Phylogeographic diversity and mosaicism of the Helicobacter pylori tfs integrative and conjugative elements.

Background: The genome of the gastric pathogen Helicobacter pylori is characterised by considerable variation of both gene sequence and content, much of which is contained within three large genomic islands comprising the cag pathogenicity island (cagPAI) and two mobile integrative and conjugative elements (ICEs) termed tfs3 and tfs4. All three islands are implicated as virulence factors, although whereas the cagPAI is well characterised, understanding of how the tfs elements influence H. pylori interactions with different human hosts is significantly confounded by limited definition of their distribution, diversity and structural representation in the global H. pylori population. Results: To gain a global perspective of tfs ICE population dynamics we established a bioinformatics workflow to extract and precisely define the full tfs pan-gene content contained within a global collection of 221 draft and complete H. pylori genome sequences. Complete (ca. 35-55kbp) and remnant tfs ICE clusters were reconstructed from a dataset comprising >12,000 genes, from which orthologous gene complements and distinct alleles descriptive of different tfs ICE types were defined and classified in comparative analyses. The genetic variation within defined ICE modular segments was subsequently used to provide a complete description of tfs ICE diversity and a comprehensive assessment of their phylogeographic context. Our further examination of the apparent ICE modular types identified an ancient and complex history of ICE residence, mobility and interaction within particular H. pylori phylogeographic lineages and further, provided evidence of both contemporary inter-lineage and inter-species ICE transfer and displacement. Conclusions: Our collective results establish a clear view of tfs ICE diversity and phylogeographic representation in the global H. pylori population, and provide a robust contextual framework for elucidating the functional role of the tfs ICEs particularly as it relates to the risk of gastric disease associated with different tfs ICE genotypes

A role for the tfs3 ICE-encoded type IV secretion system in pro-inflammatory signalling by the Helicobacter pylori Ser/Thr kinase, CtkA

Two distinct type IV secretion systems (T4SSs) can be identified in certain Helicobacter pylori strains, encoded on mobile genetic elements termed tfs3 and tfs4. Although their function remains unknown, both have been implicated in clinical outcomes of H. pylori infection. Here we provide evidence that the Tfs3 T4SS is required for activity of the pro-inflammatory Ser/Thr kinase protein, CtkA, in a gastric epithelial cell infection model. Previously, purified recombinant CtkA protein has been shown to upregulate NF-kappaB signalling and induce TNF-alpha and IL-8 cytokine secretion from cultured macrophages suggesting that it may potentiate the H. pylori-mediated inflammatory response. In this study, we show that CtkA expressed from its native host, H. pylori has a similar capacity for stimulation of a pro-inflammatory response from gastric epithelial cells. CtkA interaction was found to be dependent upon a complement of tfs3 T4SS genes, but independent of the T4SSs encoded by either tfs4 or the cag pathogenicity island. Moreover, the availability of CtkA for host cell interaction was shown to be conditional upon the carboxyl-terminus of CtkA, encoding a putative conserved secretion signal common to other variably encoded Tfs3 proteins. Collectively, our observations indicate a role for the Tfs3 T4SS in CtkA-mediated pro-inflammatory signalling by H. pylori and identify CtkA as a likely Tfs3 T4SS secretion substrate

CtkA expressed from <i>H</i>. <i>pylori</i> stimulates pro-inflammatory cytokine secretion from gastric epithelial cells.

<p>Wild-type <i>H</i>. <i>pylori</i>, <i>cagE-</i>deficient strains and isogenic derivatives constitutively expressing GSK-CtkA were assessed for their ability to induce IL-8 secretion from MKN28 cells in co-culture. Supernatants were sampled 48 h post infection and assayed for IL-8 secretion. In comparison with the parent <i>cagE</i> mutants alone, all <i>tfs3</i>(+) strains expressing GSK-CtkA (AB31, 10A, AB5) showed a trend towards induction of elevated levels of IL-8 when expressing GSK-CtkA, whereas a <i>tfs3</i>(-) strain (64) did not. The difference in levels of induced IL-8 due to GSK-CtkA expression was significant when expressed from strain AB5. Levels of IL-8 attributable to GSK-CtkA were modest relative to <i>cag</i>PAI-mediated responses from all wild-type strains. Graph shows means and SDs from three independent experiments, each performed in triplicate.</p

Sequence alignments showing conservation of C-terminal sequence between <i>tfs3</i> ICE-encoded proteins.

