Autor(es): Ferreira, Rute Vanessa Novais
Data: 2024
Identificador Persistente: https://hdl.handle.net/1822/95438
Origem: RepositóriUM - Universidade do Minho
Assunto(s): Helicobacter pylori; Bacteriophages; Metagenomics; Phageome; Phage therapy; Bacteriófagos; Metagenómica; Fagoma; Terapia fágica
Helicobacter pylori causes chronic gastric infection, affecting a significant proportion of the population worldwide. Current treatments rely on antibiotics but face reduced efficacy due to increasing antibiotic resistance, highlighting the urgent need for alternative therapies. Bacteriophages (phages) represent a promising option for the biocontrol of H. pylori. To date, only H. pylori prophages have been identified, and their characterisation remains largely incomplete. To address this limitation, this thesis aimed to isolate and characterise novel H. pylori phages, thereby expanding our current understanding of their role and potential therapeutic applications. To achieve this objective, a new PCR-based method was first developed to screen H. pylori clinical strains for the presence of prophage genes. Twelve intact prophages were identified in silico, with genomes containing genes putatively homologous to virulence factors. Moreover, phylogenetic analysis demonstrated that these prophages, along with those previously reported, exhibited strong similarities, despite their diverse geographical origins. With the information collected by the phage genes identification, prophage-positive strains were subjected to treatments to induce the release of prophages. This resulted in the isolation of phages Hpy1R and HPy2R, both of which exhibited high stability across a pH range between 3 and 11 and at 37 °C, suggesting their adaptation to the human stomach environment. Importantly, no antibiotic resistance genes were identified in the genomes of these phages, and they successfully reduced bacterial counts in the host strain across various multiplicities of infection (MOIs). Additionally, when AGS cells were co-infected with H. pylori and phage HPy2R at different MOIs it resulted in a significant increase in host cell viability and a decrease in interleukin (IL)- 8 expression, compared to non-phage-treated cells. Finally, the development of a mock phage community and synthetic phage samples, combined with a tailored metagenomics pipeline and a custom database, allowed for the precise characterisation of the phageome with minimal false-positives, even in samples with low microbial biomass. Overall, this work focused on the isolation and characterisation of novel H. pylori phages, highlighting their therapeutic potential. Additionally, the development of a bioinformatics workflow for phage content analysis marks a significant advancement in phageome research, particularly for the analysis of human tissue specimens. These findings establish a base for further research on phage therapy in H. pylori infections.
