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IntroductionS-layer anchoring in Paenibacillus alvei is enabled by a non-covalent interaction between an S-layer homology domain trimer and a secondary cell wall polymer (SCWP), ensuring the structural integrity of the bacterial cell wall. Within the SCWP repeat, pyruvylated ManNAc serves as the ligand and the UDP-GlcNAc-2-epimerase MnaA supplies UDP-ManNAc to SCWP biosynthesis.MethodsTo better understand SCWP biosynthesis and identify strategies for inhibiting pathogens with comparable cell wall architecture, like Bacillus anthracis, MnaA and rational variants were produced in E. coli and their kinetic constants determined. The effect of UDP-GlcNAc as a predicted allosteric activator and tunicamycin as a potential inhibitor of MnaA was tested in vitro supported by molecular docking experiments. Additionally, wild-type MnaA was crystallized.ResultsWe present the crystal structure of unliganded P. alvei MnaA resolved at 2.20 Å. It adopts a GT-B fold consistent with other bacterial non-hydrolyzing UDP-GlcNAc 2-epimerases. A comparison of amino acid sequences reveals conservation of putative and known catalytic and allosteric-site residues in MnaA, which was confirmed through analysis of Q42A, Q69A, E135A and H241A MnaA variants. The kinetic parameters KM and kcat of MnaA were determined to be 3.91 mM and 33.44 s-1 for the forward, and 2.41 mM and 6.02 s-1 for the reverse reaction. While allosteric regulation by UDP-GlcNAc has been proposed as a mechanism for enzyme activation, UDP-GlcNAc was not found to be essential for UDP-ManNAc epimerization by P. alvei MnaA. However, the reaction rate doubled upon addition of 5% UDP-GlcNAc. Unexpectedly, the UDP-GlcNAc analog tunicamycin did not inhibit MnaA. Molecular docking experiments comparing tunicamycin binding of P. alvei MnaA and Staphylococcus aureus MnaA, which is inhibited by tunicamycin, revealed different residues exposed to the antibiotic excluding, those at the predicted allosteric site of P. alvei MnaA, corroborating tunicamycin resistance.ConclusionThe unliganded crystal structure of P. alvei MnaA reveals an open conformation characterized by an accessible cleft between the N- and C-terminal domains. Despite the conservation of residues involved in binding the allosteric activator UDP-GlcNAc, the enzyme is not strictly regulated by the substrate. Unlike S. aureus MnaA, the activity of P. alvei MnaA remains unaffected by tunicamycin.

IntroductionDegradation of host proteins by bacterial proteases leads to the subversion of the host response and disruption of oral epithelial integrity, which is considered an essential factor in the progression of periodontitis. High-temperature requirement A (HtrA) protease, which is critical for bacterial survival and environmental adaptation, is found in several oral bacteria, including the periodontal pathogen Tannerella forsythia. This study investigated the proteolytic activity of HtrA from T. forsythia and its ability to modulate the host response.MethodsHtrA of T. forsythia was identified bioinformatically and produced as a recombinant protein. T. forsythia mutants with depleted and restored HtrA production were constructed. The effect of T. forsythia wild-type, mutants and recombinant HtrA on the degradation of casein and E-cadherin was tested in vitro. Additionally, the responses of human gingival fibroblasts and U937 macrophages to the different HtrA-stimuli were investigated and compared to those triggered by the HtrA-deficient mutant.ResultsT. forsythia wild-type producing HtrA as well as the recombinant enzyme exhibited proteolytic activity towards casein and E-cadherin. No cytotoxic effect of either the wild-type, T. forsythia mutants or rHtrA on the viability of host cells was found. In hGFB and U937 macrophages, both T. forsythia species induced an inflammatory response of similar magnitude, as indicated by gene and protein expression of interleukin (IL)-1β, IL-6, IL-8, tumour necrosis factor α and monocyte chemoattractant protein (MCP)-1. Recombinant HtrA had no significant effect on the inflammatory response in hGFBs, whereas in U937 macrophages, it induced a transient inflammatory response at the early stage of infection.ConclusionHtrA of T. forsythia exhibit proteolytic activity towards the host adhesion molecule E-cadherin and has the potential to influence the host response. Its role in the progression of periodontitis needs further clarification.

