11 research outputs found
Genexpressionsanalysen der frĂŒhen angeborenen Immunantwort des Haushuhns induziert durch eine Infektion mit Salmonella enteritidis mit Hilfe der Microarray Technologie
Salmonella infections in humans arise through chicken-based food such as eggs, egg-products, or chicken meat. The most common cause for these infections is Salmonella enteritidis, and the aim of this study has been to analyze the early innate immune response of chickens induced via this pathogen. Note that S. enteritidis is a host-adapted serovar, which only causes clinical findings in young chickens during their first week of life; adult chickens do not get sick, but may nevertheless act as inapparent infected carriers.
We studied the reaction from the chicken immune system on S. enteritidis, using macrophage cultures as well as tissue samples of infected adult chickens. The gene expression studies were carried out by an âAgilent 4x44K chicken microarrayâ method.
In our in vitro studies, we infected primary macrophages with S. enteritidis for 4 hours, using a MOI of 10. The gene expression studies resulted in the inductions of interleukins (IL1ÎČ, IL6, IL12p40, IL18), of chemokines (CCL1, CCL4 (K203), CCL20, CXCL8 (IL8), CXCL13), of some members of the tumor-nekrose-factor-superfamily (TNFSF), and of some toll-like receptors (TLR). Hence the cells have an inflammatory reaction. Particularly prominent were the expression changes of K60 (IL8 homolog), K203 (chCCLi2, MIP-1ÎČ), CCL20, and TL1a (TNFSF15). Finally, infected macrophages expressed a group of typical Th1-cytokines, including IL12p40, IL18, and IFN-Îł.
In further analysis of our data, we focused on cytokines, chemokines, and members of the TNF-superfamily. In the ceca we found similar expression patterns within these three groups as was previously found for them in the macrophages study.
In our in vivo studies, we infected chickens that were 8 weeks old and already had a well developed immune system. They were infected in the crop using a dose of 107 salmonella. At 5, 12, 24, and 48 hours of infection, we sampled the ceca and cecal tonsils for the bacterial, histological, and gene expression analyses. Already at 5 hours p.i., we were (for all but one animal) able to isolate bacteria from the ceca-tissue. The bacterial load reached its maximum at 12 hours p.i.. The infection of the cecal-tissues was confirmed in the histology, both by the detection of bacteria and by the occurrence of inflammatory cells. However, using histology, we could not detect any bacteria in cecal tonsils, which suggests that no infection was present in these organs. This suggestion was confirmed in gene expression analyses. When comparing the gene expression studies of cecal tonsils and ceca, the former showed lower counts of differential regulated genes (Tab. 11). Both their count maxima occurred at 12 hour p.i though. Moreover, at this time 41 significant regulated pathways had been identified..
In summary, the in vitro and the in vivo experiment both resulted in an initial inflammatory reaction, as well as in a typical Th1-cytokines reaction. To investigate functional characterisation of named candidate genes, in the first instance CCL20, CXCL8, K60, K203, and TL1a, future analyses of the innate immune response should involve them. This may contribute to a better understanding of the successful defense mechanisms against S. enteritidis in chicken, which may help to contain the amount of salmonellosis in humans
A comprehensive analysis of Helicobacter pylori plasticity zones reveals that they are integrating conjugative elements with intermediate integration specificity
Background: The human gastric pathogen Helicobacter pylori is a paradigm for chronic bacterial infections. Its persistence in the stomach mucosa is facilitated by several mechanisms of immune evasion and immune modulation, but also by an unusual genetic variability which might account for the capability to adapt to changing environmental conditions during long-term colonization. This variability is reflected by the fact that almost each infected individual is colonized by a genetically unique strain. Strain-specific genes are dispersed throughout the genome, but clusters of genes organized as genomic islands may also collectively be present or absent. Results: We have comparatively