectins exhibit a wide range of biological properties that make them an important subject of scientific investigation. Various studies have reported that lectin possess antitumor, immunomodulatory and antiinsects, antifungal, antibacterial, anti HIV and antigenic activities, analgesic activity, antioxidant, antiproliferative and many more (24, 12, 27). The biological activity of lectins is largely associated with their ability to recognize and bind specific carbohydrate residues such as mannose, galactose, lactose, N-acetylglucosamine, N-acetylgalactosamine, fucose, and rhamnose (12).

By interacting with carbohydrate structures present on glycoproteins, glycolipids, and polysaccharides located on cell surfaces, lectins participate in numerous biological processes including cell–cell communication, immune responses, and host defense mechanisms (4, 25).

Goldstein et al. stated that lectins are carbohydrate- binding proteins or glycoproteins of non immune origin that agglutinate cells. Among their various properties, hemagglutination is one of the most widely studied property of lectins (5, 26). Hemagglutination occurs when lectins bind to carbohydrate residues located on the surface of erythrocytes. Because lectins are multivalent proteins possessing multiple carbohydrate recognition domains, they can simultaneously bind several red blood cells. This results in cross-linking of erythrocytes and formation of visible aggregates. Due to this property, lectins have been explored as potential alternatives to commercial antisera used in ABO blood group typing. Lectin-based systems offer advantages such as natural origin, lower cost, and ease of production. Consequently, lectins have found important applications in laboratory diagnostics.

In addition to their diagnostics value, lectins have attracted considerable researches attention because of their antimicrobial properties (6). Several lectins have been reported to exhibit antibacterial, antifungal, and antiviral activities (15). These effects are primarily attributed to the interaction of lectins with carbohydrate components present on microbial cell surfaces, including those located on the cell wall and cell membrane. Binding of lectins to these carbohydrate structures may interfere with microbial growth, disrupt membrane integrity, inhibit microbial adhesion, or prevent biofilm formation.

Because of these properties, lectins are increasingly being considered as potential natural antimicrobial agents, particularly in the context of rising antibiotic resistance among pathogenic microorganisms. Despite the considerable progress made in lectin research, many aspects of their biological functions and mechanisms of action remain to be explored.

Therefore, the present study was designed to extract and characterize lectins from selected plant and fungal sources and to evaluate their hemagglutination activity as well as their antibacterial properties. The finding of this study may contribute to the development of lectin- based cost effective diagnostic tools and novel natural antimicrobial compounds.

Materials and Methods

Study Design

This experimental study was designed to extract, partially purify, and characterize lectins obtained from selected plant and fungal sources and to evaluate their biological activities like hemagglutination and antibacterial activity. Hemagglutination assays were used to observe lectin potential in ABO blood group typing, and antibacterial activity against selected bacterial culture.

Biological Materials

Plants including Lablab purpureus, Pisum sativum, and Vigna unguiculata were used. The seeds of these plants were collected from the local market.

Fungal cultures of Aspergillus niger and Penicillium sp. were obtained from the Microbiology Laboratory of Dolat Usha Institute of Applied Sciences.

Fresh fruiting bodies of Mushroom Pleurotus ostreatus were collected from Super Mushroom Farm, Dharampur, and transported to the laboratory for immediate processing.

Human blood samples were collected in EDTA tubes from Riddhi Advance Laboratory, Valsad. Standard clinical procedures were followed for the collection and with the consent. These blood samples (Blood group A, B and O) were used for hemagglutination and blood grouping experiments.

Culture Media and Reagents

Culture media including Potato Dextrose Agar (PDA), Czapek’s Dox broth, Mueller–Hinton agar, and Nutrient broth were used for fungal and bacterial culture. Phosphate buffer saline (PBS, pH 7.2), ammonium sulphate, folin–ciocalteu reagent, alkaline copper reagent, and bovine serum albumin (BSA), normal saline (0.9%) and distilled water were used throughout the experimental procedures.

Equipment

The experiments were performed using standard laboratory equipment present in microbiological lab including a centrifuge, microcentrifuge, incubator, colorimeter (Equiptronics EQ-650A), micropipettes, magnetic stirrer, analytical weighing balance, grinder, and standard laboratory glassware.

Extraction of Lectins

Extraction of lectins from Fungal Cultures

Fungal cultures of Aspergillus niger and Penicillium sp. were initially grown on Potato Dextrose Agar (culture medium) slants at room temperature for 5–7 days to obtain sufficient biomass. Then the developed fungal biomass were transferred into Czapek’s Dox broth, a production medium and incubated for 7–10 days under static condition. After incubation, the production broth was filtered using whatsman filter paper, filtrate was collected, centrifuged at 2000 rpm for 15 minutes, supernatant was used as the crude fungal lectin extract (28, 20).

