Exploring the role of multiple tonB transporters in Tannerella forsythia

Periodontitis, a chronic inflammatory disease, leads to the loss of gum, bone, and tooth. Periodontitis is characterised by a dysbiosis in which levels of certain anaerobes increase later during the biofilm development, and the most prominent of these anaerobes are the so-called red complex bacteria, namely, Tannerella forsythia (T. forsythia), Treponema denticola (T. denticola), and Porphyromonas gingivalis (P. gingivalis). T. forsythia, a periodontal pathobiont, utilises a dedicated transport and catabolic mechanism to utilise the host-derived sugar sialic acid, which is necessary for its biofilm growth and cell interactions. The best-studied member of the sialic acids is 5-N-acetylneuraminic acid (Neu5Ac) which can be found in saliva, mucus, and gingival crevicular fluid. The nan operon of T. forsythia encodes for transport, scavenging, and catabolic proteins and enzymes for sialic acid utilisation, which in Tannerella is dependent on the TonB system (tonB-exbB-exbD) and is involved in molecular uptake in a range of bacterial species (e.g., ions, B12, etc.). Crucially, this also requires the involvement of TonB-Dependent Transport proteins (TBDT) specific for sialic acid, namely NanO and NanU.
In contrast to model species like Escherichia coli (E. coli), bioinformatics revealed that T. forsythia has four putative tonB genes that were identified largely based on identifying a conserved TonB signature domain. These four tonB genes are present in all the T. forsythia genomes sequenced so far. Here, the function of the multiple putative tonB genes to transport Neu5Ac was tested by a mutagenesis programme alongside cloning of tonB and sialic acid uptake genes nanO and nanU for testing of their transport role in a heterologous expression and transport system in an E. coli transport assay strain, which lacks it is own tonB, as well as the outer membrane sialic acid transporters. As a result, we found that tonB BFO_0233 was preferentially required to utilise Neu5Ac or the heavily sialylated salivary glycoprotein mucin, indicating a similar situation to the oral cavity. In each observed phenotype, no significant difference was found between all deletion mutants in the presence of NAM.
During a bioinformatics study, we aimed to establish the extent of TonB-dependent transporters (TBDTs) in 13 genomes of T. forsythia. Therefore, the TBDTs were identified and characterised bioinformatically, and their presence or absence as part of putative PUL, and which TBDT domains were present. In ATCC 43037, for instance, we found 62 TBDTs and predicted their domain structures, 42 of which were associated with (PUL complexes). In contrast, the BU0063, periodontal health-associate isolate, showed a presence of only 35 TBDTs. This study was also able to compare the TonB box of these TBDTs and of the NanO sialic acid porin and assess the ability of this TBDT to be inhibited by targeting its conserved TonB box via peptides provided exogenously. The resulting data showed that the designed TonB box GASVVE affected not only the uptake of sialic acid, but also the biofilm formation when sialic acid or mucin was utilised as a growth substrate, which may provide a route for future therapeutic treatment for periodontitis.
Additional bioinformatic analysis was used to compare the presence of all virulence factors between 13 genomes in Tannerella after re-sequencing T. forsythia ATCC 43037 strain. Furthermore, it was recognised that T. forsythia ATCC 43037 has a unique outer membrane transporting Neu5Ac and NeuGc, but it was not known whether this porin of sialic acid was generic to nonulosonic acid (Sia-like sugars). We found during bioinformatical analysis that there were higher identities between the sialic acid outer membrane of ATCC 43037 strain (NanO) and other sialic acid outer membrane of 12 Tannerella’s strains based on nucleotide acid and amino acid sequences.
Bacteria with potential nan operon feeding pathways for sialic acid were bioinformatically investigated. The distribution of these nan operons among 2500 bacterial species from NCBI and PULDB databases was examined. This analysis showed genomes from these bacterial species that represent 12 orders, eight classes, and four phyla. These bacteria phylum are Bacteroidetes, Proteobacteria, Verrucomicrobia, and Gemmatimonadetes. Of the four phyla, this study concluded the presence of nan operons in 222 bacterial species, which belonged to 31 bacterial families. The dissemination of 222 nan operons isolated from human microbes was 67 compared to 155 non-human microbes. This study elucidates the importance of TonB, nan operons, and TBDTs in Neu5A acquisition, which lays the foundation for future studies considering the survival mechanisms of T. forsythia in Neu5Ac-restricted environments.
After the role of four tonB and nanO genes have been investigated along with the potential peptide for their inhibitions, the change of the whole transcriptional profile remains undefined under Neu5Ac and mucin conditions. Further investigation was conducted to examine the whole transcriptional profile of T. forsythia and the expression of the genes associated with both Neu5Ac and mucin conditions, imitating their survival and influence within the oral biofilm. Transcriptomic data concluded upregulation of 379 and 200 genes in response to Neu5Ac and mucin conditions, respectively. The entire transcriptional profile of T. forsythia in response to Neu5Ac and mucin conditions was described for the first time, which could provide an important basis and experimental direction for additional research into the mechanisms of periodontitis.