analysed such clusters, which are commonly termed plasticity zones, in a high number of H. pylori strains of varying geographical origin. We show that these regions contain fixed gene sets, rather than being true regions of genome plasticity, but two different types and several subtypes with partly diverging gene content can be distinguished. Their genetic diversity is incongruent with variations in the rest of the genome, suggesting that they are subject to horizontal gene transfer within H. pylori populations. We identified 40 distinct integration sites in 45 genome sequences, with a conserved heptanucleotide motif that seems to be the minimal requirement for integration. Conclusions: The significant number of possible integration sites, together with the requirement for a short conserved integration motif and the high level of gene conservation, indicates that these elements are best described as integrating conjugative elements (ICEs) with an intermediate integration site specificity
CagI Is an Essential Component of the Helicobacter pylori Cag Type IV Secretion System and Forms a Complex with CagL
Helicobacter pylori, the causative agent of type B gastritis, peptic ulcers, gastric adenocarcinoma and MALT lymphoma, uses the Cag type IV secretion system to induce a strong proinflammatory response in the gastric mucosa and to inject its effector protein CagA into gastric cells. CagA translocation results in altered host cell gene expression profiles and cytoskeletal rearrangements, and it is considered as a major bacterial virulence trait. Recently, it has been shown that binding of the type IV secretion apparatus to integrin receptors on target cells is a crucial step in the translocation process. Several bacterial proteins, including the Cag-specific components CagL and CagI, have been involved in this interaction. Here, we have examined the localization and interactions of CagI in the bacterial cell. Since the cagI gene overlaps and is co-transcribed with the cagL gene, the role of CagI for type IV secretion system function has been difficult to assess, and conflicting results have been reported regarding its involvement in the proinflammatory response. Using a marker-free gene deletion approach and genetic complementation, we show now that CagI is an essential component of the Cag type IV secretion apparatus for both CagA translocation and interleukin-8 induction. CagI is distributed over soluble and membrane-associated pools and seems to be partly surface-exposed. Deletion of several genes encoding essential Cag components has an impact on protein levels of CagI and CagL, suggesting that both proteins require partial assembly of the secretion apparatus. Finally, we show by co-immunoprecipitation that CagI and CagL interact with each other. Taken together, our results indicate that CagI and CagL form a functional complex which is formed at a late stage of secretion apparatus assembly
Human Practice. Digital Ecologies. Our Future. : 14. Internationale Tagung Wirtschaftsinformatik (WI 2019) : Tagungsband
Erschienen bei: universi - UniversitÀtsverlag Siegen. - ISBN: 978-3-96182-063-4Aus dem Inhalt:
Track 1: Produktion & Cyber-Physische Systeme
Requirements and a Meta Model for Exchanging Additive Manufacturing Capacities
Service Systems, Smart Service Systems and Cyber- Physical SystemsâWhatâs the difference? Towards a Unified Terminology
Developing an Industrial IoT Platform â Trade-off between Horizontal and Vertical Approaches
Machine Learning und Complex Event Processing: Effiziente Echtzeitauswertung am Beispiel Smart Factory
Sensor retrofit for a coffee machine as condition monitoring and predictive maintenance use case
Stakeholder-Analyse zum Einsatz IIoT-basierter Frischeinformationen in der Lebensmittelindustrie
Towards a Framework for Predictive Maintenance Strategies in Mechanical Engineering - A Method-Oriented Literature Analysis
Development of a matching platform for the requirement-oriented selection of cyber physical systems for SMEs
Track 2: Logistic Analytics
An Empirical Study of Customersâ Behavioral Intention to Use Ridepooling Services â An Extension of the Technology Acceptance Model
Modeling Delay Propagation and Transmission in Railway Networks
What is the impact of company specific adjustments on the acceptance and diffusion of logistic standards?