Extraction of lectin from Pleurotus ostreatus

Fresh fruiting bodies of mushroom Pleurotus ostreatus were washed thoroughly with distilled water. Approximately 20 g of mushroom tissue was taken and cut into small pieces and then homogenized in phosphate buffer saline (PBS). The homogenate was filtered through muslin cloth filtrate was collected, centrifuged at 2000 rpm for 15 minutes, supernatant was used as the crude fungal lectin extract (29,16).

Extraction lectins from Plant Seeds

Plant sources for lectin Lablab purpureus, Pisum sativum, and Vigna unguiculata were collected from market. Peel were removed and seeds are collected. Seed were then washed and ground into a fine paste using a grinder. This paste were mixed in some amount of phosphate buffer. The mixture was filtered through muslin cloth and filtrate was collected, centrifuged it at 2000 rpm for 15 minutes, The supernatant obtained was collected as crude plant lectin extract (8).

Partial Purification of Lectin

Partial purification of lectins was performed by ammonium sulphate precipitation. Solid ammonium sulphate was measured according to the how much lectin had and how much saturation is needed. Then ammonium sulphate was gradually added to the crude extract with continuous stirring. The mixture was incubated in refrigerator at 4°C overnight. Next day the sample was centrifuged , mixture was collected and resuspended it with phosphate buffer. Filled it in dialysis bag and placed it on magnetic stirrer in PBS for 24 hours. (23).

Protein Estimation

The protein content of lectin extracts was determined by using the Folin–Lowry method. Bovine serum albumin (BSA) was used as the standard protein. Different dilution of lectin was prepared. Then were treated with alkaline copper reagent after incubated for 15 minutes followed by Folin–Ciocalteu reagent, incubate it at room temperature for 30 minutes to develop color. The blank was set and absorbance of all the tubes were measured at 750 nm using a colorimeter, and protein concentration were calculated from the standard curve prepared using BSA (18).

Hemagglutination Assay

Hemagglutination activity of lectin extracts were done by using washed human red blood cells (RBCs). The RBCs were washed approx. three times with normal saline by centrifugation at 1500 rpm for 5 minutes. A 2% RBC suspension was prepared. Serial two-fold dilutions of lectin samples were prepared in microtiter plates by using 0.9% normal saline and equal volumes of RBC suspension were added to each well. The plates were incubated at room temperature for 60 minutes. Hemagglutination assay result were observed visually, and recorded as the hemagglutination titer (28, 5).

ABO Blood Group Typing

For blood group identification, blood sample from individuals with known ABO blood groups were collected. Drop of blood and a drop of lectin were mixed on a clear slide and observed the agglutination reactions were observed. The results were recorded to determine the specificity of lectin extracts for different ABO blood group antigens (5,14).

Antibacterial Activity

The antibacterial activity of lectin extracts were evaluated using disc diffusion method i.e Kirby–Bauer disc diffusion method (3). The different bacterial cultures were inoculated into nutrient broth and incubated at 37°C until visible turbity is observed. The bacterial suspension was swabbed uniformly on Mueller–Hinton agar plates using a sterile cotton swab. Sterile filter paper discs (6 mm diameter) which was impregnated with lectin extracts were placed on the agar surface. PBS was used as a negative control. The plates were incubated in the incubator at 37°C for 24 hours. After incubation, the plates were examined and measured for zones of inhibition.

Fourier Transform Infrared (FTIR) spectroscopy

FTIR of the crude lectin sample was performed at the Center of Excellence to obtain the infrared spectrum and confirm the presence of characteristic functional groups.

Data Analysis

Protein concentration, hemagglutination titers, antibacterial inhibition zones and result of FTIR were recorded and comparison is done to evaluate the biological activity of the extracted lectin.

Results and Discussion

Extraction of Lectins from Different Biological Sources

Lectin was extracted from six different biological sources including fungal species (Aspergillus niger, Penicillium sp., Pleurotus ostreatus) and plant sources (Lablab purpureus, Pisum sativum, and Vigna unguiculata). To confirm lectin activity, blood grouping experiments were performed based on lectins as a hemagglutinin and agglutinate erythrocyte by binding with its specific carbohydrate present on the surface of RBCs.