Tannerella forsythia, a member of the “red complex” bacteria implicated in severe periodontitis, employs various survival strategies and virulence factors to interact with the host. It thrives as a late colonizer in the oral biofilm, relying on its unique adaptation mechanisms for persistence. Essential to its survival are the type 9 protein secretion system and O-glycosylation of proteins, crucial for host interaction and immune evasion. Virulence factors of T. forsythia, including sialidase and proteases, facilitate its pathogenicity by degrading host glycoproteins and proteins, respectively. Moreover, cell surface glycoproteins like the S-layer and BspA modulate host responses and bacterial adherence, influencing colonization and tissue invasion. Outer membrane vesicles and lipopolysaccharides further induce inflammatory responses, contributing to periodontal tissue destruction. Interactions with specific host cell types, including epithelial cells, polymorphonuclear leukocytes macrophages, and mesenchymal stromal cells, highlight the multifaceted nature of T. forsythia's pathogenicity. Notably, it can invade epithelial cells and impair PMN function, promoting dysregulated inflammation and bacterial survival. Comparative studies with periodontitis-associated Porphyromonas gingivalis reveal differences in protease activity and immune modulation, suggesting distinct roles in disease progression. T. forsythia's potential to influence oral antimicrobial defense through protease-mediated degradation and interactions with other bacteria underscores its significance in periodontal disease pathogenesis. However, understanding T. forsythia's precise role in host-microbiome interactions and its classification as a keystone pathogen requires further investigation. Challenges in translating research data stem from the complexity of the oral microbiome and biofilm dynamics, necessitating comprehensive studies to elucidate its clinical relevance and therapeutic implications in periodontitis management.

Streptococci are primary colonizers of the oral cavity where they are ubiquitously present and an integral part of the commensal oral biofilm microflora. The role oral streptococci play in the interaction with the host is ambivalent. On the one hand, they function as gatekeepers of homeostasis and are a prerequisite for the maintenance of oral health - they shape the oral microbiota, modulate the immune system to enable bacterial survival, and antagonize pathogenic species. On the other hand, also recognized pathogens, such as oral Streptococcus mutans and Streptococcus sobrinus, which trigger the onset of dental caries belong to the genus Streptococcus. In the context of periodontitis, oral streptococci as excellent initial biofilm formers have an accessory function, enabling late biofilm colonizers to inhabit gingival pockets and cause disease. The pathogenic potential of oral streptococci fully unfolds when their dissemination into the bloodstream occurs; streptococcal infection can cause extra-oral diseases, such as infective endocarditis and hemorrhagic stroke. In this review, the taxonomic diversity of oral streptococci, their role and prevalence in the oral cavity and their contribution to oral health and disease will be discussed, focusing on the virulence factors these species employ for interactions at the host interface.

Abstract Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic β-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and K M and k cat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.

Abstract Background The Gram-negative oral pathogen Tannerella forsythia strictly depends on the external supply of the essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) for survival because of the lack of the common MurNAc biosynthesis enzymes MurA/MurB. The bacterium thrives in a polymicrobial biofilm consortium and, thus, it is plausible that it procures MurNAc from MurNAc-containing peptidoglycan (PGN) fragments (muropeptides) released from cohabiting bacteria during natural PGN turnover or cell death. There is indirect evidence that in T. forsythia, an AmpG-like permease (Tanf_08365) is involved in cytoplasmic muropeptide uptake. In E. coli, AmpG is specific for the import of N-acetylglucosamine (GlcNAc)-anhydroMurNAc(−peptides) which are common PGN turnover products, with the disaccharide portion as a minimal requirement. Currently, it is unclear which natural, complex MurNAc sources T. forsythia can utilize and which role AmpG plays therein. Results We performed a screen of various putative MurNAc sources for T. forsythia mimicking the situation in the natural habitat and compared bacterial growth and cell morphology of the wild-type and a mutant lacking AmpG (T. forsythia ΔampG). We showed that supernatants of the oral biofilm bacteria Porphyromonas gingivalis and Fusobacterium nucleatum, and of E. coli ΔampG, as well as isolated PGN and defined PGN fragments obtained after enzymatic digestion, namely GlcNAc-anhydroMurNAc(−peptides) and GlcNAc-MurNAc(−peptides), could sustain growth of T. forsythia wild-type, while T. forsythia ΔampG suffered from growth inhibition. In supernatants of T. forsythia ΔampG, the presence of GlcNAc-anhMurNAc and, unexpectedly, also GlcNAc-MurNAc was revealed by tandem mass spectrometry analysis, indicating that both disaccharides are substrates of AmpG. The importance of AmpG in the utilization of PGN fragments as MurNAc source was substantiated by a significant ampG upregulation in T. forsythia cells cultivated with PGN, as determined by quantitative real-time PCR. Further, our results indicate that PGN-degrading amidase, lytic transglycosylase and muramidase activities in a T. forsythia cell extract are involved in PGN scavenging. Conclusion T. forsythia metabolizes intact PGN as well as muropeptides released from various bacteria and the bacterium’s inner membrane transporter AmpG is essential for growth on these MurNAc sources, and, contrary to the situation in E. coli, imports both, GlcNAc-anhMurNAc and GlcNAc-MurNAc fragments.

