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The recently identified bacterium Tannerella serpentiformis is the closest phylogenetic relative of Tannerella forsythia, whose presence in oral biofilms is associated with periodontitis. Conversely, T. serpentiformis is considered health-associated. This discrepancy was investigated in a comparative study of the two Tannerella species. The biofilm behavior was analyzed upon their addition and of Porphyromonas gingivalis-each bacterium separately or in combinations-to an in vitro five-species oral model biofilm. Biofilm composition and architecture was analyzed quantitatively using real-time PCR and qualitatively by fluorescence in situ hybridization/confocal laser scanning microscopy, and by scanning electron microscopy. The presence of T. serpentiformis led to a decrease of the total cell number of biofilm bacteria, while P. gingivalis was growth-promoting. This effect was mitigated by T. serpentiformis when added to the biofilm together with P. gingivalis. Notably, T. serpentiformis outcompeted T. forsythia numbers when the two species were simultaneously added to the biofilm compared to biofilms containing T. forsythia alone. Tannerella serpentiformis appeared evenly distributed throughout the multispecies biofilm, while T. forsythia was surface-located. Adhesion and invasion assays revealed that T. serpentiformis was significantly less effective in invading human gingival epithelial cells than T. forsythia. Furthermore, compared to T. forsythia, a higher immunostimulatory potential of human gingival fibroblasts and macrophages was revealed for T. serpentiformis, based on mRNA expression levels of the inflammatory mediators interleukin 6 (IL-6), IL-8, monocyte chemoattractant protein-1 and tumor necrosis factor α, and production of the corresponding proteins. Collectively, these data support the potential of T. serpentiformis to interfere with biological processes relevant to the establishment of periodontitis.

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. forsythiaO-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. forsythiaO-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.

Glycans play many important roles in bacterial biology and the complexity of the glycan structures requires biochemical assays in place to help characterize the biosynthetic pathways. Our focus has been on the use of enzymes from pathogens which make molecular mimics of host glycans. We have been examining glycosyltransferases that make strategic linkages in biologically active glycans which can be also exploited for potential therapeutic glycoconjugate synthesis. This chapter will provide details on assays for a variety of bacterial glycosyltransferases that we and others have used for the characterization of pathogen glycoconjugate biosynthetic pathways, and for the in vitro synthesis of human-like glycans produced by bacterial pathogens. The methods presented here should enable other assays to be developed for new pathway characterization.

Glycans play many important roles in bacterial biology and the complexity of the glycan structures requires biochemical assays in place to help characterize the biosynthetic pathways. Our focus has been on the use of enzymes from pathogens which make molecular mimics of host glycans. We have been examining glycosyltransferases that make strategic linkages in biologically active glycans which can be also exploited for potential therapeutic glycoconjugate synthesis. This chapter will provide details on assays for a variety of bacterial glycosyltransferases that we and others have used for the characterization of pathogen glycoconjugate biosynthetic pathways, and for the in vitro synthesis of human-like glycans produced by bacterial pathogens. The methods presented here should enable other assays to be developed for new pathway characterization.

Polysialyltransferases (polySTs) are glycosyltransferases that synthesize polymers of sialic acid found in vertebrates and some bacterial pathogens. Bacterial polySTs have utility in the modification of therapeutic proteins to improve serum half-life, and the potential for tissue engineering. PolySTs are membrane-associated proteins and as recombinant proteins suffer from inherently low solubility, low expression levels and poor thermal stability. To improve their physicochemical and biochemical properties, we applied a directed evolution approach using a FACS-based ultrahigh-throughput assay as a simple, robust and reliable screening method. We were able to enrich a large mutant library and, in combination with plate-based high-throughput secondary screening, we discovered mutants with increased enzymatic activity and improved stability compared to the wildtype enzyme. This work presents a powerful strategy for the screening of directed evolution libraries of bacterial polySTs to identify better catalysts for in vitro polysialylation of therapeutics.

