Bacteria, NanB sialidase Gene and Ligand
Neu5Acα(2-6)Gal ligand was downloaded from the Protein Data Bank database web server (https://pubchem.ncbi.nlm.nih.gov) in 3D and optimized with Open Babel. The 3D structure of NanB sialidase from Pasteurella multocida isolate 86-1913 (Accession number AF274868) was obtained by entering the amino acid to Raptor X program. Then the molecular weight prediction of the NanB sialidase protein was carried out by entering the amino acid sequence at https://www.bioinformatics.org/sms/prot mw.html. Meanwhile, to isolate pure NanB sialidase, screening was carried out on Pasteurella multocida, a local isolate archive of PT. Medika Satwa Laboratoris which only has one type of sialidase, namely NanB sialidase.
Analysis of the bonding of NanB sialidase Pasteurella multocida with Neu5Ac2-6Gal by in silico test
NanB and Neu5Acα(2-6)Gal was prepared using AutoDockTools 1.5.6 by removing water molecules and adding nonpolar hydrogens, charges, and atoms. The grid was arranged by making a gridbox that covers the target protein's surface followed with treatment by the auto grid program linked to the application. The molecular docking process of the ligand with NanB sialidase from Pasteurella multocida was carried out using the auto grid program. The docking output was the docked ligand's structure on the enzyme active site and its respective affinity score. Analysis of the docking results was carried out on residues interacting with the ligands, Gibbs free binding energy (∆G), structural conformation, affinity, and hydrogen bonding between NanB sialidase and Neu5Acα(2-6)Gal ligands (Morris et al. 2009).
Visualization of molecular docking results between ligands and proteins was carried out using Edu PyMOL and LIGPLOT software. Visualization using Edu PyMOL software aimed to clarify the binding site of the ligand to the protein. LIGPLOT+ software was used to determine the interaction of the number and distance of hydrogen bonds and amino acid residues involved in the interaction between ligands and proteins based on the 3D structure (Morris et al. 2009).
Culture, Identification, and Confirmation of Pasteurella multocida
A total of nine archival isolates were re-cultured using Brain Heart Infusion (BHI) Broth media and subsequently with Blood agar (BA) media. Incubation was carried out overnight at 37℃. The growing colonies were observed macroscopically and microscopically, followed by the catalase test (Panna et al. 2015), oxidase test (Grehn and Müller 1989), indole test (Hunt et al. 2001), and molecular confirmation through Polymerase Chain Reaction (PCR) assay.
Pure colonies were then extracted using PrestoTM Mini gDNA Bacteria Kit (Geneaid) according to the relevant protocols to obtain pure DNA. The primer used in the confirmation PCR test for Pasteurella multocida was an ompH-specific primer pair with a target amplicon of 1000 bp (Table 1). The PCR process was carried out using the KAPA2G Fast Hotstart Readymix PCR Kit (Merck) according to the relevant procedures with a final reaction volume of 10 μl and annealing temperature optimized for 55℃ for 40 cycles. The amplified sample was then visualized by electrophoresis using 1.5% agarose gel and stained using 0.5 g/ml ethidium bromide. The marker used was 100 bp (VC 100 bp Plus DNA Ladder Vivantis) as a standard measure. Isolates identified as Pasteurella multocida through PCR testing were then tested on the CapA and CapB genes (Table 1) to differentiate the identified serotypes of Pasteurella multocida (Kurnia et al. 2018).
Identification and Molecular Characterization of Sialidase-Coding Genes Pasteurella multocida
Identification of the presence of the gene encoding Pasteurella multocida sialidase was carried out on isolates that had been identified as Pasteurella multocida type A. Screening for the presence of the sialidase gene was carried out using the NanB and NanH primer (Table 1). The KAPA2G Fast Hotstart Readymix PCR Kit was used in the PCR process with a total PCR reaction of 50 μl and an annealing temperature of 56℃ (Silaen et al. 2020). The sequencing of amplicons that only showed positive NanB were performed by First Base sequencing service agency, Malaysia. Sequencing data were analyzed using MEGA X and Bioedit software to compare the genetics of the study isolate NanB with the NanB gene belonging to Pasteurella multocida 86-1913.
Native Production of NanB Sialidase Pasteurella multocida
Pasteurella multocida bacteria containing only the gene encoding NanB sialidase was propagated in the main batch of Brain Heart Infusion (BHI) broth 1000 ml and then centrifuged at 4℃ at a speed of 4000 x g for 45 minutes. The bacterial pellet was used for the subsequent production of NanB sialidase (Natalia and Priadi 1998).