Robust Route Planning in Intermodal Urban Traffic
Track 3: Unternehmensmodellierung & Informationssystemgestaltung (Enterprise Modelling & Information Systems Design)
Work System Modeling Method with Different Levels of Specificity and Rigor for Different Stakeholder Purposes
Resolving Inconsistencies in Declarative Process Models based on Culpability Measurement
Strategic Analysis in the Realm of Enterprise Modeling â On the Example of Blockchain-Based Initiatives for the Electricity Sector
Zwischenbetriebliche Integration in der Möbelbranche: Konfigurationen und Einflussfaktoren
Novicesâ Quality Perceptions and the Acceptance of Process Modeling Grammars
Entwicklung einer Definition fĂŒr Social Business Objects (SBO) zur Modellierung von Unternehmensinformationen
Designing a Reference Model for Digital Product Configurators
Terminology for Evolving Design Artifacts
Business Role-Object Specification: A Language for Behavior-aware Structural Modeling of Business Objects
Generating Smart Glasses-based Information Systems with BPMN4SGA: A BPMN Extension for Smart Glasses Applications
Using Blockchain in Peer-to-Peer Carsharing to Build Trust in the Sharing Economy
Testing in Big Data: An Architecture Pattern for a Development Environment for Innovative, Integrated and Robust Applications
Track 4: Lern- und Wissensmanagement (e-Learning and Knowledge Management)
eGovernment Competences revisited â A Literature Review on necessary Competences in a Digitalized Public Sector
Say Hello to Your New Automated Tutor â A Structured Literature Review on Pedagogical Conversational Agents
Teaching the Digital Transformation of Business Processes: Design of a Simulation Game for Information Systems Education
Conceptualizing Immersion for Individual Learning in Virtual Reality
Designing a Flipped Classroom Course â a Process Model
The Influence of Risk-Taking on Knowledge Exchange and Combination
Gamified Feedback durch Avatare im Mobile Learning
Alexa, Can You Help Me Solve That Problem? - Understanding the Value of Smart Personal Assistants as Tutors for Complex Problem Tasks
Track 5: Data Science & Business Analytics
Matching with Bundle Preferences: Tradeoff between Fairness and Truthfulness
Applied image recognition: guidelines for using deep learning models in practice
Yield Prognosis for the Agrarian Management of Vineyards using Deep Learning for Object Counting
Reading Between the Lines of Qualitative Data â How to Detect Hidden Structure Based on Codes
Online Auctions with Dual-Threshold Algorithms: An Experimental Study and Practical Evaluation
Design Features of Non-Financial Reward Programs for Online Reviews: Evaluation based on Google Maps Data
Topic Embeddings â A New Approach to Classify Very Short Documents Based on Predefined Topics
Leveraging Unstructured Image Data for Product Quality Improvement
Decision Support for Real Estate Investors: Improving Real Estate Valuation with 3D City Models and Points of Interest
Knowledge Discovery from CVs: A Topic Modeling Procedure
Online Product Descriptions â Boost for your Sales?
EntscheidungsunterstĂŒtzung durch historienbasierte Dienstreihenfolgeplanung mit Pattern
A Semi-Automated Approach for Generating Online Review Templates
Machine Learning goes Measure Management: Leveraging Anomaly Detection and Parts Search to Improve Product-Cost Optimization
Bedeutung von Predictive Analytics fĂŒr den theoretischen Erkenntnisgewinn in der IS-Forschung
Track 6: Digitale Transformation und Dienstleistungen
Heuristic Theorizing in Software Development: Deriving Design Principles for Smart Glasses-based Systems
Mirroring E-service for Brick and Mortar Retail: An Assessment and Survey
Taxonomy of Digital Platforms: A Platform Architecture Perspective
Value of Star Players in the Digital Age
Local Shopping Platforms â Harnessing Locational Advantages for the Digital Transformation of Local Retail Outlets: A Content Analysis
A Socio-Technical Approach to Manage Analytics-as-a-Service â Results of an Action Design Research Project
Characterizing Approaches to Digital Transformation: Development of a Taxonomy of Digital Units
Expectations vs. Reality â Benefits of Smart Services in the Field of Tension between Industry and Science
Innovation Networks and Digital Innovation: How Organizations Use Innovation Networks in a Digitized Environment
Characterising Social Reading Platformsâ A Taxonomy-Based Approach to Structure the Field
Less Complex than Expected â What Really Drives IT Consulting Value
