Systematic site-directed mutagenesis of the Helicobacter pylori CagL protein of the Cag type IV secretion system identifies novel functional domains

The Cag Type IV secretion system, which contributes to inflammation and cancerogenesis during chronic infection, is one of the major virulence factors of the bacterial gastric pathogen Helicobacter pylori. We have generated and characterized a series of non-marked site-directed chromosomal mutants in H. pylori to define domains of unknown function of the essential tip protein CagL of the Cag secretion system. Characterizing the CagL mutants, we determined that their function to activate cells and transport the effector CagA was reduced to different extents. We identified three novel regions of the CagL protein, involved in its structural integrity, its possible interaction with the CagPAIT4SS pilus protein CagI, and in its binding to integrins and other host cell ligands. In particular two novel variable CagL motifs were involved in integrin binding, TSPSA, and TASLI, which is located opposite of its integrin binding motif RGD. We thereby defined functionally important subdomains within the CagL structure, which can be used to clarify CagL contributions in the context of other CagPAI proteins or for inhibition of the CagT4SS. This structure-function correlation of CagL domains can also be instructive for the functional characterization of other potential VirB5 orthologs whose structure is not yet known

Systematic site-directed mutagenesis of the Helicobacter pylori CagL protein of the Cag type IV secretion system identifies novel functional domains

The Cag Type IV secretion system, which contributes to inflammation and cancerogenesis during chronic infection, is one of the major virulence factors of the bacterial gastric pathogen Helicobacter pylori. We have generated and characterized a series of non-marked site-directed chromosomal mutants in H. pylori to define domains of unknown function of the essential tip protein CagL of the Cag secretion system. Characterizing the CagL mutants, we determined that their function to activate cells and transport the effector CagA was reduced to different extents. We identified three novel regions of the CagL protein, involved in its structural integrity, its possible interaction with the CagPAIT4SS pilus protein CagI, and in its binding to integrins and other host cell ligands. In particular two novel variable CagL motifs were involved in integrin binding, TSPSA, and TASLI, which is located opposite of its integrin binding motif RGD. We thereby defined functionally important subdomains within the CagL structure, which can be used to clarify CagL contributions in the context of other CagPAI proteins or for inhibition of the CagT4SS. This structure-function correlation of CagL domains can also be instructive for the functional characterization of other potential VirB5 orthologs whose structure is not yet known

RNAseq analysis of Aspergillus fumigatus in blood reveals a just wait and see resting stage behavior

Background: Invasive aspergillosis is started after germination of Aspergillus fumigatus conidia that are inhaled by susceptible individuals. Fungal hyphae can grow in the lung through the epithelial tissue and disseminate hematogenously to invade into other organs. Low fungaemia indicates that fungal elements do not reside in the bloodstream for long. Results: We analyzed whether blood represents a hostile environment to which the physiology of A. fumigatus has to adapt. An in vitro model of A. fumigatus infection was established by incubating mycelium in blood. Our model allowed to discern the changes of the gene expression profile of A. fumigatus at various stages of the infection. The majority of described virulence factors that are connected to pulmonary infections appeared not to be activated during the blood phase. Three active processes were identified that presumably help the fungus to survive the blood environment in an advanced phase of the infection: iron homeostasis, secondary metabolism, and the formation of detoxifying enzymes. Conclusions: We propose that A. fumigatus is hardly able to propagate in blood. After an early stage of sensing the environment, virtually all uptake mechanisms and energy-consuming metabolic pathways are shut-down. The fungus appears to adapt by trans-differentiation into a resting mycelial stage. This might reflect the harsh conditions in blood where A. fumigatus cannot take up sufficient nutrients to establish self-defense mechanisms combined with significant growth

On a Recent Construction of "Vacuum-like" Quantum Field States in Curved Spacetime

Afshordi, Aslanbeigi and Sorkin have recently proposed a construction of a distinguished "S-J state" for scalar field theory in (bounded regions of) general curved spacetimes. We establish rigorously that the proposal is well-defined on globally hyperbolic spacetimes or spacetime regions that can be embedded as relatively compact subsets of other globally hyperbolic spacetimes, and also show that, whenever the proposal is well-defined, it yields a pure quasifree state. However, by explicitly considering portions of ultrastatic spacetimes, we show that the S-J state is not in general a Hadamard state. In the specific case where the Cauchy surface is a round 3-sphere, we prove that the representation induced by the S-J state is generally not unitarily equivalent to that of a Hadamard state, and indeed that the representations induced by S-J states on nested regions of the ultrastatic spacetime also fail to be unitarily equivalent in general. The implications of these results are discussed.Comment: 25pp, LaTeX. v2 References added, typos corrected. To appear in Class Quantum Gravit