Some of the methods used are the chloroform method with a slight modification of (Ames et al. 1984), the glycine method (Kaderbhai et al. 1997), freeze-thaw (Lall et al. 1989), and osmotic shock (Neu and Heppel 1965). In this study, several modifications of osmotic shock were used to divide them into the original method, the addition of Ca2+, the addition of lysozyme, and a combination of Ca2+ and lysozyme. Based on the results of sialidase production, the method that gave the highest specific activity value of sialidase was used as the sialidase production method, followed by anion exchange chromatography and affinity chromatography.
Anion exchange chromatography was performed using Q-sepharose with the resulting fractions labeled as F0, F1, F2, F3, F4, and F5, respectively. The fraction showing the highest specific activity was purified by column (5 ml) affinity chromatography using N-(p-Aminophenyl)oxamic acid–Agarose (Sigma-Aldrich, Germany) according to the relevant procedures. The estimated molecular weight of protein sialidase was carried out using SDS PAGE at 12% separating gel and 4% stacking gel (Tarigan et al. 2013). At each stage of purification, protein count was performed using the Bradford method and tested for sialidase activity using the Neuraminidase assay kit (Sigma-Aldrich) according to the relevant procedures.
Optimum pH, Temperature and Sialidase Incubation Period Tests
To find the optimum temperature, 80 μl of the Neuraminidase assay kit (Sigma-Aldrich) reaction mixture and 20 μl of NanB sialidase protein fraction were mixed and then incubated at different temperatures, namely 20℃, 25℃, 30℃, 37℃, 40℃, 45℃ and 50℃. The sialidase activity was subsequently calculated (Islam and Roy 2018).
The determination of the optimum pH was tested by incubating 20 µl of sialidase in pH 3 to pH 10 (1:1) in 80 µl of the Neuraminidase assay kit (Sigma-Aldrich) reaction mixture at 37℃. The sialidase activity was subsequently calculated as Units/ml in the same manner as the previous protocol. Meanwhile, the determination of sialidase activity in a certain incubation period was carried out by incubating sialidase at 37℃ and then calculating its activity at 24, 48, and 72 hours of incubation (Islam and Roy 2018).
Sialidase Toxicity Test on Red Blood Cells
NanB sialidase toxicity test was carried out on chicken and rabbit red blood cells. A total of 500 μl of sialidase in graded doses (0%, 12.5%, 25%, 50%, and 100%) was added to 500 μl of chicken and rabbit red blood cells with a concentration of 1% each and then incubated at 37°C for 2 hours. Centrifugation was carried out at a speed of 6000 x g for 3 minutes, then transferred the supernatant to a microplate and read using a microplate reader with a wavelength of 562 nm. As a lysis control, lysis buffer (Geneaid) was used. Toxicity was calculated using the following formula: 100 x (OD of sample - OD of negative control/ OD of lysis control - OD of negative control) (Gao et al. 2018).
Sialidase Specificity Test for Sialic Acid in Chicken and Rabbit Red Blood Cells
Red blood cells of chickens and rabbits were treated with the addition of graded sialidase concentrations (0%, 12.5%, 25%, 50%, and 100%) for 2 hours at 37°C. Cells were washed three times with PBS and fixed with 0.05% glutaraldehyde in PBS. Cell-based enzyme-linked lectin assay measured the amounts of (2,6)-linked sialic acid and (2,3)-linked sialic acid. The fixed cells were blocked with 3% bovine serum albumin (BSA) in PBS and streptavidin-biotin blocking reagent (Vector Laboratories, Burlingame, CA) to block endogenous streptavidin- and biotin-binding sites. Cells were rinsed once with PBS-0.1% Tween 20 (PBST) and incubated with two µg biotinylated SNA lectin (Vector Laboratory)/ml and 20 µg biotinylated MAA lectin (Vector Laboratory)/ml at 37°C. SNA (Sambucus nigra) is specific for Neu5Acα(2-6)Gal while MAA (Maackia amurensis) is specific for Neu5Acα(2-3)Gal. The cells were washed four times with PBST. Secondary detection of bound lectins was carried out by incubating 5 g streptavidin-HRP/ml for 1 hour at 37°C. Cells were washed five times in PBST, added in tetramethylbenzidine (TMB, Sigma), and suspended in 1 M H2SO4. The absorbance was measured at 450 nm, and the percentage of sialic acid remaining was calculated using the following calculation: 100% × [(absorbance of treated cells - background) / (absorbance of treated cells - background)]. Cells treated with streptavidin-HRP alone without lectins were used as a background control (Malakhov et al. 2006).