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.
Recyclable markers based on site-specific recombination allow repetitive gene targeting in filamentous fungi. Here we describe for the first time functionality of the bacterial recombination system employing  serine recombinase acting on six recognition sequences (-rec/six) in a fungal host, the human pathogen Aspergillus fumigatus, and its use in establishing a self-excising resistance marker cassette for serial gene replacement.
The Helicobacter pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) involved in host interaction and pathogenicity. Previously, seven cag PAI proteins were identified as homologs of Agrobacterium tumefaciens Vir proteins, which form a paradigm T4SS. The T pilus composed of the processed VirB2 pilin is an external structural part of the A. tumefaciens T4SS. In H. pylori, cag-dependent assembly of pili has not been observed so far, nor has a pilin (VirB2) ortholog been characterized. We have here identified, using a motif-based search, an H. pylori cag island protein (HP0546) that possesses sequence and predicted structural similarities to VirB2-like pilins of other T4SSs. The HP0546 protein displays interstrain variability in its terminal domains. HP0546 was expressed as a FLAG-tagged fusion protein in Escherichia coli, A. tumefaciens, and H. pylori and was detected as either two or three bands of different molecular masses in the insoluble fraction, indicating protein processing. As reported previously, isogenic H. pylori mutants in the putative cag pilin gene had reduced abilities to induce cag PAI-dependent interleukin-8 secretion in gastric epithelial cells. Fractionation analysis of H. pylori, using a specific antiserum raised against an N-terminal HP0546 peptide, showed that the protein is partially surface exposed and that its surface localization depended upon an intact cag system. By immunoelectron microscopy, HP0546 was localized in surface appendages, with surface exposure of an N-terminal epitope. Pronounced strain-to-strain variability of this predicted surfaceexposed part of HP0546 indicates a strong selective pressure for variation in vivo.
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 CagPAI T4SS 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.
SummaryMoulds are characterized by their saprophytic lifestyle that is based on osmotrophy. Among them, Aspergillus fumigatus has emerged as the leading cause of fungal infections in the presence of an underlying immunodeficiency. To assess the role of its nutritional versatility for virulence, transcriptional profiling studies in the presence of varying sources of nitrogen were carried out and revealed an extensive reprogramming of the fungal transcriptome when shifting to a proteinaceous growth substrate. Transcripts encoding metabolic activities were predominantly upregulated, as were proteinases and transport activities. To probe whether fundamental aspects of its osmotrophic lifestyle, that is, extracellular proteolysis and uptake of oligopeptides, are required for A. fumigatus pathogenicity, serial gene replacements were carried out, which eventually yielded an octuple deletion mutant ablated for the opt gene family. This strain displayed no growth defect on various substrates, but supplementary reduction of extracellular proteolytic activity by additional deletion of the prtT gene revealed a synthetic phenotype on porcine lung tissue agar. Virulence studies in a murine model of pulmonary aspergillosis did not disclose any attenuation in virulence of these deletants. Our data emphasize a high degree of redundancy encoded by the A. fumigatus genome that secures nutrient supply for growth and, therefore, virulence.
BackgroundInvasive 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.ResultsWe 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi10.1186/s12864-015-1853-1) contains supplementary material, which is available to authorized users.
Die Kombination massiv-paralleler long-read-Sequenzierungstechnologie mit short-read-Technologie eröffnet neue Wege in der Identifikation und quantitativen Analyse von mRNA-Transkripten und hilft so beim Verständnis von Genexpression.Useful combination of massive parallel long-read sequencing technology and short-read technology opens new ways for identification and quantitative analysis of mRNA transcripts and helps understanding gene expression.ó Die hoch dynamische Entwicklung der Sequenzierungstechnologien, ehemals angetrieben von der Sequenzierung ganzer Genome, eröffnet immer neue Analysemöglichkeiten. Die am Markt verfügbaren Sequenziergeräte haben systembedingte Unterschiede, die man bei der Transkriptionsanalyse kombinieren und sinnvoll nutzen kann. Die Anwendungsmöglichkeiten, die Band-breite und die Detailtiefe der Ergebnisse möchten wir hier anhand einer neu entwickelten Methode darstellen.Abb. 1: Schematische Darstellung der de novo-Expressionsanalyse mittels kombinierter Sequenzierung zweier unterschiedlicher cDNA-Banken auf verschiedenen Systemen. Weitere Erläuterungen siehe Text.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.