Tannerella forsythia and Porphyromonas gingivalis target distinct virulence factors bearing a structurally conserved C-terminal domain (CTD) to the type IX protein secretion system (T9SS). The T9SS comprises an outer membrane translocation complex which works in concert with a signal peptidase for CTD cleavage. Among prominent T9SS cargo linked to periodontal diseases are the TfsA and TfsB components of T. forsythia’s cell surface (S-) layer, the bacterium’s BspA surface antigen and a set of cysteine proteinases (gingipains) from P. gingivalis. To assess the overall role of the bacterial T9SS in the host response, human macrophages and human gingival fibroblasts were stimulated with T. forsythia and P. gingivalis wild-type bacteria and T9SS signal peptidase-deficient mutants defective in protein secretion, respectively. The immunostimulatory potential of these bacteria was compared by analyzing the mRNA expression levels of the pro-inflammatory mediators IL-6, IL-8, MCP-1 and TNF-α by qPCR and by measuring the production of the corresponding proteins by ELISA. Shot-gun proteomics analysis of T. forsythia and P. gingivalis outer membrane preparations confirmed that several CTD-bearing virulence factors which interact with the human immune system were depleted from the signal peptidase mutants, supportive of effective T9SS shut-down. Three and, more profoundly, 16 hours post stimulation, the T. forsythia T9SS mutant induced significantly less production of cytokines and the chemokine in human cells compared to the corresponding parent strain, while the opposite was observed for the P. gingivalis T9SS mutant. Our data indicate that T9SS shut-down translates into an altered inflammatory response in periodontal pathogens. Thus, the T9SS as a potential novel target for periodontal therapy needs further evaluation.

Abstract Background Tannerella forsythia is a bacterial pathogen implicated in periodontal disease. Numerous virulence-associated T. forsythia genes have been described, however, it is necessary to expand the knowledge on T. forsythia’s genome structure and genetic repertoire to further elucidate its role within pathogenesis. Tannerella sp. BU063, a putative periodontal health-associated sister taxon and closest known relative to T. forsythia is available for comparative analyses. In the past, strain confusion involving the T. forsythia reference type strain ATCC 43037 led to discrepancies between results obtained from in silico analyses and wet-lab experimentation. Results We generated a substantially improved genome assembly of T. forsythia ATCC 43037 covering 99% of the genome in three sequences. Using annotated genomes of ten Tannerella strains we established a soft core genome encompassing 2108 genes, based on orthologs present in > = 80% of the strains analysed. We used a set of known and hypothetical virulence factors for comparisons in pathogenic strains and the putative periodontal health-associated isolate Tannerella sp. BU063 to identify candidate genes promoting T. forsythia’s pathogenesis. Searching for pathogenicity islands we detected 38 candidate regions in the T. forsythia genome. Only four of these regions corresponded to previously described pathogenicity islands. While the general protein O-glycosylation gene cluster of T. forsythia ATCC 43037 has been described previously, genes required for the initiation of glycan synthesis are yet to be discovered. We found six putative glycosylation loci which were only partially conserved in other bacteria. Lastly, we performed a comparative analysis of translational bias in T. forsythia and Tannerella sp. BU063 and detected highly biased genes. Conclusions We provide resources and important information on the genomes of Tannerella strains. Comparative analyses enabled us to assess the suitability of T. forsythia virulence factors as therapeutic targets and to suggest novel putative virulence factors. Further, we report on gene loci that should be addressed in the context of elucidating T. forsythia’s protein O-glycosylation pathway. In summary, our work paves the way for further molecular dissection of T. forsythia biology in general and virulence of this species in particular.