Modularity Canvas â A Framework for Visualizing Potentials of Service Modularity
Towards a Conceptualization of Capabilities for Innovating Business Models in the Industrial Internet of Things
A Taxonomy of Barriers to Digital Transformation
Ambidexterity in Service Innovation Research: A Systematic Literature Review
Design and success factors of an online solution for cross-pillar pension information
Track 7: IT-Management und -Strategie
A Frugal Support Structure for New Software Implementations in SMEs
How to Structure a Company-wide Adoption of Big Data Analytics
The Changing Roles of Innovation Actors and Organizational Antecedents in the Digital Age
Bewertung des Kundennutzens von Chatbots fĂŒr den Einsatz im Servicedesk
Understanding the Benefits of Agile Software Development in Regulated Environments
Are Employees Following the Rules? On the Effectiveness of IT Consumerization Policies
Agile and Attached: The Impact of Agile Practices on Agile Team Membersâ Affective Organisational Commitment
The Complexity Trap â Limits of IT Flexibility for Supporting Organizational Agility in Decentralized Organizations
Platform Openness: A Systematic Literature Review and Avenues for Future Research
Competence, Fashion and the Case of Blockchain
The Digital Platform Otto.de: A Case Study of Growth, Complexity, and Generativity
Track 8: eHealth & alternde Gesellschaft
Security and Privacy of Personal Health Records in Cloud Computing Environments â An Experimental Exploration of the Impact of Storage Solutions and Data Breaches
Patientenintegration durch Pfadsysteme
Digitalisierung in der StressprĂ€vention â eine qualitative Interviewstudie zu Nutzenpotenzialen
User Dynamics in Mental Health Forums â A Sentiment Analysis Perspective
Intent and the Use of Wearables in the Workplace â A Model Development
Understanding Patient Pathways in the Context of Integrated Health Care Services - Implications from a Scoping Review
Understanding the Habitual Use of Wearable Activity Trackers
On the Fit in Fitness Apps: Studying the Interaction of Motivational Affordances and Usersâ Goal Orientations in Affecting the Benefits Gained
Gamification in Health Behavior Change Support Systems - A Synthesis of Unintended Side Effects
Investigating the Influence of Information Incongruity on Trust-Relations within Trilateral Healthcare Settings
Track 9: Krisen- und KontinuitÀtsmanagement
Potentiale von IKT beim Ausfall kritischer Infrastrukturen: Erwartungen, Informationsgewinnung und Mediennutzung der Zivilbevölkerung in Deutschland
Fake News Perception in Germany: A Representative Study of Peopleâs Attitudes and Approaches to Counteract Disinformation
Analyzing the Potential of Graphical Building Information for Fire Emergency Responses: Findings from a Controlled Experiment
Track 10: Human-Computer Interaction
Towards a Taxonomy of Platforms for Conversational Agent Design
Measuring Service Encounter Satisfaction with Customer Service Chatbots using Sentiment Analysis
Self-Tracking and Gamification: Analyzing the Interplay of Motivations, Usage and Motivation Fulfillment
Erfolgsfaktoren von Augmented-Reality-Applikationen: Analyse von Nutzerrezensionen mit dem Review-Mining-Verfahren
Designing Dynamic Decision Support for Electronic Requirements Negotiations
Who is Stressed by Using ICTs? A Qualitative Comparison Analysis with the Big Five Personality Traits to Understand Technostress
Walking the Middle Path: How Medium Trade-Off Exposure Leads to Higher Consumer Satisfaction in Recommender Agents
Theory-Based Affordances of Utilitarian, Hedonic and Dual-Purposed Technologies: A Literature Review
Eliciting Customer Preferences for Shopping Companion Apps: A Service Quality Approach
The Role of Early User Participation in Discovering Software â A Case Study from the Context of Smart Glasses
The Fluidity of the Self-Concept as a Framework to Explain the Motivation to Play Video Games
Heart over Heels? An Empirical Analysis of the Relationship between Emotions and Review Helpfulness for Experience and Credence Goods
Track 11: Information Security and Information Privacy
Unfolding Concerns about Augmented Reality Technologies: A Qualitative Analysis of User Perceptions
To (Psychologically) Own Data is to Protect Data: How Psychological Ownership Determines Protective Behavior in a Work and Private Context
Understanding Data Protection Regulations from a Data Management Perspective: A Capability-Based Approach to EU-GDPR
On the Difficulties of Incentivizing Online Privacy through Transparency: A Qualitative Survey of the German Health Insurance Market
