Mycobacteria are classified into tuberculous mycobacteria and non-tuberculous mycobacteria (NTM). Most NTM are environmental bacteria found in soil and water sources; they are saprophytic organisms that are generally non-pathogenic to humans. However, pathological conditions such as cystic fibrosis or chronic lung diseases can promote the onset of NTM infections. In particular, Mycobacterium abscessus and Mycobacterium avium are the main contributors to the increasing incidence of NTM infections. The lungs represent the most commonly affected site, although NTM can also cause ocular, cutaneous, and disseminated infections. Mycobacteria exhibit a natural resistance to many antibiotic molecules, due to the peculiar structure of their cell wall, their ability to form biofilms, their potential intracellular lifestyle, and the formation of granulomas in the host’s lungs, all of which reduce drug efficacy. In this way, bacteria can replicate within macrophages, evading the immune response. Furthermore, they induce an inflammatory response that leads to the recruitment of immune cells at the site of infection and promotes granuloma formation. These structures may subsequently break down, resulting in a massive extracellular release of bacteria and consequent tissue damage. The complexity of treatment calls for new therapeutic approaches; one of the most promising is phage therapy. This approach relies on the use of bacteriophages capable of infecting and lysing bacterial cells. To this end, lytic phages lacking genes associated with antibiotic resistance and virulence are selected. However, mycobacteriophages are generally lysogenic and tend to integrate their genome into the bacterial chromosome. To overcome this limitation, lysogenic phages can be genetically engineered to remove genes involved in lysogeny, making them strictly lytic and therefore suitable for therapeutic use. Phage therapy is currently authorized under compassionate use and has shown promising results in the treatment of patients with multidrug-resistant infections, for which conventional antibiotic therapy is ineffective. The overall aim of this thesis was to contribute to the study of mycobacteriophages potentially applicable in therapeutic settings against M. abscessus, through the analysis of phages already available at the Microbiology Laboratory of the Department of Biology of the University of Pisa, as well as the isolation and characterization of new mycobacteriophages. The first part of this work focused on the isolation and genotypic and phenotypic characterization of four new mycobacteriophages: Amelia, John Lord, Kurisu, and Okabe. The ultimate goal of this section was to expand the laboratory’s phage collection and to define the main biological characteristics of newly isolated mycobacteriophages, providing a useful foundation for their potential therapeutic application. In the second part of the thesis, the phenotypic characterization of the mycobacteriophage Pisa4, previously isolated in the laboratory, was completed. Previous analyses of host range and efficiency of plating (EOP) had shown that phage Pisa4 was active against several clinical strains of M. abscessus; in some cases, the presence of lysis halos had also been observed, indicating lytic activity not necessarily associated with productive phage replication. Based on these observations, the mechanisms of phage–bacterium interaction and the interaction between the phage and cells of the innate immune system were further investigated, analyzing NET release and the modulation of cytokine expression (IL-6, IFN-γ, and TNF-α) by neutrophils and peripheral blood mononuclear cells.
I micobatteri sono classificati in micobatteri tubercolari e micobatteri non tubercolari (NTM). La maggior parte degli NTM sono batteri ambientali presenti nel suolo e nelle fonti d’acqua; si tratta di organismi saprofiti generalmente non patogeni per l’uomo. Tuttavia, condizioni patologiche come la fibrosi cistica o malattie polmonari possono favorire l’insorgenza di infezioni da NTM. In particolare, Mycobacterium abscessus e Mycobacterium avium sono i principali responsabili dell’aumento delle infezioni da NTM. Il sito di infezione più colpito sono i polmoni, ma gli NTM possono causare anche infezioni oculari, cutanee e disseminate. I micobatteri presentano una naturale resistenza a molte molecole antibiotiche, dovuta alla peculiare struttura della loro parete cellulare, alla produzione di biofilm, al possibile stile di vita intracellulare e alla formazione di granulomi nei polmoni dell’ospite, che riducono l’efficacia dei farmaci. In questo modo, i batteri possono replicarsi all’interno dei macrofagi, evitando la risposta immunitaria. Inoltre, inducono una risposta infiammatoria che porta al reclutamento di cellule immunitarie nel sito di infezione e alla formazione di granulomi. Queste strutture possono rompersi, determinando un massiccio rilascio extracellulare di batteri e danno tissutale. La complessità del trattamento richiede nuovi approcci terapeutici; uno dei più promettenti è la terapia fagica. Essa si basa sull’impiego di batteriofagi in grado di infettare e lisare le cellule batteriche. A questo fine, vengono selezionati fagi litici privi di geni associati alla resistenza agli antibiotici e alla virulenza. Tuttavia, i micobatteriofagi sono generalmente lisogeni e tendono a integrare il loro genoma nel cromosoma batterico. Per superare questo limite, i fagi lisogeni possono essere ingegnerizzati geneticamente per rimuovere i geni coinvolti nella lisogenia, rendendoli litici e quindi idonei all’uso terapeutico. La terapia fagica è attualmente autorizzata per uso compassionevole e ha mostrato risultati promettenti nel trattamento di pazienti con infezioni multi-resistenti ai farmaci, per