Abstract Background Tannerella forsythia is a Gram-negative oral pathogen. Together with Porphyromonas gingivalis and Treponema denticola it constitutes the “red complex” of bacteria, which is crucially associated with periodontitis, an inflammatory disease of the tooth supporting tissues that poses a health burden worldwide. Due to the absence of common peptidoglycan biosynthesis genes, the unique bacterial cell wall sugar N-acetylmuramic acid (MurNAc) is an essential growth factor of T. forsythia to build up its peptidoglycan cell wall. Peptidoglycan is typically composed of a glycan backbone of alternating N-acetylglucosamine (GlcNAc) and MurNAc residues that terminates with anhydroMurNAc (anhMurNAc), and short peptides via which the sugar backbones are cross-linked to build up a bag-shaped network. Results We investigated T. forsythia’s peptidoglycan structure, which is an essential step towards anti-infective strategies against this pathogen. A new sensitive radioassay was developed which verified the presence of MurNAc and anhMurNAc in the cell wall of the bacterium. Upon digest of isolated peptidoglycan with endo-N-acetylmuramidase, exo-N-acetylglucosaminidase and muramyl-L-alanine amidase, respectively, peptidoglycan fragments were obtained. HPLC and mass spectrometry (MS) analyses revealed the presence of GlcNAc-MurNAc-peptides and the cross-linked dimer with retention-times and masses, respectively, equalling those of control digests of Escherichia coli and P. gingivalis peptidoglycan. Data were confirmed by tandem mass spectrometry (MS2) analysis, revealing the GlcNAc-MurNAc-tetra-tetra-MurNAc-GlcNAc dimer to contain the sequence of the amino acids alanine, glutamic acid, diaminopimelic acid (DAP) and alanine, as well as a direct cross-link between DAP on the third and alanine on the fourth position of the two opposite stem peptides. The stereochemistry of DAP was determined by reversed-phase HPLC after dabsylation of hydrolysed peptidoglycan to be of the meso-type. Conclusion T. forsythia peptidoglycan is of the A1γ-type like that of E. coli. Additionally, the classification of P. gingivalis peptidoglycan as A3γ needs to be revised to A1γ, due to the presence of meso-DAP instead of LL-DAP, as reported previously.

Human periodontal ligament stromal cells (hPDLSCs) and gingival mesenchymal stromal cells (hGMSCs) are resident mesenchymal stromal cells (MSCs) of the periodontal tissue. The lipopolysaccharide (LPS) from Porphyromonas gingivalis is structurally distinct from that of other Gram-negative bacteria, and earlier studies linked this structural difference to a distinct virulence activity and the ability to activate toll-like receptor 2 (TLR-2), besides TLR-4 as commonly occurring upon LPS challenge. Later studies, in contrast, argue that TLR-2 activation by P. gingivalis LPS is due to lipoprotein contamination. In the present study, we aimed to define the influence of structure versus purity of P. gingivalis LPS on the immune response of hPDLSCs and hGMSCs. Cells were stimulated with commercially available “standard” P. gingivalis LPS, “ultrapure” P. gingivalis LPS, or “ultrapure” Escherichia coli LPS, and the expression of interleukin- (IL-) 8, IL-6, monocyte chemoattractant protein- (MCP-) 1, TLR-2, and TLR-4 was evaluated. The contribution of TLR-4 to the LPS-induced response was assessed using the specific TLR-4 inhibitor TAK-242. “Standard” P. gingivalis LPS induced significantly higher IL-8, IL-6, and MCP-1 production compared to the “ultrapure” LPS preparations, with no significant difference detectable for “ultrapure” LPS from P. gingivalis and E. coli. By using TAK-242, the response of hPDLSCs and hGMSCs to “ultrapure” LPS preparations was effectively inhibited to the levels comparable to those of nonstimulated controls. In contrast, high levels of response to “standard” LPS were observed, even in the presence of TAK-242. Our data show that the response of MSCs from periodontal tissue to LPS depends more on the purity of the LPS preparation than on the LPS source. Even a small amount of contaminating lipoproteins can drastically enhance the hPDLSCs’ and hGMSCs; responsiveness to P. gingivalis LPS, which might also contribute to the progression of periodontal disease.

Gram-positive bacterial envelopes comprise proteinaceous surface layers (S-layers) important for survival and virulence that are often anchored to the cell wall through secondary cell wall polymers. Here the authors use a structural and biophysical approach to define the molecular mechanism of this important interaction.