Peanut allergy is an IgE-mediated severe hypersensitivity disorder. The lack of a treatment of this potentially fatal allergy has led to intensive research on vaccine development. Here, we describe the design and initial characterization of a carrier-bound peptide derived from the most potent peanut allergen, Ara h 2, as a candidate vaccine. Based on the adjuvant capability of bacterial surface (S-) layers, a fusion protein of the S-layer protein SlpB from Lactobacillus buchneri CD034 and the Ara h 2-derived peptide AH3a42 was produced. This peptide comprised immunodominant B-cell epitopes as well as one T cell epitope. The fusion protein SlpB-AH3a42 was expressed in E. coli, purified, and tested for its IgE binding capacity as well as for its ability to activate sensitized rat basophil leukemia (RBL) cells. The capacity of Ara h 2-specific IgG rabbit-antibodies raised against SlpB-AH3a42 or Ara h 2 to inhibit IgE-binding was determined by ELISA inhibition assays using sera of peanut allergic patients sensitized to Ara h 2. IgE specific to the SlpB-AH3a42 fusion protein was detected in 69% (25 of 36) of the sera. Despite the recognition by IgE, the SlpB-AH3a42 fusion protein was unable to induce β-hexosaminidase release from sensitized RBL cells at concentrations up to 100 ng per ml. The inhibition of IgE-binding to the natural allergen observed after pre-incubation of the 20 sera with rabbit anti-SlpB-AH3a42 IgG was more than 30% for four sera, more than 20% for eight sera, and below 10% for eight sera. In comparison, anti-Ara h 2 rabbit IgG antibodies inhibited binding to Ara h 2 by 48% ±13.5%. Our data provide evidence for the feasibility of this novel approach towards the development of a peanut allergen peptide-based carrier-bound vaccine. Our experiments further indicate that more than one allergen-peptide will be needed to induce a broader protection of patients allergic to Ara h 2.

The occurrence of nonulosonic acids in bacteria is wide-spread and linked to pathogenicity. However, the knowledge of cognate nonulosonic acid transferases is scarce. In the periodontopathogen Tannerella forsythia, several proposed virulence factors carry strain-specifically either a pseudaminic or a legionaminic acid derivative as terminal sugar on an otherwise structurally identical, protein-bound oligosaccharide. This study aims to shed light on the transfer of either nonulosonic acid derivative on a proximal N-acetylmannosaminuronic acid residue within the O-glycan structure, exemplified with the bacterium's abundant S-layer glycoproteins. Bioinformatic analyses provided the candidate genes Tanf_01245 (strain ATCC 43037) and TFUB4_00887 (strain UB4), encoding a putative pseudaminic and a legionaminic acid derivative transferase, respectively. These transferases have identical C-termini and contain motifs typical of glycosyltransferases (DXD) and bacterial sialyltransferases (D/E-D/E-G and HP). They share homology to type B glycosyltransferases and TagB, an enzyme catalyzing glycerol transfer to an N-acetylmannosamine residue in teichoic acid biosynthesis. Analysis of a cellular pool of nucleotide-activated sugars confirmed the presence of the CMP-activated nonulosonic acid derivatives, which are most likely serving as substrates for the corresponding transferase. Single gene knock-out mutants targeted at either transferase were analyzed for S-layer O-glycan composition by ESI-MS, confirming the loss of the nonulosonic acid derivative. Cross-complementation of the mutants with the nonnative nonulosonic acid transferase was not successful indicating high stringency of the enzymes. This study identified plausible candidates for a pseudaminic and a legionaminic acid derivative transferase; these may serve as valuable tools for engineering of novel sialoglycoconjugates.

