Analysis of the cell surface layer ultrastructure of the oral pathogen Tannerella forsythia

The Gram-negative oral pathogen Tannerella forsythia is decorated with a 2D crystalline surface (S-) layer, with two different S-layer glycoprotein species being present. Prompted by the predicted virulence potential of the S-layer, this study focused on the analysis of the arrangement of the individual S-layer glycoproteins by a combination of microscopic, genetic, and biochemical analyses. The two S-layer genes are transcribed into mRNA and expressed into protein in equal amounts. The S-layer was investigated on intact bacterial cells by transmission electron microscopy, by immune fluorescence microscopy, and by atomic force microscopy. The analyses of wild-type cells revealed a distinct square S-layer lattice with an overall lattice constant of 10.1 ± 0.7 nm. In contrast, a blurred lattice with a lattice constant of 9.0 nm was found on S-layer single-mutant cells. This together with in vitro self-assembly studies using purified (glyco)protein species indicated their increased structural flexibility after self-assembly and/or impaired self-assembly capability. In conjunction with TEM analyses of thin-sectioned cells, this study demonstrates the unusual case that two S-layer glycoproteins are co-assembled into a single S-layer. Additionally, flagella and pilus-like structures were observed on T. forsythia cells, which might impact the pathogenicity of this bacterium

Austria

In Austria, per capita consumption of wood products is comparatively high and has increased considerably during the last decades. One reason is the high apparent consumption of wood by Austrian intermediary products producers. It is also an effect of an increasingly wood friendly culture in wood construction, a society that has overall positive attitude to wood as a material, amongst others. Consumption of wood products is to some parts dependent on the image of these products in the eyes of consumers, especially vis-à-vis substitution products. Here, the shift to urban societies, technological developments and competitive behaviour of substitute producers have for some time left wood with a not-so-favourable image of being old-fashioned. Recent PR campaigns have successfully tackled this problem. The fact that Austria has a diverse landscape ranging from plains to high alpine regions creates a wide range of recreational, environmental and protective services. However, these are generally not exploited on a commercial basis but embedded in legal and customary rights and often provided by the state

Glycobiology Aspects of the Periodontal Pathogen Tannerella forsythia

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\u27s 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

Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees.

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

Adhesion of <i>P. larvae</i> 04-309 Δ<i>spl</i>A and <i>P. larvae</i> 04-309 wild-type to primary pupal gut cells.

<p>(A) Vitality of primary pupal gut cells after 6 days of cultivation was analyzed using Mitotracker Red FM (Invitrogen) to specifically label active mitochondria indicative for live cells. Cells were microscopically analyzed using Differential Interference Contrast (DIC) (left) as well as TexRed (mitochondria) and DAPI (nuclei) filters to visualize fluorescence signals (right). (B) Percentage of <i>P. larvae</i> 03-189 wild-type (03-189 wt; ERIC I, naturally SplA-deficient) and <i>P. larvae</i> 04-309 Δ<i>spl</i>A (SplA knockout mutant) associated to pupal gut cells after 60 min of incubation, extensive washing, and cell lysis; results are presented as mean ± SEM of three independent-experiments and are related to cell-associated bacteria of the wild-type strain <i>P. larvae</i> 04-309 (04-309 wt; ERIC II), i.e. the amount of <i>P. larvae</i> 04-309 cell-associated bacteria in each experiment equalled 100%. Data were analyzed by one-way analysis of variance (ANOVA) followed by the Bonferroni post-hoc test (***p<0.001).</p

Analysis of the genomic and protein sequence of <i>P. larvae</i> SplA.

<p>(A) In contrast to <i>P. larvae</i> ERIC II representatives, all <i>P. larvae</i> ERIC I strains investigated showed an additional adenin at position 894 (bold and highlighted by a star), leading to a frameshift mutation causing a premature translational stop at TAA (bold and underlined). This obviously results in the observed lack of a functional SplA in representatives of <i>P. larvae</i> ERIC I. (B) For SLH-domain prediction of SplA the CDD (Conserved domains and protein classification, NCBI) was applied. Two predicted domains with homology to SLH domains of other <i>Bacillaceae</i> (<i>B. anthracis</i>, <i>B. weihenstephanensis</i>) but also to SLH domains of other genera were identified. <i>P. larvae</i> SLH 1 domain of SplA (upper panel) includes residues 117–164 of SplA and exhibits homologies to several SLH domains of <i>Bacillaceae</i>. <i>P. larvae</i> SLH 2 domain of SplA (lower panel) includes residues 188–220 and exhibits homologies not only to other <i>Bacillaceae</i> but also to SLH domains of other genera. Identical amino acids are marked in black, conserved amino acids are highlighted in dark grey and blocks of similar residues are presented with a light grey background. Strains showing homologous domains are listed alphabetically.</p

TEM micrographs of negatively stained self-assembly products of recombinant purified S-layer protein SplA.

<p>(A) Cylindrical self-assembly products of the recombinant purified S-layer protein SplA of <i>P. larvae</i> 04-309 (ERIC II); (B) Enlarged region of a cylinder showing the regular lattice; (C) Power spectrum of the S-layer patch indicated in (B); (D) S-layer lattice reconstruction.</p