Understanding Glycans and Glycosidases in Infection and Biofilm Formation as a Route to Novel Antimicrobials

Periodontitis is a chronic bacterially induced disease characterized by inflammation of the gingivae and subsequent destruction of the tissues and supporting structures of the periodontium, which can lead to tooth loss. The red complex pathobionts: Porphyromonas gingivalis and Tannerella forsythia are mostly associated with periodontitis and are shown to modulate the host’s innate immune system. These oral pathogens secrete sialidase enzymes that they use to scavenge sialic acids found at the terminus of host glycoprotein chains for nutrition or to evade host immune responses. Activities of these pathogens, therefore, poses a problem to public health as such, necessitates the need to carry out research aimed at understanding this important enzyme as a route for the development of novel inhibitors of periodontitis and other immune-modulatory diseases. Over-expressed P. gingivalis (SiaPG) and T. forsythia (NanH) sialidase enzymes were purified using HisTag low-affinity chromatography, and the sialidase activity was tested using 4-methylumbelliferyl N-acetyl-α-D-neuraminic acid sodium salt (MUNANA), as substrate. Inhibition studies using whole cells and the purified sialidases of T. forsythia (NanH) and P. gingivalis (SiaPg), showed ECG as the best plant-derived inhibitor, while 2e3aDFNeu5Ac9N3 as the most potent of all the screened compounds. Additionally, Palmatine and Berberine chloride synergistically inhibited almost a 100 % NanH sialidase activity, with mechanisms of action (MOA) showing Palmatine, Berberine chloride, and 2e3aDFNeu5Ac9N3, as non-competitive, uncompetitive and competitive inhibitors of NanH sialidase, respectively. Furthermore, sialidase promotes the growth of P. gingivalis and T. forsythia, and supports host-pathogen interactions via adhesion and invasion of oral epithelial cells (H357). In addition, the role of P. gingivalis and T. forsythia sialidases on host innate immune modulation in the presence or absence of 2e3aDFNeu5Ac9N3 was also assessed using flow cytometry. NanH sialidase of T. forsythia appears to upregulate the secretion of pro-inflammatory cytokines such as interleukin-6 (IL-6), IL-8, and IL1-β in the cell supernatants, which was abrogated significantly by 2e3aDFNeu5Ac9N3 with minimal cytotoxic effects on the oral epithelial cells. Also, molecular docking of several inhibitors into the active-site pockets of NanH-apo and SiaPg using AutoDock Vina and PyMol suggested that Oseltamivir, Siastatin B, DANA, and 2e3aDFNeu5Ac9N3 coordinate the arginine triad (Arg423, Arg487, Arg212 and Arg194, Arg213, Arg460), of NanH and SIaPg of T. forsythia and P. gingivalis, respectively. Also, the nucleophilic dyad tyrosine and glutamate interact with the anomeric carbon as well as the acid/base aspartate residue Asp237/Asp280 interacting with the N-acetyl group of respective inhibitors. The superior inhibitory properties of 2e3aDFNeu5Ac9N3 on both the purified and whole cell sialidase activities of P. gingivalis and T. forsythia make it a promising compound, and can further be developed as a novel immunomodulatory agent of periodontitis or other inflammatory diseases.