What is Your Selfie Worth? A Field Study on Individualsâ Valuation of Personal Data
Justification of Mass Surveillance: A Quantitative Study
An Exploratory Study of Risk Perception for Data Disclosure to a Network of Firms
Track 12: Umweltinformatik und nachhaltiges Wirtschaften
KommunikationsfĂ€den im Nadelöhr â Fachliche Prozessmodellierung der Nachhaltigkeitskommunikation am Kapitalmarkt
Potentiale und Herausforderungen der Materialflusskostenrechnung
Computing Incentives for User-Based Relocation in Carsharing
Sustainabilityâs Coming Home: Preliminary Design Principles for the Sustainable Smart District
Substitution of hazardous chemical substances using Deep Learning and t-SNE
A Hierarchy of DSMLs in Support of Product Life-Cycle Assessment
A Survey of Smart Energy Services for Private Households
Door-to-Door Mobility Integrators as Keystone Organizations of Smart Ecosystems: Resources and Value Co-Creation â A Literature Review
Ein EntscheidungsunterstĂŒtzungssystem zur ökonomischen Bewertung von Mieterstrom auf Basis der Clusteranalyse
Discovering Blockchain for Sustainable Product-Service Systems to enhance the Circular Economy
Digitale RĂŒckverfolgbarkeit von Lebensmitteln: Eine verbraucherinformatische Studie
Umweltbewusstsein durch audiovisuelles Content Marketing? Eine experimentelle Untersuchung zur Konsumentenbewertung nachhaltiger Smartphones
Towards Predictive Energy Management in Information Systems: A Research Proposal
A Web Browser-Based Application for Processing and Analyzing Material Flow Models using the MFCA Methodology
Track 13: Digital Work - Social, mobile, smart
On Conversational Agents in Information Systems Research: Analyzing the Past to Guide Future Work
The Potential of Augmented Reality for Improving Occupational First Aid
Prevent a Vicious Circle! The Role of Organizational IT-Capability in Attracting IT-affine Applicants
Good, Bad, or Both? Conceptualization and Measurement of Ambivalent User Attitudes Towards AI
A Case Study on Cross-Hierarchical Communication in Digital Work Environments
âShow Me Your People Skillsâ - Employing CEO Branding for Corporate Reputation Management in Social Media
A Multiorganisational Study of the Drivers and Barriers of Enterprise Collaboration Systems-Enabled Change
The More the Merrier? The Effect of Size of Core Team Subgroups on Success of Open Source Projects
The Impact of Anthropomorphic and Functional Chatbot Design Features in Enterprise Collaboration Systems on User Acceptance
Digital Feedback for Digital Work? Affordances and Constraints of a Feedback App at InsurCorp
The Effect of Marker-less Augmented Reality on Task and Learning Performance
Antecedents for Cyberloafing â A Literature Review
Internal Crowd Work as a Source of Empowerment - An Empirical Analysis of the Perception of Employees in a Crowdtesting Project
Track 14: GeschÀftsmodelle und digitales Unternehmertum
Dividing the ICO Jungle: Extracting and Evaluating Design Archetypes
Capturing Value from Data: Exploring Factors Influencing Revenue Model Design for Data-Driven Services
Understanding the Role of Data for Innovating Business Models: A System Dynamics Perspective
Business Model Innovation and Stakeholder: Exploring Mechanisms and Outcomes of Value Creation and Destruction
Business Models for Internet of Things Platforms: Empirical Development of a Taxonomy and Archetypes
Revitalizing established Industrial Companies: State of the Art and Success Principles of Digital Corporate Incubators
When 1+1 is Greater than 2: Concurrence of Additional Digital and Established Business Models within Companies
Special Track 1: Student Track
Investigating Personalized Price Discrimination of Textile-, Electronics- and General Stores in German Online Retail
From Facets to a Universal Definition â An Analysis of IoT Usage in Retail
Is the Technostress Creators Inventory Still an Up-To-Date Measurement Instrument? Results of a Large-Scale Interview Study
Application of Media Synchronicity Theory to Creative Tasks in Virtual Teams Using the Example of Design Thinking
TrustyTweet: An Indicator-based Browser-Plugin to Assist Users in Dealing with Fake News on Twitter
Application of Process Mining Techniques to Support Maintenance-Related Objectives
How Voice Can Change Customer Satisfaction: A Comparative Analysis between E-Commerce and Voice Commerce
Business Process Compliance and Blockchain: How Does the Ethereum Blockchain Address Challenges of Business Process Compliance?
