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Chapter I: Introduction

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DOCTORAL SCHOOL IN BIOLOGY Biology Applied to Human Health XXVII Cycle DUAL RNA-SEQUENCING APPROACH FOR DISSECTING NONTYPEABLE HAEMOPHILUS INFLUENZAE AND HOST CELL TRANSCRIPTOMES Presented by: Buket Baddal Supervisors: Dr. Alfredo Pezzicoli Dr. Marco Soriani Director: Prof. Paolo Visca I dedicate this thesis to my beloved parents who inspired and supported me unconditionally in countless ways. II ABSTRACT Characterization of host-pathogen interactions is critical for the development of next-generation therapies and vaccines. Classical approaches involve the use of transformed cell lines and/or animal models which may not reflect the complexity and response of the human host. The Gram-negative bacterium nontypeable Haemophilus influenzae (NTHi) commonly resides as a commensal in the human nasopharynx from where it can disseminate to local organs to cause a wide spectrum of diseases including otitis media, chronic obstructive pulmonary disease, cystic fibrosis and bronchitis. Successful colonization by NTHi depends on its ability to adhere and adapt to the respiratory tract mucosa, which serves as a frontline defense against respiratory pathogens. In opportunistic infections, colonization is followed by either a paracellular route across the epithelial barrier or invasion of non-phagocytic and epithelial cells. However, the temporal events associated to a successful colonization are far from being fully characterized. Recent improvements in tissue engineering techniques including the development of differentiated primary cell cultures and organotypic 3D cellular models have significantly increased our understanding of microbial pathogenesis by providing physiologically relevant representations of human upper airway tissue. Bridging of these techniques with the currently available next-generation sequencing technologies is a conceptually novel approach for studying infection-linked transcriptome alterations in such systems. Massively parallel cDNA sequencing (RNA-seq) offers the possibility of comprehensive and simultaneous whole genome transcriptional profiling of both host and invading pathogen, and overcomes the existing technical and economical limitations of probe-dependent methods. Taking advantage of the technological advances, we reconstituted the ciliated human bronchial epithelium in vitro using primary bronchial epithelial cells to simultaneously monitor the infection-linked global changes in NTHi and infected host epithelia gene expression by dual RNA-seq. Acquisition of a total of nearly 2,5 billion sequences allowed construction of high-resolution strand-specific transcriptome maps of NTHi during infection of host mucosal surface, and monitoring of metabolic as well as stress-induced host-adaptation strategies of this pathogen. The initial stage of colonization was characterized by the binding of NTHi to cilia. Temporal analysis of host mRNA signatures revealed consequent remodeling of target cell cytoskeleton and junction complexes elicited by bacterial infection, with a profound effect on intermediate filament network of bronchial epithelium. At later stage of infection when bacteria start to internalize, NTHi down-regulated the central metabolism and increased the expression of transporters indicating alterations in the bacterial metabolic regime due to the evolving substrate availability. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms including the transport of exogenous glutathione and the activation of the toxin-antitoxin system. Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs regulated during human host infection in Haemophilus species. Our data by providing a robust and comprehensive catalogue of regulatory and adaptive responses reflecting the complex crosstalk between the host and invading pathogen, may provide important insights into NTHi pathogenesis and the development of efficacious preventive strategies. tissue engineering | host-pathogen interaction | dual RNA-seq | NTHi transcriptome | host response III RIASSUNTO La caratterizzazione delle interazioni ospite-patogeno è fondamentale per lo sviluppo di nuove terapie e vaccini di ultima generazione. Approcci classici prevedono l'utilizzo di linee cellulari trasformate e/o modelli animali che non possono riflettere la complessità e la risposta dell'ospite umano. Il batterio Gram-negativo nontypeable Haemophilus influenzae (NTHi) risiede comunemente come commensale nel rinofaringe umano da dove può diffondere ai tessuti adiacenti provocando un ampio spettro di malattie, tra cui otite media, COPD, fibrosi cistica e bronchite. La capacità di colonizzazione di NTHi dipende dalla sua abilità di aderire ed adattarsi alla mucosa delle vie respiratorie, che