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

Diverse members of the Bacteroidetes phylum have general protein O-glycosylation systems that are essential for processes such as host colonization and pathogenesis. Here, we analyzed the function of a putative fucosyltransferase (FucT) family that is widely encoded in Bacteroidetes protein O-glycosylation genetic loci. We studied the FucT orthologs of three Bacteroidetes species—Tannerella forsythia, Bacteroides fragilis, and Pedobacter heparinus. To identify the linkage created by the FucT of B. fragilis, we elucidated the full structure of its nine-sugar O-glycan and found that l-fucose is linked β1,4 to glucose. Of the two fucose residues in the T. forsythia O-glycan, the fucose linked to the reducing-end galactose was shown by mutational analysis to be l-fucose. Despite the transfer of l-fucose to distinct hexose sugars in the B. fragilis and T. forsythia O-glycans, the FucT orthologs from B. fragilis, T. forsythia, and P. heparinus each cross-complement the B. fragilis ΔBF4306 and T. forsythia ΔTanf_01305 FucT mutants. In vitro enzymatic analyses showed relaxed acceptor specificity of the three enzymes, transferring l-fucose to various pNP-α-hexoses. Further, glycan structural analysis together with fucosidase assays indicated that the T. forsythia FucT links l-fucose α1,6 to galactose. Given the biological importance of fucosylated carbohydrates, these FucTs are promising candidates for synthetic glycobiology.

The cell walls of Gram-positive bacteria contain a variety of glycopolymers (CWGPs), a significant proportion of which are covalently linked to the peptidoglycan (PGN) scaffolding structure. Prominent CWGPs include wall teichoic acids of Staphylococcus aureus, streptococcal capsules, mycobacterial arabinogalactan, and rhamnose-containing polysaccharides of lactic acid bacteria. CWGPs serve important roles in bacterial cellular functions, morphology, and virulence. Despite evident differences in composition, structure and underlaying biosynthesis pathways, the final ligation step of CWGPs to the PGN backbone involves a conserved class of enzymes—the LytR-CpsA-Psr (LCP) transferases. Typically, the enzymes are present in multiple copies displaying partly functional redundancy and/or preference for a distinct CWGP type. LCP enzymes require a lipid-phosphate-linked glycan precursor substrate and catalyse, with a certain degree of promiscuity, CWGP transfer to PGN of different maturation stages, according to in vitro evidence. The prototype attachment mode is that to the C6-OH of N-acetylmuramic acid residues via installation of a phosphodiester bond. In some cases, attachment proceeds to N-acetylglucosamine residues of PGN—in the case of the Streptococcus agalactiae capsule, even without involvement of a phosphate bond. A novel aspect of LCP enzymes concerns a predicted role in protein glycosylation in Actinomyces oris. Available crystal structures provide further insight into the catalytic mechanism of this biologically important class of enzymes, which are gaining attention as new targets for antibacterial drug discovery to counteract the emergence of multidrug resistant bacteria.

The cell surface of the oral pathogen Tannerella forsythia is heavily glycosylated with a unique, complex decasaccharide that is O-glycosidically linked to the bacterium’s abundant surface (S-) layer, as well as other proteins. The S-layer glycoproteins are virulence factors of T. forsythia and there is evidence that protein O-glycosylation underpins the bacterium’s pathogenicity. To elucidate the protein O-glycosylation pathway, genes suspected of encoding pathway components were first identified in the genome sequence of the ATCC 43037 type strain, revealing a 27-kb gene cluster that was shown to be polycistronic. Using a gene deletion approach targeted at predicted glycosyltransferases (Gtfs) and methyltransferases encoded in this gene cluster, in combination with mass spectrometry of the protein-released O-glycans, we show that the gene cluster encodes the species-specific part of the T. forsythia ATCC 43037 decasaccharide and that this is assembled step-wise on a pentasaccharide core. The core was previously proposed to be conserved within the Bacteroidetes phylum, to which T. forsythia is affiliated, and its biosynthesis is encoded elsewhere on the bacterial genome. Next, to assess the prevalence of protein O-glycosylation among Tannerella sp., the publicly available genome sequences of six T. forsythia strains were compared, revealing gene clusters of similar size and organization as found in the ATCC 43037 type strain. The corresponding region in the genome of a periodontal health-associated Tannerella isolate showed a different gene composition lacking most of the genes commonly found in the pathogenic strains. Finally, we investigated whether differential cell surface glycosylation impacts T. forsythia’s overall immunogenicity. Release of proinflammatory cytokines by dendritic cells (DCs) upon stimulation with defined Gtf-deficient mutants of the type strain was measured and their T cell-priming potential post-stimulation was explored. This revealed that the O-glycan is pivotal to modulating DC effector functions, with the T. forsythia-specific glycan portion suppressing and the pentasaccharide core activating a Th17 response. We conclude that complex protein O-glycosylation is a hallmark of pathogenic T. forsythia strains and propose it as a valuable target for the design of novel antimicrobials against periodontitis.