Improving Business Model Configuration through a Question-based Approach
The Influence of Situational Factors and Gamification on Intrinsic Motivation and Learning
Evaluation von ITSM-Tools fĂŒr Integration und Management von Cloud-Diensten am Beispiel von ServiceNow
How Software Promotes the Integration of Sustainability in Business Process Management
Criteria Catalog for Industrial IoT Platforms from the Perspective of the Machine Tool Industry
Special Track 3: Demos & Prototyping
Privacy-friendly User Location Tracking with Smart Devices: The BeaT Prototype
Application-oriented robotics in nursing homes
Augmented Reality for Set-up Processe
Mixed Reality for supporting Remote-Meetings
Gamification zur Motivationssteigerung von Werkern bei der Betriebsdatenerfassung
Automatically Extracting and Analyzing Customer Needs from Twitter: A âNeedminingâ Prototype
GaNEsHA: Opportunities for Sustainable Transportation in Smart Cities
TUCANA: A platform for using local processing power of edge devices for building data-driven services
Demonstrator zur Beschreibung und Visualisierung einer kritischen Infrastruktur
Entwicklung einer alltagsnahen persuasiven App zur Bewegungsmotivation fĂŒr Ă€ltere Nutzerinnen und Nutzer
A browser-based modeling tool for studying the learning of conceptual modeling based on a multi-modal data collection approach
Exergames & Dementia: An interactive System for People with Dementia and their Care-Network
Workshops
Workshop Ethics and Morality in Business Informatics (Workshop Ethik und Moral in der Wirtschaftsinformatik â EMoWIâ19)
Model-Based Compliance in Information Systems - Foundations, Case Description and Data Set of the MobIS-Challenge for Students and Doctoral Candidates
Report of the Workshop on Concepts and Methods of Identifying Digital Potentials in Information Management
Control of Systemic Risks in Global Networks - A Grand Challenge to Information Systems Research
Die Mitarbeiter von morgen - Kompetenzen kĂŒnftiger Mitarbeiter im Bereich Business Analytics
Digitaler Konsum: Herausforderungen und Chancen der Verbraucherinformati
CagI is an essential component of the Cag type IV secretion apparatus.
<p>(A) AGS cells were infected for 4 h with the indicated strains at a multiplicity of infection of 100, and infection lysates were examined by immunoblot for the presence of CagA and CagA tyrosine phosphorylation (PTyr). Infected cells were also examined by light microscopy for induction of the hummingbird phenotype. (B) Culture supernatants of AGS cells infected with the indicated <i>H. pylori</i> strains as above were tested for their IL-8 content. IL-8 values induced by wild-type P12 were set to 100%, and other values are given in relation to wild-type levels. Data shown are average values of at least three independent experiments with standard deviations.</p
Localization of CagI and CagL in bacterial cell fractions.