ha la funzione di prima linea difensiva contro gli agenti patogeni del tratto respiratorio. Durante le infezioni opportunistiche, la colonizzazione è seguita da un attraversamento paracellulare della barriera epiteliale o dall’invasione di cellule non fagocitiche ed epiteliali. Tuttavia gli eventi temporali associati ad una colonizzazione produttiva non sono stati ancora completamente caratterizzati. Recenti miglioramenti delle tecniche di ingegneria tissutale in vitro, tra cui lo sviluppo di colture cellulari primarie differenziate e modelli cellulari 3D organotipici, hanno aumentato significativamente la nostra comprensione della patogenesi microbica fornendo rappresentazioni fisiologicamente rilevanti di tessuto delle vie aeree superiori umani. La combinazione di queste tecniche con le tecnologie di sequenziamento di nuova generazione è un approccio concettualmente innovativo per studiare le alterazioni del trascrittoma durante un’infezione. Il sequenziamento tramite RNA-seq offre la possibilità di analizzare simultaneamente il trascrittoma di ospite e patogeno, superando le limitazioni tecniche relative alla separazione dei campioni eucariotici da quelli procariotici. In questo studio l'epitelio ciliato bronchiale umano è stato ricostituito in vitro utilizzando cellule epiteliali bronchiali primarie ed usato come modello di infezione per NTHi. Successivamente, tramite dual-RNA sequencing, sono state analizzate le variazioni dell’espressione genica di entrambi ospite e patogeno. L’acquisizione di un totale di quasi 2,5 miliardi di sequenze ha permesso la costruzione di mappe ad alta risoluzione del trascrittoma di NTHi durante l'infezione dell’epitelio, la caratterizzazione dell’adattamento metabolico nonché dei pathways di adattamento allo stress cellulare. Nella fase iniziale della colonizzazione è stata osservata l’adesione di NTHi alle cellule ciliate. L’analisi temporale del trascrittoma dell’epitelio ha evidenziato il rimodellamento del citoscheletro, in particolare come riarrangiamento della rete dei filamenti intermedi. Nelle fasi successive dell’infezione, quando i batteri iniziano ad essere internalizzati, NTHi down- regola il metabolismo centrale e aumenta l'espressione di trasportatori specifici per alcuni substrati disponibili nel nuovo ambiente. Contemporaneamente l'ambiente ossidativo generato dalle cellule infettate induce l’espressione batterica di meccanismi di difesa dallo stress fra cui il trasporto di glutatione esogeno e l'attivazione dei sistemi tossina/anti-tossina. Abbiamo inoltre identificato una serie di nuovi small RNAs batterici fortemente regolati durante l'infezione. Questo lavoro fornisce la caratterizzazione dei trascrittomi dell’ospite e del patogeno nelle prime fasi d’infezione, e suggerisce lo sviluppo di strategie mirate per prevenire le infezioni di NTHi. ingegneria tissutale | interazione ospite-patogeno | dual RNA-seq | NTHi trascrittoma | risposta dell'ospite IV ACKNOWLEDGEMENTS This work was conducted at Novartis Vaccines, Siena in In Vitro Cell Biology Group, headed by Dr. Marco Soriani. I am very thankful for his welcoming in the lab, providing me with the opportunity to work at the frontier of current vaccine development and cutting-edge technologies, but most importantly for always being there to help. I equally thank Prof. Christoph Tang who has awarded me with Marie Curie Fellowship and allowed me to perform my thesis project under the framework of European Community's Seventh Framework Programme “EIMID ITN” [European Institute of Microbiology and Infectious Diseases Initial Training Network, FP7-PEOPLE-2010-264388], as well as his enriching collaboration and hosting me in this laboratory in University of Oxford. I am extremely grateful to my supervisor, Dr. Alfredo Pezzicoli for his guidance over the course of my PhD, for his never-wavering optimism, giving me the initiative to grow as an independent research scientist and above all, for teaching me how much fun microscopy can be. I also thank Prof. Paolo Visca from University of Roma Tre for all the support and valuable advice he has given me during my PhD. I would like to acknowledge all my lab members (past and present) of the In Vitro Cell Biology group at Novartis, particularly Lucia Lapazio, Maria Valeri, Pasquale Marrazzo, Silvia Rossi Paccani and Alessandra Greco for the friendly atmosphere they created in the lab, their scientific feedback, and moral support during tough days. I am especially grateful to my precious friends Christina Merakou, Tanja Dapa, Cristina Faralla, Magdalena Kasendra, Riccardo Barrile and Giacomo Golfieri for their kindhearted help and care, all the fun we had together, keeping
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  • Aberrant Sialylation in Cancer: Biomarker and Potential Target for Therapeutic Intervention?

