Examining c-di-GMP and possible quorum sensing regulation in Pseudomonas fluorescens SBW25:links between intra and inter-cellular regulation benefits community cooperative activities such as biofilm formation

Bacterial success in colonizing complex environments requires individual response to micro-scale conditions as well as community-level cooperation to produce large-scale structures such as biofilms. Connecting individual and community responses could be achieved by linking the intracellular sensory and regulatory systems mediated by bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) and other compounds of individuals with intercellular quorum sensing (QS) regulation controlling populations. There is growing evidence to suggest that biofilm formation by many pseudomonads is regulated by both intra and intercellular systems, though in the case of the model Pseudomonas fluorescens SBW25 Wrinkly Spreader in which mutations increasing c-di-GMP levels result in the production of a robust cellulose-based air-liquid interface biofilm, no evidence for the involvement of QS regulation has been reported. However, our recent review of the P. fluorescens SBW25 genome has identified a potential QS regulatory pathway and other QS–associated genes linked to c-di-GMP homeostasis, and QS signal molecules have also been identified in culture supernatants. These findings suggest a possible link between c-di-GMP and QS regulation in P. fluorescens SBW25 which might allow a more sophisticated and responsive control of cellulose production and biofilm formation when colonising the soil and plant-associated environments P. fluorescens SBW25 normally inhabits.Анализ ц-ди-ГМФ и возможного чувства кворума у Pseudomonas fluorescens SBW 25: связь между внутри и межклеточной регуляцией способствует кооперативному поведению в сообществе и формированию биоплёнкиУспешность бактериальной колонизации сложных экониш требует индивидуального ответа на изменения условий на микроуровне равно как и кооперации на уровне сообщества для продукции таких крупно масштабных структур как биоплёнки. Координация индивидуальных ответ ов и ответов сообщества может быть достигнута путем связывания внутриклеточных сенсорных и регуляторных систем, опосредуемых бис-(3',5')-циклическим димерным гуанозинмонофосфатом (ц-ди-ГМФ) и другими соединениями индивидуумов с межклеточной регуляцией - чувством кворума (ЧК), контролирующем популяци ю. Накапливается всё больше доказательств того, что формирование биопленки многими псевдомонадами регулируется как внутри клеточными, так и меж клеточными регуляторными системами, хотя в случае модельной Pseudomonas fluorescens SBW25 Wrinkly Spreader, у которой мутации, повышающ ие уровни ц-ди-ГМФ, приводят к созданию прочной целлюлозной биоплёнки на границе раздела фаз воздух-жидкость, не было обнаружено ни ка кого свидетельства вовлечения кворум-зависимой регуляции. Однако наш недавний обзор генома P. fluorescens SBW25 выявил потенциальный ЧК-зависимый регуляторный пу ть и другие ЧК-зависимые гены, связанные с гомеостазом ц-ди-ГМФ, а молекулы ЧК-сигналинга были идентифицированы в культуре. Эти данные свидетельствуют о возможной связи между ц-ди-ГМФ-регуляцией и ЧК у P. fluorescens SBW25, что позволяет более сложный и гибкий контроль над продукцией целлюлозы и образовани ем биопленки при колонизации почв и экониш, aссоциированных с растениям и, - естественными средами обитания P. fluorescens SBW25

Comparative genomic analysis of novel Acinetobacter symbionts : A combined systems biology and genomics approach

Acknowledgements This work was supported by University of Delhi, Department of Science and Technology- Promotion of University Research and Scientific Excellence (DST-PURSE). V.G., S.H. and U.S. gratefully acknowledge the Council for Scientific and Industrial Research (CSIR), University Grant Commission (UGC) and Department of Biotechnology (DBT) for providing research fellowship.Peer reviewedPublisher PD

Pseudomonas aeruginosa infection and persistence mechanisms in cystic fibrosis patients