<p>(A) <i>H. pylori</i> cells grown in liquid culture for 24â48 hours were lysed by ultrasonication and subsequently fractionated into soluble and insoluble proteins by ultracentrifugation. Ultracentrifugation pellets containing membrane-associated and other insoluble proteins were extracted with 1% triton X-100 to separate outer membrane-associated (TX100 insol) from inner membrane-associated (TX100 sol) proteins. Comparable amounts of each fraction were analysed by immunoblot for their CagI and CagL content. As controls, immunoblots against the outer membrane-associated proteins CagX and AlpB, and the partly soluble and partly inner membrane-associated protein RecA were used. Representative immunoblots are shown. CagI and CagL bands are indicated by arrowheads. (B) Ultracentrifugation pellets were resuspended and subjected to isopycnic density gradient centrifugation on 25â60% sucrose gradients. Fractions were collected from the gradients and analyzed by immunoblotting with the indicated antisera. CagI and CagL bands are indicated by arrowheads. (C) Bacteria were subjected to limited proteolytic digestion by proteinase K. Equal amounts of untreated control cells (P12) and proteinase K-treated bacteria (P12 PK) were analyzed by immunoblot with the indicated antisera.</p
Presence of several Cag components influences CagI and CagL protein levels.
<p>Whole cell lysates of equal amounts of the wild-type strain P12 and of isogenic mutants in single <i>cag</i> genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035341#pone-0035341-t001" target="_blank">Table 1</a>) were separated by SDS-PAGE and examined by immunoblotting with the anti-CagI and anti-CagL antisera, respectively. Representative immunoblots are shown. Note that the <i>cagI</i> mutant shown here was generated with plasmid pWS327. Arrowheads indicate the positions of CagI and CagL protein bands, respectively.</p
Operon organization of the <i>cagH</i>, <i>cagI</i> and <i>cagL</i> genes.
<p>(A) Gene arrangement of the <i>cag</i> pathogenicity island in <i>H. pylori</i> strain P12. Genes are represented as arrows, and their designations are indicated below. The operon containing <i>cagI</i> may comprise five (<i>cagF</i>, <i>cagG</i>, <i>cagH</i>, <i>cagI</i> and <i>cagL</i>) or even more genes, but <i>cagH</i>, <i>cagI</i> and <i>cagL</i> are particularly tightly associated since they have overlapping reading frames or Shine-Dalgarno (SD) sequences. Translational starts are indicated by arrows. (B) A <i>cagI</i> deletion mutant containing a terminatorless chloramphenicol resistance cassette <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035341#pone.0035341-Fischer2" target="_blank">[16]</a> was generated by transformation with plasmid pSO171. A polyclonal rabbit antiserum raised against CagI was used for immunoblot analysis of whole cell lysates of the P12 wild-type strain or isogenic <i>cagA</i> or <i>cagI</i> mutants. The same lysates were examined by Western blot for the presence of CagL using the polyclonal rabbit antiserum AK271. CagI and CagL protein bands, respectively, are marked by arrowheads. Note that both antisera recognize cross-reactive bands, but anti-CagI does not react with CagL or vice versa (data not shown). (C) Plasmid constructs used for generation and complementation of <i>cagI</i> mutants. A counterselection strategy was used for generating marker-free deletion mutants. Mutants generated with plasmids pWS320 and pWS326 retain the 3âČ part of <i>cagI</i>, whereas the mutant generated with plasmid pWS327 has a complete deletion without any <i>cagI</i> traces. For complementation of <i>cagI</i> mutants <i>in trans</i>, the <i>cagI</i> gene was cloned under the control of the <i>cagA</i> promoter and integrated into the <i>recA</i> gene (plasmid pWS322).</p
Analysis of CagI and CagL production, and of <i>cagL</i> transcription, in <i>cagI</i> mutants and complemented strains.
<p>(A) Whole cell lysates of the P12 wild-type strain and the indicated <i>cagI</i> mutants or complemented mutants were examined for CagI and CagL production by immunoblot analysis. Numbers in brackets indicate the plasmids used for mutant generation or complementation. (B) The indicated strains were grown in liquid culture to mid-exponential phase, and harvested for total RNA preparation and cDNA synthesis. Transcript levels of <i>cagL</i> were analyzed by quantitative real-time PCR (qPCR) and normalized to levels of 16S-rRNA for each cDNA sample. Bars represent mean values and standard deviations of <i>cagL</i> transcript levels in relation to wild-type levels for at least three independent RNA preparations.</p