    Aberrant Sialylation in Cancer: Biomarker and Potential Target for Therapeutic Intervention?

    cancers Review Aberrant Sialylation in Cancer: Biomarker and Potential Target for Therapeutic Intervention? Silvia Pietrobono * and Barbara Stecca * Tumor Cell Biology Unit, Core Research Laboratory, Institute for Cancer Research and Prevention (ISPRO), Viale Pieraccini 6, 50139 Florence, Italy * Correspondence: [email protected] (S.P.); [email protected] (B.S.); Tel.: +39-055-7944568 (S.P.); +39-055-7944567 (B.S.) Simple Summary: Sialylation is a post-translational modification that consists in the addition of sialic acid to growing glycan chains on glycoproteins and glycolipids. Aberrant sialylation is an established hallmark of several types of cancer, including breast, ovarian, pancreatic, prostate, colorectal and lung cancers, melanoma and hepatocellular carcinoma. Hypersialylation can be the effect of increased activity of sialyltransferases and results in an excess of negatively charged sialic acid on the surface of cancer cells. Sialic acid accumulation contributes to tumor progression by several paths, including stimulation of tumor invasion and migration, and enhancing immune evasion and tumor cell survival. In this review we explore the mechanisms by which sialyltransferases promote cancer progression. In addition, we provide insights into the possible use of sialyltransferases as biomarkers for cancer and summarize findings on the development of sialyltransferase inhibitors as potential anti-cancer treatments. Abstract: Sialylation is an integral part of cellular function, governing many biological processes Citation: Pietrobono, S.; Stecca, B. including cellular recognition, adhesion, molecular trafficking, signal transduction and endocytosis. Aberrant Sialylation in Cancer: Sialylation is controlled by the levels and the activities of sialyltransferases on glycoproteins and Biomarker and Potential Target for lipids. Altered gene expression of these enzymes in cancer yields to cancer-specific alterations of Therapeutic Intervention? Cancers glycoprotein sialylation.
  • Histidinol Dehydrogenase from Neurospora Crassa

    Histidinol Dehydrogenase from Neurospora Crassa

    Fungal Genetics Reports Volume 3 Article 1 Histidinol dehydrogenase from Neurospora crassa E. H. Creaser R. B. Drysdale Follow this and additional works at: https://newprairiepress.org/fgr This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. Recommended Citation Creaser, E. H., and R.B. Drysdale (1963) "Histidinol dehydrogenase from Neurospora crassa," Fungal Genetics Reports: Vol. 3, Article 1. https://doi.org/10.4148/1941-4765.2139 This Research Note is brought to you for free and open access by New Prairie Press. It has been accepted for inclusion in Fungal Genetics Reports by an authorized administrator of New Prairie Press. For more information, please contact [email protected]. Histidinol dehydrogenase from Neurospora crassa Abstract Histidinol dehydrogenase from Neurospora crassa This research note is available in Fungal Genetics Reports: https://newprairiepress.org/fgr/vol3/iss1/1 RESEARCH NOTES Creaser, E.H. and R. B. Drysdale. Histidinol It is thought that the locus histidine-3 controls hvo --__ dehydrogenase from Neurosooro w. functions in Neurospora, the first of these being in the early stages ot histidine biosynthesis and largely unknown at present. The second function is to direct the formation of the terminal enzyme in the sequence- histidinol dehydrogenase. We have studied the purification and some properties of this enzyme. The enzyme con be extracted from wet mycelium by grinding with gloss powder or from dried mycelium by extraction with pH 9. I Tris buffer. The extract is treated with 0.05M MnC12 to prec:pitate nucleic acids and unwonted proteins. Ammonium sulphate is added to 50% saturation and the precipitate discorded.