<p>Sequence alignment demonstrates conservation in the last 25 C-terminal amino acid residues of CtkA, PZ39 and Fic proteins encoded in <i>tfs3</i> gene clusters from a representative selection of <i>H</i>. <i>pylori</i> strains.</p

Signalling pathways involved in CtkA-mediated stimulation of IL-8 secretion.

<p>MKN28 cell monolayers remained untreated (-), or were treated with chemical inhibitors for NF-κB (6 amino-4-(4-phenoxyphenylethylamino) quinazoline), JNK (SP600125) and MEK1 (U0126) for 1 h before and during infection with <i>H</i>. <i>pylori</i> strain AB5Δ<i>cagE</i>::<i>gsk</i>-<i>ctkA</i>. IL-8 levels were subsequently assessed by ELISA. Basal levels of IL-8 detected in uninfected supernatants are also indicated for reference (‘Cells’). Graph shows means and SDs from three independent experiments, each performed in triplicate. <i>p</i> values indicate significant differences in the presence of inhibitor compared with infection alone.</p

Pro-inflammatory signalling in response to CtkA requires the Tfs3 type IV secretion system and the C-terminus of CtkA.

<p>MKN28 cells were co-cultured with <i>H</i>. <i>pylori</i> AB5Δ<i>cagE</i> derivative strains (MOI = 20) for 48 or 72 h prior to determination of IL-8 concentrations in supernatants by ELISA. Both inactivation of (A) <i>tfs3 virB9</i> and (B) deletion of the last 23 C-terminal amino acid residues of CtkA in strain AB5Δ<i>cagE</i>::gsk-<i>ctkA</i>(1–906) resulted in abrogation of IL-8 secretion. (C) Similar effects were observed following co-culture of the complement of AB5 strains with gastric AGS cells although IL-8 responses for all strains were markedly lower. Graph shows means and SDs from three independent experiments, each performed in triplicate. <i>p</i> values indicate significant differences in IL-8 levels compared to the AB5Δ<i>cagE</i>::<i>gsk-ctkA</i> strain. (D) Western immunoblot analysis of 100X concentrated supernatants from <i>H</i>. <i>pylori</i> strains AB5Δ<i>cagE</i>, AB5Δ<i>cagE</i>:<i>gsk</i>-<i>ctkA</i>, AB5Δ<i>virB9</i>Δ<i>cagE</i>:<i>gsk</i>-<i>ctkA</i> and AB5Δ<i>cagE</i>:<i>gsk</i>-<i>ctkA</i>_1-906 (Lanes 1–4 respectively) grown in RPMI 1640. Blots were probed with anti-GSK, anti-GAPDH and anti-CagA specific antiserum. Detection of GAPDH and CagA in supernatants indicates non-secretory release of intracellular protein in all strain backgrounds.</p

Spatial scales of marine conservation management for breeding seabirds

Knowing the spatial scales at which effective management can be implemented is fundamental for conservation planning. This is especially important for mobile species, which can be exposed to threats across large areas, but the space use requirements of different species can vary to an extent that might render some management approaches inefficient. Here the space use patterns of seabirds were examined to provide guidance on whether conservation management approaches should be tailored for taxonomic groups with different movement characteristics. Seabird tracking data were synthesised from 5419 adult breeding individuals of 52 species in ten families that were collected in the Atlantic Ocean basin between 1998 and 2017. Two key aspects of spatial distribution were quantified, namely how far seabirds ranged from their colony, and to what extent individuals from the same colony used the same areas at sea. There was evidence for substantial differences in patterns of space-use among the ten studied seabird families, indicating that several alternative conservation management approaches are needed. Several species exhibited large foraging ranges and little aggregation at sea, indicating that area-based conservation solutions would have to be extremely large to adequately protect such species. The results highlight that short-ranging and aggregating species such as cormorants, auks, some penguins, and gulls would benefit from conservation approaches at relatively small spatial scales during their breeding season. However, improved regulation of fisheries, bycatch, pollution and other threats over large spatial scales will be needed for wide-ranging and dispersed species such as albatrosses, petrels, storm petrels and frigatebirds