Pseudomonas aeruginosa is an environmentally-ubiquitous organism which causes opportunistic infections in humans, with a predisposition towards chronically infecting the cystic fibrosis lung. Its high phenotypic and genotypic plasticity facilitates long-term survival, and chronic P. aeruginosa respiratory infections are associated with increased morbidity and mortality. Understanding the drivers and mechanisms of P. aeruginosa adaptation in relation to disease progression is critical for the development of novel detection methods and treatment strategies. This thesis studies the phenotypic and genotypic adaptation of P. aeruginosa isolates from CF patients, exploring how bacterial characteristics relate to infection stage and severity. By performing whole genome sequence analysis of ten isolates from diverse CF infection backgrounds, common genetic adaptations are identified which occur regardless of infection stage, and analysis of non-coding sequence mutation rate upstream of quorum sensing-regulated genes suggests that certain intragenic sequences may be selectively mutated. This work also investigates whether acute pulmonary exacerbation (or treatment thereof) drives behavioural adaptation of P. aeruginosa, and whether P. aeruginosa phenotypes during these acute periods of infection are similar between patients. Additionally, this work explores the value and implications of detecting P. aeruginosa quorum sensing molecules in respiratory samples. A DNA-encoded biosensor system is optimised for the detection of the P. aeruginosa quorum sensing molecule 3-oxododecanoyl-homoserine lactone (3OC12-HSL) in CF sputum, and an inverse correlation between sputum 3OC12-HSL levels and P. aeruginosa isolate antibiotic resistance is identified. We also report a correlation between quorum sensing and cyclic-di-GMP signalling, relating this to the regulation of biofilm formation and the acute-to-chronic lifestyle switch. Overall, this thesis investigates certain P. aeruginosa lifestyles and adaptations in the context of CF respiratory infection, with a particular focus on the use of quorum sensing signalling – both by the bacterium as a mechanism of lifestyle adjustment, and by us for the monitoring of infections.Open Acces

Knowledge as the Currency of Managing Risk: a Novel Framework to Unite Quality Risk Management and Knowledge Management

In a manner of speaking, knowledge is the currency of managing risk and an organization that is risk-focused will want to apply the best of what it knows to assess those risks, identify appropriate risk controls and evaluate the performance of those controls. An organization that effectively manages knowledge should be able to recognize and proactively apply new learnings to better anticipate risks. This is particularly important in the manufacture of medicinal products. Since the publication of ICH Q10 in 2010, QRM and KM have been positioned as coenablers to the Pharmaceutical Quality System. However, in practice these two disciplines have remained largely distinct and disconnected. This paper presents a novel way to consider Quality Risk Management (QRM) and Knowledge Management (KM) which represents their true interdependencies, and which has the potential to deliver more effective and risk-based control strategies in a more synergistic and effective manner. This paper advocates for the need to strengthen the relationship between QRM and KM. In order to better understand the synergistic relationship between QRM and KM, a Knowledge Management process model is first proposed to envision KM akin to the familiar representation of QRM in ICH Q9 . Following this model, a framework is presented in the form of a Risk-Knowledge Infinity Cycle which serves to visualise and understand the QRM-KM relationship. It is the authors belief that treating QRM and KM in this way has a variety of potential benefits for biopharmaceutical companies, including improved risk-based decision making, facilitating evidence-based risk reduction and increased process knowledge, leading to less uncertainty and subjectivity in QRM outputs. This should ultimately result in more effective risk-based control strategies and more reliable manufacturing processes, which potentially lead to increased protection – and other benefits including product availability and value – for patients

Report of the NATO Advanced Research Workshop Piestany, Slovakia: 18-20 May 2000

Ye

Genetic Dissection of the Regulatory Network Associated with High c-di-GMP Levels in Pseudomonas putida KT2440

