Screening and purification of NanB sialidase from Pasteurella multocida with activity in hydrolyzing sialic acid Neu5Acα(2-6)Gal and Neu5Acα(2-3)Gal

Abstract

Study on sialidases as antiviral agents has been widely performed, but many types of sialidase have not been tested for their antiviral activity. Pasteurella multocida NanB sialidase is one such sialidase that has never been isolated for further research. In this study, the activity of NanB sialidase was investigated in silico by docking the NanB sialidase of Pasteurella multocida to the Neu5Acα(2-6)Gal and Neu5Acα(2-3)Gal ligands. Additionally, some local isolates of Pasteurella multocida, which had the NanB gene were screened, and the proteins were isolated for further testing regarding their activity in hydrolyzing Neu5Acα(2-6)Gal and Neu5Acα(2-3)Gal. Silico studies showed that the NanB sialidase possesses an exceptional affinity towards forming a protein-ligand complex with Neu5Acα(2-6)Gal and Neu5Acα(2-3)Gal. NanB sialidase of Pasteurella multocida B018 at 0.129 U/mL and 0.258 U/mL doses can hydrolyze Neu5Acα(2-6)Gal and Neu5Acα(2-3)Gal better than other doses. In addition, those doses can inhibit effectively H9N2 viral binding to red blood cells. This study suggested that the NanB sialidase of Pasteurella multocida B018 has a potent antiviral activity because can hydrolyze sialic acid on red blood cells surface and inhibit the H9N2 viral binding to the cells.

Figure 1

Some of the structures required in the docking process. (A) NanB sialidase protein generated from the Raptor X program; (B) 2D structure of Neu5Acα(2–6)Gal; (C) Neu5Acα(2–6)Gal 3D structure.

Figure 2

Visualization of interactions between Neu5Acα(2–6)Gal and NanB sialidase using Pymol showing hydrogen bonds, residues and their interaction distance with Neu5Acα(2–6)Gal in 3D.

Figure 3

PCR test results confirm Pasteurella multocida and its serotypes. (A) amplification of the ompH gene from all isolates showed a position of 460 bp; (B) PCR test results to distinguish serotypes from Pasteurella multocida. The positive control is the result of DNA extraction from locale isolates of Pasteurella multocida types A and B mixed that showed two bands of the gel electrophoresis.

Figure 4

The detection results of NanB and NanH sialidase genes from six isolates confirmed as Pasteurella multocida type A. NanB is indicated by the 554 bp, while NanH in the 360 bp.

Figure 5

Results of the Pasteurella multocida isolate selection test to be used in purification of protein sialidase. (A) Phylogenetic tree showing the relationship between Pasteurella multocida isolates; (B) Alignment of amino acids with the Bioedit program to determine points of difference in amino acids.

Figure 6

Results of SDS Page on protein at each stage of NanB sialidase Pasteurella multocida B018 purification. (1) Markers; (2) crude sel of Pasteurella multocida before chloroform method; (3) protein after Affinity chromatography; (4) protein after anion exchange chromatography; (5) initial protein supernatant crude sialidase.

Figure 7

Test results for optimum temperature, pH and incubation period of NanB sialidase Pasteurella multocida B018. (A) Optimum temperature; (B) optimum pH; (C) incubation period.

Figure 8

Results of sialidase toxicity test on red blood cells of chicken and rabbit. (A) chicken red blood cells; (B) rabbit red blood cells.

Figure 9

Results of NanB sialidase specificity test for two different sialic acids and two different blood types. (A) Neu5Acα(2–3)Gal, chicken red blood cells; (B) Neu5Acα(2–6)Gal, chicken red blood cells; (C) Neu5Acα(2–3)Gal, rabbit red blood cells; and (D) Neu5Acα(2–6)Gal rabbit red blood cells.

Figure 10

Viral copy number of H9 genes of the viruses that bind to red blood cells. (A) chicken red blood cells and (B) rabbit red blood cells.