Glycoconjugates are the most diverse biomolecules of life. Mostly located at the cell surface, they translate into cell-specific “barcodes” and offer a vast repertoire of functions, including support of cellular physiology, lifestyle, and pathogenicity. Functions can be fine-tuned by non-carbohydrate modifications on the constituting monosaccharides. Among these modifications is pyruvylation, which is present either in enol or ketal form. The most commonly best-understood example of pyruvylation is enol-pyruvylation of N-acetylglucosamine, which occurs at an early stage in the biosynthesis of the bacterial cell wall component peptidoglycan. Ketal-pyruvylation, in contrast, is present in diverse classes of glycoconjugates, from bacteria to algae to yeast—but not in humans. Mild purification strategies preventing the loss of the acid-labile ketal-pyruvyl group have led to a collection of elucidated pyruvylated glycan structures. However, knowledge of involved pyruvyltransferases creating a ring structure on various monosaccharides is scarce, mainly due to the lack of knowledge of fingerprint motifs of these enzymes and the unavailability of genome sequences of the organisms undergoing pyruvylation. This review compiles the current information on the widespread but under-investigated ketal-pyruvylation of monosaccharides, starting with different classes of pyruvylated glycoconjugates and associated functions, leading to pyruvyltransferases, their specificity and sequence space, and insight into pyruvate analytics.

Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated sorting of cell-surface proteins and concomitantly engage a pyruvylated secondary cell-wall polymer as a cell-wall ligand. Specifically, pyruvate ketal linked to β-D-ManNAc is regarded as an indispensable epitope in this cell-surface display mechanism. That secondary cell wall polymer (SCWP) pyruvylation and SLH domain-containing proteins are functionally coupled is supported by the presence of an ortholog of the predicted pyruvyltransferase CsaB in bacterial genomes, such as those of Bacillus anthracis and Paenibacillus alvei. The P. alvei SCWP, consisting of pyruvylated disaccharide repeats [→4)-β-D-GlcNAc-(1→3)-4,6-Pyr-β-D-ManNAc-(1→] serves as a model to investigate the widely unexplored pyruvylation reaction. Here, we reconstituted the underlying enzymatic pathway in vitro in combination with synthesized compounds, used mass spectrometry, and nuclear magnetic resonance spectroscopy for product characterization, and found that CsaB-catalyzed pyruvylation of β-D-ManNAc occurs at the stage of the lipid-linked repeat. We produced the P. alvei TagA (PAV_RS07420) and CsaB (PAV_RS07425) enzymes as recombinant, tagged proteins, and using a synthetic 11-phenoxyundecyl-diphosphoryl-α-GlcNAc acceptor, we uncovered that TagA is an inverting UDP-α-D-ManNAc:GlcNAc-lipid carrier transferase, and that CsaB is a pyruvyltransferase, with synthetic UDP-α-D-ManNAc and phosphoenolpyruvate serving as donor substrates. Next, to substitute for the UDP-α-D-ManNAc substrate, the recombinant UDP-GlcNAc-2-epimerase MnaA (PAV_RS07610) of P. alvei was included in this in vitro reconstitution system. When all three enzymes, their substrates and the lipid-linked GlcNAc primer were combined in a one-pot reaction, a lipid-linked SCWP repeat precursor analog was obtained. This work highlights the biochemical basis of SCWP biosynthesis and bacterial pyruvyl transfer.