Cosmological horizons and reconstruction of quantum field theories

As a starting point, we state some relevant geometrical properties enjoyed by the cosmological horizon of a certain class of Friedmann-Robertson-Walker backgrounds. Those properties are generalised to a larger class of expanding spacetimes $M$ admitting a geodesically complete cosmological horizon \scrim common to all co-moving observers. This structure is later exploited in order to recast, in a cosmological background, some recent results for a linear scalar quantum field theory in spacetimes asymptotically flat at null infinity. Under suitable hypotheses on $M$, encompassing both the cosmological de Sitter background and a large class of other FRW spacetimes, the algebra of observables for a Klein-Gordon field is mapped into a subalgebra of the algebra of observables \cW(\scrim) constructed on the cosmological horizon. There is exactly one pure quasifree state $\lambda$ on \cW(\scrim) which fulfils a suitable energy-positivity condition with respect to a generator related with the cosmological time displacements. Furthermore $\lambda$ induces a preferred physically meaningful quantum state $\lambda_M$ for the quantum theory in the bulk. If $M$ admits a timelike Killing generator preserving \scrim, then the associated self-adjoint generator in the GNS representation of $\lambda_M$ has positive spectrum (i.e. energy). Moreover $\lambda_M$ turns out to be invariant under every symmetry of the bulk metric which preserves the cosmological horizon. In the case of an expanding de Sitter spacetime, $\lambda_M$ coincides with the Euclidean (Bunch-Davies) vacuum state, hence being Hadamard in this case. Remarks on the validity of the Hadamard property for $\lambda_M$ in more general spacetimes are presented.Comment: 32 pages, 1 figure, to appear on Comm. Math. Phys., dedicated to Professor Klaus Fredenhagen on the occasion of his 60th birthda

Comparative Genomics of Helicobacter pylori Strains of China Associated with Different Clinical Outcome

In this study, a whole-genome CombiMatrix Custom oligonucleotide tiling microarray with 90000 probes covering six sequenced Helicobacter pylori (H. pylori) genomes was designed. This microarray was used to compare the genomic profiles of eight unsequenced strains isolated from patients with different gastroduodenal diseases in Heilongjiang province of China. Since significant genomic variation was found among these strains, an additional 76 H. pylori strains associated with different clinical outcomes were isolated from various provinces of China. These strains were tested by polymerase chain reaction to demonstrate this distinction. We identified several highly variable regions in strains associated with gastritis, gastric ulceration, and gastric cancer. These regions are associated with genes involved in the bacterial type I, type II, and type III R-M systems. They were also associated with the virB gene, which lies on the well-studied cag pathogenic island. While previous studies have reported on the diverse genetic characterization of this pathogenic island, in this study, we find that it is conserved in all strains tested by microarray. Moreover, a number of genes involved in the type IV secretion system, which is related to horizontal DNA transfer between H. pylori strains, were identified in the comparative analysis of the strain-specific genes. These findings may provide insight into new biomarkers for the prediction of gastric diseases

Genomic structure and insertion sites of Helicobacter pylori prophages from various geographical origins

We present the full genomic sequences, insertion sites and phylogenetic analysis of 28 prophages found in H. pylori isolates from patients of distinct disease types, ranging from gastritis to gastric cancer, and geographic origins, covering most continents. The gentic diversity of H pylori is known to be influenced by these genomic elements including prophages who’s geneomes range from 22.6 to 33.0 Kbp. There was a high conservation of integration site shared in over 50% of cases with greater than 40% or prophage genomes harbouring insertion sequences (IS). Furthermore prophage genomes present a robust phylogeographic pattern, revealing four distinct clusters: one African, one Asian and two European prophage populations. There was evidence of recombination within the genome of some prophages, which resulted in genome mosaics composed by different populations, which may yield additional H. pylori phenotypes

A Global Overview of the Genetic and Functional Diversity in the Helicobacter pylori cag Pathogenicity Island

The Helicobacter pylori cag pathogenicity island (cagPAI) encodes a type IV secretion system. Humans infected with cagPAI–carrying H. pylori are at increased risk for sequelae such as gastric cancer. Housekeeping genes in H. pylori show considerable genetic diversity; but the diversity of virulence factors such as the cagPAI, which transports the bacterial oncogene CagA into host cells, has not been systematically investigated. Here we compared the complete cagPAI sequences for 38 representative isolates from all known H. pylori biogeographic populations. Their gene content and gene order were highly conserved. The phylogeny of most cagPAI genes was similar to that of housekeeping genes, indicating that the cagPAI was probably acquired only once by H. pylori, and its genetic diversity reflects the isolation by distance that has shaped this bacterial species since modern humans migrated out of Africa. Most isolates induced IL-8 release in gastric epithelial cells, indicating that the function of the Cag secretion system has been conserved despite some genetic rearrangements. More than one third of cagPAI genes, in particular those encoding cell-surface exposed proteins, showed signatures of diversifying (Darwinian) selection at more than 5% of codons. Several unknown gene products predicted to be under Darwinian selection are also likely to be secreted proteins (e.g. HP0522, HP0535). One of these, HP0535, is predicted to code for either a new secreted candidate effector protein or a protein which interacts with CagA because it contains two genetic lineages, similar to cagA. Our study provides a resource that can guide future research on the biological roles and host interactions of cagPAI proteins, including several whose function is still unknown