le quali la terapia antibiotica tradizionale è inefficace. L’obiettivo generale di questa tesi è stato quello di contribuire allo studio di micobatteriofagi potenzialmente utilizzabili in ambito terapeutico contro M. abscessus, attraverso un’analisi di fagi già disponibili presso il Laboratorio di Microbiologia del Dipartimento di Biologia dell’Università di Pisa e l’isolamento e la caratterizzazione di nuovi micobatteriofagi. La prima parte di questo lavoro è stata dedicata all’isolamento e alla caratterizzazione genotipica e fenotipica di quattro nuovi micobatteriofagi; Amelia, John Lord, Kurisu e Okabe. Il fine ultimo di questa parte è stato ampliare la fagoteca del laboratorio e definire le principali caratteristiche biologiche dei micobatteriofagi di nuovo isolamento, fornendo basi utili per il loro potenziale impiego in terapia. Nella seconda parte della tesi è stata completata la caratterizzazione fenotipica del micobatteriofago Pisa4, precedentemente isolato in laboratorio. Le precedenti analisi dello spettro d’ospite e dell’efficienza di piastratura (EOP) avevano evidenziato che il fago Pisa4 risultava attivo contro diversi ceppi clinici di M. abscessus; in alcuni casi era stata osservata anche la presenza di aloni di lisi, indicativi di un’attività litica non necessariamente associata a una replicazione fagica produttiva. Sulla base di queste osservazioni, sono stati approfonditi i meccanismi di interazione fago-batterio e l’interazione del fago con le cellule del sistema immunitario innato, analizzando il rilascio di NET e la modulazione dell’espressione delle citochine IL-6, IFN-γ e TNF-α da parte di neutrofili e cellule mononucleate del sangue periferico.
Caratterizzazione genotipica e fenotipica di nuovi micobatteriofagi e studio dell’interazione delle cellule umane dell’immunità innata con il fago Pisa4
MACCIONI, SARA
2024/2025
Abstract
Mycobacteria are classified into tuberculous mycobacteria and non-tuberculous mycobacteria (NTM). Most NTM are environmental bacteria found in soil and water sources; they are saprophytic organisms that are generally non-pathogenic to humans. However, pathological conditions such as cystic fibrosis or chronic lung diseases can promote the onset of NTM infections. In particular, Mycobacterium abscessus and Mycobacterium avium are the main contributors to the increasing incidence of NTM infections. The lungs represent the most commonly affected site, although NTM can also cause ocular, cutaneous, and disseminated infections. Mycobacteria exhibit a natural resistance to many antibiotic molecules, due to the peculiar structure of their cell wall, their ability to form biofilms, their potential intracellular lifestyle, and the formation of granulomas in the host’s lungs, all of which reduce drug efficacy. In this way, bacteria can replicate within macrophages, evading the immune response. Furthermore, they induce an inflammatory response that leads to the recruitment of immune cells at the site of infection and promotes granuloma formation. These structures may subsequently break down, resulting in a massive extracellular release of bacteria and consequent tissue damage. The complexity of treatment calls for new therapeutic approaches; one of the most promising is phage therapy. This approach relies on the use of bacteriophages capable of infecting and lysing bacterial cells. To this end, lytic phages lacking genes associated with antibiotic resistance and virulence are selected. However, mycobacteriophages are generally lysogenic and tend to integrate their genome into the bacterial chromosome. To overcome this limitation, lysogenic phages can be genetically engineered to remove genes involved in lysogeny, making them strictly lytic and therefore suitable for therapeutic use. Phage therapy is currently authorized under compassionate use and has shown promising results in the treatment of patients with multidrug-resistant infections, for which conventional antibiotic therapy is ineffective. The overall aim of this thesis was to contribute to the study of mycobacteriophages potentially applicable in therapeutic settings against M. abscessus, through the analysis of phages already available at the Microbiology Laboratory of the Department of Biology of the University of Pisa, as well as the isolation and characterization of new mycobacteriophages. The first part of this work focused on the isolation and genotypic and phenotypic characterization of four new mycobacteriophages: Amelia, John Lord, Kurisu, and Okabe. The ultimate goal of this section was to expand the laboratory’s phage collection and to define the main biological characteristics of newly isolated mycobacteriophages, providing a useful foundation for their potential therapeutic application. In the second part of the thesis, the phenotypic characterization of the mycobacteriophage Pisa4, previously isolated in the laboratory, was completed. Previous analyses of host range and efficiency of plating (EOP) had shown that phage Pisa4 was active against several clinical strains of M. abscessus; in some cases, the presence of lysis halos had also been observed, indicating lytic activity not necessarily associated with productive phage replication. Based on these observations, the mechanisms of phage–bacterium interaction and the interaction between the phage and cells of the innate immune system were further investigated, analyzing NET release and the modulation of cytokine expression (IL-6, IFN-γ, and TNF-α) by neutrophils and peripheral blood mononuclear cells.| File | Dimensione | Formato | |
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Descrizione: Tesi Magistrale in Biologia Sperimentale e Applicata
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https://hdl.handle.net/20.500.14239/34764