Most bacteria grow in nature forming multicellular structures named biofilms. The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key player in the regulation of the transition from planktonic to sessile lifestyles and this regulation is crucial in the development of biofilms. In Pseudomonas putida KT2440, Rup4959, a multidomain response regulator with diguanylate cyclase activity, when overexpressed causes an increment in the intracellular levels of c-di-GMP that gives rise to a pleiotropic phenotype consisting of increased biofilm formation and crinkly colony morphology. In a broad genomic screen we have isolated mutant derivatives that lose the crinkly morphology, designed as cfc (crinkle free colony). A total of 19 different genes have been identified as being related with the emergence of the cfc phenotype either because the expression or functionality of Rup4959 is compromised, or due to a lack of transduction of the c-di-GMP signal to downstream elements involved in the acquisition of the phenotype. Discernment between these possibilities was investigated by using a c-di-GMP biosensor and by HPLC-MS quantification of the second messenger. Interestingly five of the identified genes encode proteins with AAA+ ATPase domain. Among the bacterial determinants found in this screen are the global transcriptional regulators GacA, AlgU and FleQ and two enzymes involved in the arginine biosynthesis pathway. We present evidences that this pathway seems to be an important element to both the availability of the free pool of the second messenger c-di-GMP and to its further transduction as a signal for biosynthesis of biopolimers. In addition we have identified an uncharacterized hybrid sensor histidine kinase whose phosphoaceptor conserved histidine residue has been shown in this work to be required for in vivo activation of the orphan response regulator Rup4959, which suggests these two elements constitute a two-component phosphorelay system.This work was supported by grants BFU2010-17946 and BFU2013-43469-P from Plan Nacional de I+D+I (Spanish Government) and by EDFR funds. OH-R was supported by a FPI fellowship and LB-M by predoctoral fund from Junta de Andalucía. MAM was supported by the Postdoctoral Juan de la Cierva Spanish Research Program (JCI-2012-11815).Peer reviewedPeer Reviewe

Topoisomerase II inhibitors induce DNA damage-dependent interferon responses circumventing Ebola virus immune evasion

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication

An Audience with Pharmaceutical Regulators, Academia and Industry 2019: the Role of Quality Risk Management (QRM) and Knowledge Management (KM) in Medicinal Product Realisation for Patients in the 21st. Century

A monograph based on a seminar organised by the School of Chemistry & Pharmaceutical Science, Technological University Dublin, with the Health Products Regulatory Authority, and with Regulatory Science Ireland, 4th. April, 2019.https://arrow.tudublin.ie/ditpress/1009/thumbnail.jp

Qualification of academic facilities for small-scale automated manufacture of autologous cell-based products

Academic centres, hospitals and small companies, as typical development settings for UK regenerative medicine assets, are significant contributors to the development of autologous cell-based therapies. Often lacking the appropriate funding, quality assurance heritage or specialist regulatory expertise, qualifying aseptic cell processing facilities for Good Manufacturing Practice (GMP) compliance is a significant challenge. The qualification of a new Cell Therapy Manufacturing Facility (CTMF) with automated processing capability, the first of its kind in a UK academic setting, provides a unique demonstrator for the qualification of small-scale, automated facilities for GMP compliant manufacture of autologous cell-based products in these settings. This paper shares our experiences in qualifying the CTMF, focussing on our approach to streamlining the qualification effort, the challenges, project delays and inefficiencies we encountered and the subsequent lessons learned

Unique reporter-based sensor platforms to monitor signalling in cells

Introduction: In recent years much progress has been made in the development of tools for systems biology to study the levels of mRNA and protein, and their interactions within cells. However, few multiplexed methodologies are available to study cell signalling directly at the transcription factor level. <p/>Methods: Here we describe a sensitive, plasmid-based RNA reporter methodology to study transcription factor activation in mammalian cells, and apply this technology to profiling 60 transcription factors in parallel. The methodology uses two robust and easily accessible detection platforms; quantitative real-time PCR for quantitative analysis and DNA microarrays for parallel, higher throughput analysis. <p/>Findings: We test the specificity of the detection platforms with ten inducers and independently validate the transcription factor activation. <p/>Conclusions: We report a methodology for the multiplexed study of transcription factor activation in mammalian cells that is direct and not theoretically limited by the number of available reporters