Sialic acid transporter NanT participates in Tannerella forsythia biofilm formation and survival on epithelial cells

Abstract

Tannerella forsythia is a periodontal pathogen implicated in periodontitis. This gram-negative pathogen depends on exogenous peptidoglycan amino sugar N-acetylmuramic acid (NAM) for growth. In the biofilm state the bacterium can utilize sialic acid (Neu5Ac) instead of NAM to sustain its growth. Thus, the sialic acid utilization system of the bacterium plays a critical role in the growth and survival of the organism in the absence of NAM. We sought the function of a T. forsythia gene annotated as nanT coding for an inner-membrane sugar transporter located on a sialic acid utilization genetic cluster. To determine the function of this putative sialic acid transporter, an isogenic nanT-deletion mutant generated by allelic replacement strategy was evaluated for biofilm formation on NAM or Neu5Ac, and survival on KB epithelial cells. Moreover, since T. forsythia forms synergistic biofilms with Fusobacterium nucleatum, co-biofilm formation activity in mixed culture and sialic acid uptake in culture were also assessed. The data showed that the nanT-inactivated mutant of T. forsythia was attenuated in its ability to uptake sialic acid. The mutant formed weaker biofilms compared to the wild-type strain in the presence of sialic acid and as co-biofilms with F. nucleatum. Moreover, compared to the wild-type T. forsythia nanT -inactivated mutant showed reduced survival when incubated on KB epithelial cells. Taken together, the data presented here demonstrate that NanT-mediated sialic transportation is essential for sialic acid utilization during biofilm growth and survival of the organism on epithelial cells and implies sialic acid might be key for its survival both in subgingival biofilms and during infection of human epithelial cells in vivo.

Structure of the LPS O-chain from Fusobacterium nucleatum strain 10953, containing sialic acid

“…In this regard, the periodontal pathogen T. forsythia , which is found in close association with F. nucleatum in dental plaque [1], could harvest sialic acid from F. nucleatum LPS. Our previous studies have indicated that utilization of sialic acid from host and cohabiting bacterial partners such as fusobacteria likely plays a role in the survival of T. forsythia in the oral environment [26]. The identification of FucNAc4N is of interest as this monosaccharide is often found, as it is here, as a component of zwitterionic polysaccharides that may have biological implications [27, 28].…”

Peptidoglycan synthesis in Tannerella forsythia: Scavenging is the modus operandi

“…Moreover, metabolic pathways exist in some bacteria that allow them to direct sialic acid into peptidoglycan synthesis . As mentioned above, although sialic acid is nutritionally important for the growth of T. forsythia in biofilms, sialic acid utilization is also critical for the survival of T. forsythia on epithelial cells . It was observed that, compared with the parental strain, a sialic acid transport‐deficient mutant was significantly less fit to survive on oral epithelial cell monolayers .…”

“…[48][49][50][51][52][53] As mentioned above, although sialic acid is nutritionally important for the growth of T. forsythia in biofilms, 47 sialic acid utilization is also critical for the survival of T. forsythia on epithelial cells. 54 It was observed that, compared with the parental strain, a sialic acid transport-deficient mutant was significantly less fit to survive on oral epithelial cell monolayers. 54 Not only epithelial cells, but other bacteria with which T. forsythia is known to interact, such as F. nucleatum can provide sialic acid.…”

“…54 It was observed that, compared with the parental strain, a sialic acid transport-deficient mutant was significantly less fit to survive on oral epithelial cell monolayers. 54 Not only epithelial cells, but other bacteria with which T. forsythia is known to interact, such as F. nucleatum can provide sialic acid. We and others have shown that many strains of F. nucleatum express surface sialic acid, 55,56 and specifically in the case of F. nucleatum strains, sialic acid is a component of the lipopolysaccharide O-chain.…”

Sialic acid acquisition in bacteria–one substrate, many transporters

“…Homologues of NanT are found throughout a wide range of human pathogens within the Enterobacteriaceae including the genera Salmonella, Yersinia, Citrobacter and Chronobacter [12] , but none of these have been experimentally characterised. Homologues are also seen in the Bacteroidetes which have been studied including the NanT protein in the oral pathogen Tannerella forsythia by the Stafford group [13,14] , who have also demonstrated a role for sialic acid uptake for biofilm formation and also survival when exposed to epithelial cells in this organism [14] . A role for NanT in sialic acid uptake has also been demonstrated in the closely related gut bacterium Bacteroides fragilis [15] .…”