Lectin Activity of Crude Extracts

Hemagglutination activity were performed to evaluate the lectin activity present in the extracts obtained from the different fungal and plant sources. Lectin activity was checked by adding drop of crude lectin of different soucres in different blood group on clean grease free slide.

The lectin activity of crude extracts was confirm of the fungal and plants extract by ABO blood group typing by the visible agglutination of blood samples as shown in figure 3.1.

Figure 3.1: Slide showing ABO blood group typing of the extracted lectin. Panels (1-4) shows agglutination patterns of different lectins ANL, POL, LPL, and PSL respectively with different blood groups.

The results interpreted from the above experiment, that the lectin activity was successfully detected from the extracts collected from Aspergillus niger, Pleurotus ostreatus, Lablab purpureus, and Pisum sativum (8,20,21). But, extracts collected from Penicillium sp. and Vigna unguiculata did not show any detectable hemagglutination activity. The absence of detectable lectin activity may be attributed to the low lectin concentration in the crude extract,or due to growth conditions which is optimum for the growth for lectin was not obtained, or may be lectin activity is loss during the extraction and purification steps. Sharon & Lis have reported that lectin production can vary significantly depending on which species used, environmental conditions or optimum condition, and extraction procedures used for the downstream process (25).

Based on these observations, four lectin samples showing lectin activity were selected for further study are as follows:

• ANL – Lectin extracted from Aspergillus niger (Fungal source)
• POL – Lectin extracted from Pleurotus ostreatus (Fungi- mushroom source)
• LPL – Lectin extracted from Lablab purpureus (Plant seed source)
• PSL – Lectin extracted from Pisum sativum (Plant seed source)

These lectin extracts were subsequently used for further analysis for testing to evaluate its hemagglutination specificity, blood group recognition, antibacterial activity, and structural characterization.

Hemagglutination Activity of Extracted Lectins

The hemagglutination assay was performed using red blood cells of different ABO blood groups in order to determine the specificity of the lectin samples toward blood group antigens (5, 25). The assay revealed distinct agglutination patterns among the different lectins, indicating differences in carbohydrate recognition properties.

The lectin extracted from Aspergillus niger (ANL) shows selective hemagglutination toward blood group A and no visible agglutination was observed with the blood group B and O. These results suggest that lectin ANL exhibits binding affinity to carbohydrate structures associated with antigen A present on erythrocyte A membrane and not toward erythrocyte B and O (33).

Similarly, the lectin extracted from Pleurotus ostreatus (POL) shows selective agglutination toward blood group B and no visible agglutination was observed with the blood group A and O. So, we can interpret that lectin POL may have binding affinity to carbohydrate structures associated with antigen B present on erythrocyte B cell membrane and not toward erythrocyte A and O. This result suggests that the fungal lectins may have the ability for selective carbohydrate recognition domain (CRDs) for recoginition of specific carbohydrate residue on the surface of particular erythrocyte.

In the contradict, the lectins extracted from plant sources Lablab purpureus (LPL) and Pisum sativum (PSL), shows broad range of hemagglutination activity. Both LPL and PSL shows agglutination toward all type of blood groups A, B, and O (8, 10). This indicates that these lectins are non specific toward particular blood group antigen’ carbohydrate and more likely recognize carbohydrate structures that are commonly present on the surface of human red blood cells.

The hemagglutination reactions were visually confirmed using U shaped microtiter plates by Hemagglutination assay (25). If the diffuse lattice across the bottom of the well is observed then its means a positive reaction, lectin have recognize the carbohydrate residue present on the surface of erythrocyte, formed a cross- linking of cells by binding with multiple RBCs at a time. If the compact button at the cell of the well observed, its indicate that the lectin doesn’t bind with the carbohydrate present on the RBCs and RBCs are settled down in well. These observation clearly suggests that the presence of lectin activity and provided clarification of the extracted lectin’s specificity.

The patterns observed from the above experiment, These results highlight that lectin in context to carbohydrate- binding property are very diverse in nature. Lectins that can recognize and bind to specific carbohydrate present on particular antigen of RBCs, it may be used as blood group typing and have potential application in diagnostic assays. Lectins that agglutinate multiple blood groups may interact with the more common carbohydrate residue present on cell surfaces.

The ABO blood group typing of the extrated lectins were confirmed by the visible agglutination of bloods as shown in figure 3.1. and the result of Hemagglutination assay observed in microtitre plate well are shown in figure 3.2.

The hemagglutination patterns observed for each lectin sample are summarized below:

Table 3.1: Hemagglutination specificity of lectin samples toward different ABO blood groups