Tannerella forsythia is an anaerobic, Gram-negative periodontal pathogen. A unique O-linked oligosaccharide decorates the bacterium's cell surface proteins and was shown to modulate the host immune response. In our study, we investigated the biosynthesis of the nonulosonic acid (NulO) present at the terminal position of this glycan. A bioinformatic analysis of T. forsythia genomes revealed a gene locus for the synthesis of pseudaminic acid (Pse) in the type strain ATCC 43037 while strains FDC 92A2 and UB4 possess a locus for the synthesis of legionaminic acid (Leg) instead. In contrast to the NulO in ATCC 43037, which has been previously identified as a Pse derivative (5-N-acetimidoyl-7-N-glyceroyl-3,5,7,9-tetradeoxy-l-glycero-l-manno-NulO), glycan analysis of strain UB4 performed in this study indicated a 350-Da, possibly N-glycolyl Leg (3,5,7,9-tetradeoxy-d-glycero-d-galacto-NulO) derivative with unknown C5,7 N-acyl moieties. We have expressed, purified and characterized enzymes of both NulO pathways to confirm these genes’ functions. Using capillary electrophoresis (CE), CE–mass spectrometry and NMR spectroscopy, our studies revealed that Pse biosynthesis in ATCC 43037 essentially follows the UDP-sugar route described in Helicobacter pylori, while the pathway in strain FDC 92A2 corresponds to Leg biosynthesis in Campylobacter jejuni involving GDP-sugar intermediates. To demonstrate that the NulO biosynthesis enzymes are functional in vivo, we created knockout mutants resulting in glycans lacking the respective NulO. Compared to the wild-type strains, the mutants exhibited significantly reduced biofilm formation on mucin-coated surfaces, suggestive of their involvement in host-pathogen interactions or host survival. This study contributes to understanding possible biological roles of bacterial NulOs.

Flagellin glycosylation impacts, in several documented cases, the functionality of bacterial flagella. The basis of flagellin glycosylation has been studied for various Gram-negative bacteria, but less is known about flagellin glycans of Gram-positive bacteria including Paenibacillus alvei, a secondary invader of honeybee colonies diseased with European foulbrood. Paenibacillus alvei CCM 2051(T) swarms vigorously on solidified culture medium, with swarming relying on functional flagella as evidenced by abolished biofilm formation of a non-motile P. alvei mutant defective in the flagellin protein Hag. Here, the glycobiology of the polar P. alvei flagella was investigated. Analysis on purified flagellin demonstrated that the 30-kDa Hag protein (PAV_2c01710) is modified with an O-linked trisaccharide comprised of one hexose and two N-acetyl-hexosamine residues, at three sites of glycosylation. Downstream of the hag gene on the bacterial chromosome, two open reading frames (PAV_2c01630, PAV_2c01640) encoding putative glycosyltransferases were shown to constitute a flagellin glycosylation island. Mutants defective in these genes exhibited altered migration in sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as loss of extracellular flagella production and bacterial motility. This study reveals that flagellin glycosylation in P. alvei is pivotal to flagella formation and bacterial motility in vivo, and simultaneously identifies flagella glycosylation as a second protein O-glycosylation system in this bacterium, in addition to the well-investigated S-layer tyrosine O-glycosylation pathway.