Tannerella forsythia is an anaerobic, Gram-negative oral pathogen that thrives in multispecies gingival biofilms associated with periodontitis. The bacterium is auxotrophic for the commonly essential bacterial cell wall sugar N-acetylmuramic acid (MurNAc) and, thus, strictly depends on an exogenous supply of MurNAc for growth and maintenance of cell morphology. A MurNAc transporter (Tf_MurT; Tanf_08375) and an ortholog of the Escherichia coli etherase MurQ (Tf_MurQ; Tanf_08385) converting MurNAc-6-phosphate to GlcNAc-6-phosphate were recently described for T. forsythia. In between the respective genes on the T. forsythia genome, a putative kinase gene is located. In this study, the putative kinase (Tf_MurK; Tanf_08380) was produced as a recombinant protein and biochemically characterized. Kinetic studies revealed Tf_MurK to be a 6-kinase with stringent substrate specificity for MurNAc exhibiting a 6 × 104-fold higher catalytic efficiency (kcat/Km) for MurNAc than for N-acetylglucosamine (GlcNAc) with kcat values of 10.5 s-1 and 0.1 s-1 and Km values of 200 μM and 116 mM, respectively. The enzyme kinetic data suggest that Tf_MurK is subject to substrate inhibition (Ki[S] = 4.2 mM). To assess the role of Tf_MurK in the cell wall metabolism of T. forsythia, a kinase deletion mutant (ΔTf_murK::erm) was constructed. This mutant accumulated MurNAc intracellularly in the exponential phase, indicating the capability to take up MurNAc, but inability to catabolize MurNAc. In the stationary phase, the MurNAc level was reduced in the mutant, while the level of the peptidoglycan precursor UDP-MurNAc-pentapeptide was highly elevated. Further, according to scanning electron microscopy evidence, the ΔTf_murK::erm mutant was more tolerant toward low MurNAc concentration in the medium (below 0.5 μg/ml) before transition from healthy, rod-shaped to fusiform cells occurred, while the parent strain required > 1 μg/ml MurNAc for optimal growth. These data reveal that T. forsythia readily catabolizes exogenous MurNAc but simultaneously channels a proportion of the sugar into peptidoglycan biosynthesis. Deletion of Tf_murK blocks MurNAc catabolism and allows the direction of MurNAc solely to peptidoglycan biosynthesis, resulting in a growth advantage in MurNAc-depleted medium. This work increases our understanding of the T. forsythia cell wall metabolism and may pave new routes for lead finding in the treatment of periodontitis.

The periodontopathogen Tannerella forsythia has a characteristic cell surface (S-) layer modified with a unique O-glycan. This structure was analyzed for its role in biofilm formation employing an in vitro multispecies biofilm model, into which different T. forsythia strains and mutants with a modified cell surface composition were incorporated together with nine other oral species. The influence of the glycosylated T. forsythia S-layer on the bacterial composition of the biofilms was analyzed quantitatively using quantitative real-time PCR as well as qualitatively by fluorescence in situ hybridization and confocal laser scanning microscopy. It was evident that while changes of the T. forsythia cell surface did not affect the quantitative composition of the multispecies consortium, with the exception of Campylobacter rectus cell numbers, the localization of T. forsythia within the biofilm and its aggregation with Porphyromonas gingivalis were changed. Thus, the glycosylated T. forsythia S-layer might have relevance for positioning of this species within the biofilm and influence its co-localization with P. gingivalis and the prevalence of C. rectus. This might further pinpoint a pivotal role of T. forsythia cell surface structures in the virulence of this species when interacting with host tissues and immune system, from within or beyond the biofilm.

Anammox bacteria are a distinct group of Planctomycetes that are characterized by their unique ability to perform anaerobic ammonium oxidation with nitrite to dinitrogen gas in a specialized organelle. The cell of anammox bacteria comprises three membrane-bound compartments and is surrounded by a two-dimensional crystalline S-layer representing the direct interaction zone of anammox bacteria with the environment. Previous results from studies with the model anammox organism Kuenenia stuttgartiensis suggested that the protein monomers building the S-layer lattice are glycosylated. In the present study, we focussed on the characterization of the S-layer protein glycosylation in order to increase our knowledge on the cell surface characteristics of anammox bacteria. Mass spectrometry (MS) analysis showed an O-glycan attached to thirteen sites distributed over the entire 1591-amino acid S-layer protein. This glycan is composed of six monosaccharide residues, of which five are N-acetylhexosamine (HexNAc) residues. Four of these HexNAc residues have been identified as GalNAc. The sixth monosaccharide in the glycan is a putative dimethylated deoxyhexose. Two of the HexNAc residues were also found to contain a methyl group, thereby leading to an extensive degree of methylation of the glycan. This study presents the first characterization of a glycoprotein in a planctomycete and shows that the S-layer protein Kustd1514 of K. stuttgartiensis is heavily glycosylated with an O-linked oligosaccharide which is additionally modified by methylation. S-layer glycosylation clearly contributes to the diversification of the K. stuttgartiensis cell surface and can be expected to influence the interaction of the bacterium with other cells or abiotic surfaces.

