Functional characterization of sialidases from oral bacteria

Abstract

Sialidases (neuraminidases) are glycoside hydrolase enzymes that cleave sialic acid moieties from sialic acid-containing glycoconjugates. Many bacterial species utilize sialidases to acquire sialic acid from host glycoproteins or glycolipids for nutritional or other physiological purposes. Sialidases from oral bacteria have previously been shown to play key roles in colonization, biofilm formation, and are considered potential virulence factors. Here, I investigated the biochemical and biological activities of (multi-domain) sialidase homologues from Schaalia odontolytica, Schaalia meyeri and Capnocytophaga ochracea. These species are commonly found in biofilms on teeth and oral mucosal surfaces, and play key roles in dental plaque formation. Full length and truncated versions of sialidase homologues encoded by S. odontolytica CCUG 20536T (Sodo-NanH1, Sodo-NanH2, Sodo-NanH3) and S. meyeri ATCC 35568T (Smey-NanH2, Smey-NanH3) were cloned, expressed and biochemically characterized, to probe the functions of their respective domains. Sodo-NanH1 contains an N-terminal non-catalytic dual Laminin G (LamG) domain and C-terminal central sialidase domain. Sodo-NanH2 and Smey-NanH2 both contain an N-terminal signal peptide, central sialidase domain and C-terminal, non-catalytic PspC domain. Sodo-NanH3 and Smey-NanH3 comprise only a catalytic domain. Their respective enzyme kinetic parameters were determined using the fluorogenic substrate 2’-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA). 3’-sialyllactose (3’-SL) and 6’-sialyllactose (6’-SL) were used to determine α-2,3- versus α-2,6-linkage cleavage specificity. Sodo-NanH1, Sodo-NanH2 and Smey-NanH2 preferred α-2,3-linked substrates while Sodo-NanH3 and Smey-NanH3 preferred α-2,6-linked substrates. Sodo-NanH2 and Smey-NanH2 had the fastest and most efficient enzymatic activities. Results from cell culture-based assays indicated that the cell surface associated NanH2 functions as the major sialidase in S. odontolytica and S. meyeri. Truncated versions of Sodo-NanH2 and Smey-NanH2 that lacked the C-terminal PspC domain had greatly elevated sialidase activities, indicating the PspC domain negatively modulates catalytic activities. Truncations of Sodo-NanH1 lacking the dual-LamG domain exhibited negligible hydrolytic activities. The five, recombinant full-length sialidase proteins exhibited varying levels of sensitivity to three sialidase inhibitors: DANA, siastatin B and oseltamivir. DANA possessed the strongest inhibitory effects on all sialidases. Supplementation of nutritionally replete growth medium with DANA inhibited planktonic growth and biofilm formation by the type strains of S. odontolytica, S. meyeri, Actinomyces viscosus, Actinomyces naeslundii and Actinomyces oris, which are all sialidase-positive species. Supplementation with free sialic acid exhibited variable effects on growth rates and biofilm formation, dependent on the concentrations used. The single sialidase homologue encoded by C. ochracea CCUG 9716T (Coch_0016, Co-NanH) demonstrated optimal sialidase activities under acidic conditions (ca. pH 5.5 - 6.0). Co-NanH possessed potent sialidase activities (versus MUNANA) and was most sensitive to inhibition by DANA. The deletion of the nanH gene of C. ochracea led to an abrogation in sialidase activities. The C. ochracea ΔnanH strain exhibited no significant growth defects in a nutritionally replete culture medium, but demonstrated substantially lower biofilm-forming capabilities than the wild type strain. In conclusion, the NanH1, NanH2 and NanH3 sialidase homologues encoded by S. odontolytica and S. meyeri differ significantly in their respective biochemical characteristics. Their respective biological roles in oral health and disease remain to be fully-elucidated. My results highlight the key involvement of sialidases in biofilm formation.