Surface (S)-layer proteins are model systems for studying protein glycosylation in bacteria and simultaneously hold promises for the design of novel, glyco-functionalized modules for nanobiotechnology due to their 2D self-assembly capability. Understanding the mechanism governing S-layer glycan biosynthesis in the Gram-positive bacterium Paenibacillus alvei CCM 2051T is necessary for the tailored glyco-functionalization of its S-layer. Here, the putative oligosaccharyl:S-layer protein transferase WsfB from the P. alvei S-layer glycosylation gene locus is characterized. The enzyme is proposed to catalyze the final step of the glycosylation pathway, transferring the elongated S-layer glycan onto distinct tyrosine O-glycosylation sites. Genetic knock-out of WsfB is shown to abolish glycosylation of the S-layer protein SpaA but not that of other glycoproteins present in P. alvei CCM 2051T, confining its role to the S-layer glycosylation pathway. A transmembrane topology model of the 781-amino acid WsfB protein is inferred from activity measurements of green fluorescent protein and phosphatase A fused to defined truncations of WsfB. This model shows an overall number of 13 membrane spanning helices with the Wzy_C domain characteristic of O-oligosaccharyl:protein transferases (O-OTases) located in a central extra-cytoplasmic loop, which both compares well to the topology of OTases from Gram-negative bacteria. Mutations in the Wzy C motif resulted in loss of WsfB function evidenced in reconstitution experiments in P. alvei ΔWsfB cells. Attempts to use WsfB for transferring heterologous oligosaccharides to its native S-layer target protein in Escherichia coli CWG702 and Salmonella enterica SL3749, which should provide lipid-linked oligosaccharide substrates mimicking to some extent those of the natural host, were not successful, possibly due to the stringent function of WsfB. Concluding, WsfB has all features of a bacterial O-OTase, making it the most probable candidate for the oligosaccharyl:S-layer protein transferase of P. alvei, and a promising candidate for the first O-OTase reported in Gram-positives.

The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051(T) possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051(T) cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix.

The gram-positive bacterium Paenibacillus alvei CCM 2051 T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β- d -Gal p -(1[α- d -Glc p -(1→6)]→4)-β- d -Man p NAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO 2 →4-β- d -Man p NAc-(1→4)]→3)-α- l -Rha p -(1→3)-α- l -Rha p -(1→3)-α- l -Rha p -(1→3)-β- d -Gal p -(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the sgsE S-layer gene promoter of Geobacillus stearothermophilus NRS 2004/3a and on the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. As a target gene, wsfP , encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional P. alvei CCM 2051 T wsfP mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of wsfP in the glycan-deficient P. alvei mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism P. alvei CCM 2051 T .

The periodontal pathogen Tannerella forsythia expresses several glycosidases which are linked to specific growth requirements and are involved in the invasion of host tissues. α-l-Fucosyl residues are exposed on various host glycoconjugates and, thus, the α-l-fucosidases predicted in the T. forsythia ATCC 43037 genome could potentially serve roles in host-pathogen interactions. We describe the molecular cloning and characterization of the putative fucosidase TfFuc1 (encoded by the bfo_2737 = Tffuc1 gene), previously reported to be present in an outer membrane preparation. In terms of sequence, this 51-kDa protein is a member of the glycosyl hydrolase family GH29. Using an artificial substrate, p-nitrophenyl-α-fucose (KM 670 μM), the enzyme was determined to have a pH optimum of 9.0 and to be competitively inhibited by fucose and deoxyfuconojirimycin. TfFuc1 was shown here to be a unique α(1,2)-fucosidase that also possesses α(1,6) specificity on small unbranched substrates. It is active on mucin after sialidase-catalyzed removal of terminal sialic acid residues and also removes fucose from blood group H. Following knock-out of the Tffuc1 gene and analyzing biofilm formation and cell invasion/adhesion of the mutant in comparison to the wild-type, it is most likely that the enzyme does not act extracellularly. Biochemically interesting as the first fucosidase in T. forsythia to be characterized, the biological role of TfFuc1 may well be in the metabolism of short oligosaccharides in the periplasm, thereby indirectly contributing to the virulence of this organism. TfFuc1 is the first glycosyl hydrolase in the GH29 family reported to be a specific α(1,2)-fucosidase.