Glycobiology is important for the periodontal pathogen Tannerella forsythia, affecting the bacterium’s cellular integrity, its life-style, and virulence potential. The bacterium possesses a unique Gram-negative cell envelope with a glycosylated surface (S-) layer as outermost decoration that is proposed to be anchored via a rough lipopolysaccharide. The S-layer glycan has the structure 4‑MeO-b-ManpNAcCONH2-(1→3)-[Pse5Am7Gc-(2→4)-]-b-ManpNAcA-(1→4)-[4-MeO-a-Galp-(1→2)-]-a-Fucp-(1→4)-[-a-Xylp-(1→3)-]-b-GlcpA-(1→3)-[-b-Digp-(1→2)-]-a-Galp and is linked to distinct serine and threonine residues within the D(S/T)(A/I/L/M/T/V) amino acid motif. Also several other Tannerella proteins are modified with the S‑layer oligosaccharide, indicating the presence of a general O‑glycosylation system. Protein O‑glycosylation impacts the life-style of T. forsythia since truncated S-layer glycans present in a defined mutant favor biofilm formation. While the S‑layer has also been shown to be a virulence factor and to delay the bacterium's recognition by the innate immune system of the host, the contribution of glycosylation to modulating host immunity is currently unraveling. Recently, it was shown that Tannerella surface glycosylation has a role in restraining the Th17-mediated neutrophil infiltration in the gingival tissues. Related to its asaccharolytic physiology, T. forsythia expresses a robust enzymatic repertoire, including several glycosidases, such as sialidases, which are linked to specific growth requirements and are involved in triggering host tissue destruction. This review compiles the current knowledge on the glycobiology of T. forsythia.

BackgroundSwarming and biofilm formation have been studied for a variety of bacteria. While this is well investigated for Gram-negative bacteria, less is known about Gram-positive bacteria, including Paenibacillus alvei, a secondary invader of diseased honeybee colonies infected with Melissococcus pluton, the causative agent of European foulbrood (EFB).MethodologyPaenibacillus alvei CCM 2051(T) is a Gram-positive bacterium which was recently shown to employ S-layer homology (SLH) domains as cell wall targeting modules to display proteins on its cell surface. This study deals with the newly identified 1335-amino acid protein SlhA from P. alvei which carries at the C‑terminus three consecutive SLH-motifs containing the predicted binding sequences SRGE, VRQD, and LRGD instead of the common TRAE motif. Based on the proof of cell surface location of SlhA by fluorescence microscopy using a SlhA-GFP chimera, the binding mechanism was investigated in an in vitro assay. To unravel a putative function of the SlhA protein, a knockout mutant was constructed. Experimental data indicated that one SLH domain is sufficient for anchoring of SlhA to the cell surface, and the SLH domains of SlhA recognize both the peptidoglycan and the secondary cell wall polymer in vitro. This is in agreement with previous data from the S-layer protein SpaA, pinpointing a wider utilization of that mechanism for cell surface display of proteins in P. alvei. Compared to the wild-type bacterium ΔslhA revealed changed colony morphology, loss of swarming motility and impaired biofilm formation. The phenotype was similar to that of the flagella knockout Δhag, possibly due to reduced EPS production influencing the functionality of the flagella of ΔslhA.ConclusionThis study demonstrates the involvement of the SLH domain-containing protein SlhA in swarming and biofilm formation of P. alvei CCM 2051(T).

The gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis.

The amazing repertoire of glycoconjugates present on bacterial cell surfaces includes lipopolysaccharides, capsular polysaccharides, lipooligosaccharides, exopolysaccharides, and glycoproteins. While the former are constituents of Gram-negative cells, we review here the cell surface S-layer glycoproteins of Gram-positive bacteria. S-layer glycoproteins have the unique feature of self-assembling into 2D lattices providing a display matrix for glycans with periodicity at the nanometer scale. Typically, bacterial S-layer glycans are O-glycosidically linked to serine, threonine, or tyrosine residues, and they rely on a much wider variety of constituents, glycosidic linkage types, and structures than their eukaryotic counterparts. As the S-layer glycome of several bacteria is unravelling, a picture of how S-layer glycoproteins are biosynthesized is evolving. X-ray crystallography experiments allowed first insights into the catalysis mechanism of selected enzymes. In the future, it will be exciting to fully exploit the S-layer glycome for glycoengineering purposes and to link it to the bacterial interactome.