Paenibacillus alvei CCM 2051(T) cells are decorated with a two-dimensional (2D) crystalline array comprised of the glycosylated S-layer protein SpaA. At its N terminus, SpaA possesses three consecutive surface layer (S-layer) homology (SLH) domains containing the amino acid motif TRAE, known to play a key role in cell wall binding, as well as the TVEE and TRAQ variations thereof. SpaA is predicted to be anchored to the cell wall by interaction of the SLH domains with a peptidoglycan (PG)-associated, nonclassical, pyruvylated secondary cell wall polymer (SCWP). In this study, we have analyzed the role of the three predicted binding motifs within the SLH domains by mutating them into TAAA motifs, either individually, pairwise, or all of them. Effects were visualized in vivo by homologous expression of chimeras made of the mutated S-layer proteins and enhanced green fluorescent protein and in an in vitro binding assay using His-tagged SpaA variants and native PG-containing cell wall sacculi that either contained SCWP or were deprived of it. Experimental data indicated that (i) the TRAE, TVEE, and TRAQ motifs are critical for the binding function of SLH domains, (ii) two functional motifs are sufficient for cell wall binding, regardless of the domain location, (iii) SLH domains have a dual-recognition function for the SCWP and the PG, and (iv) cell wall anchoring is not necessary for SpaA glycosylation. Additionally, we showed that the SLH domains of SpaA are sufficient for in vivo cell surface display of foreign proteins at the cell surface of P. alvei.

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., Author Summary Paenibacillus larvae is the most devastating bacterial pathogen of honey bees. However, the molecular interactions between infected larvae and P. larvae are poorly understood and little more than speculation exist concerning virulence factors. Recently, a putative S-layer protein has been identified in P. larvae. We here demonstrate that only representatives of P. larvae genotype ERIC II harbor a functional splA-gene and that SplA is a true S-layer protein with self-assembly capability. The presence of a functional S-layer protein is novel for P. larvae. When elucidating the biological function of SplA we broke new ground by establishing primary cell culture for pupal gut cells and by developing a genetic system for disruption of gene expression in this important honey bee pathogen. By using these novel methods we were able to prove that SplA serves as a shape-determining factor, mediates adhesion to host cells, and is a key virulence factor of P. larvae ERIC II. These results present an immense advancement in our understanding of P. larvae pathogenesis. Furthermore, we propose P. larvae as a model system for the analysis of the in vivo functions of S-layer proteins because P. larvae SlpA knockout-mutants retain viability and are thus suitable for functional studies.

Glycosylation is a frequent and heterogeneous posttranslational protein modification occurring in all domains of life. While protein N-glycosylation at asparagine and O-glycosylation at serine, threonine or hydroxyproline residues have been studied in great detail, only few data are available on O-glycosidic attachment of glycans to the amino acid tyrosine. In this study, we describe the identification and characterization of a bacterial protein tyrosine O-glycosylation system. In the Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051(T), a polysaccharide consisting of [-->3)-beta-d-Galp-(1[alpha-d-Glcp-(1-->6)] -->4)-beta-d-ManpNAc-(1-->] repeating units is O-glycosidically linked via an adaptor with the structure -[GroA-2-->OPO(2)-->4-beta-d-ManpNAc-(1-->4)] -->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->3)-alpha-l-Rhap-(1-->3)-beta-d-Galp-(1--> to specific tyrosine residues of the S-layer protein SpaA. A +AH4-24.3-kb S-layer glycosylation (slg) gene cluster encodes the information necessary for the biosynthesis of this glycan chain within 18 open reading frames (ORF). The corresponding translation products are involved in the biosynthesis of nucleotide-activated monosaccharides, assembly and export as well as in the transfer of the completed polysaccharide chain to the S-layer target protein. All ORFs of the cluster, except those encoding the nucleotide sugar biosynthesis enzymes and the ATP binding cassette (ABC) transporter integral transmembrane proteins, were disrupted by the insertion of the mobile group II intron Ll.LtrB, and S-layer glycoproteins produced in mutant backgrounds were analyzed by mass spectrometry. There is evidence that the glycan chain is synthesized in a process comparable to the ABC-transporter-dependent pathway of the lipopolysaccharide O-polysaccharide biosynthesis. Furthermore, with the protein WsfB, we have identified an O-oligosaccharyl:protein transferase required for the formation of the covalent beta-d-Gal-->Tyr linkage between the glycan chain and the S-layer protein.