Publicação
Reprogramming Pseudomonas aeruginosa: an in depth study on phage-host interaction
| Resumo: | Phages are bacterial viruses with high interest for science and technology due to their applicability in human health, biotechnology, agriculture, and veterinary, as well as in synthetic biology. As therapeutic agents, phages can be used to treat life-threatening bacterial infections, such as Pseudomonas aeruginosa infections, although its approval in human medicine is mainly limited by the lack of knowledge about phage mode of action under in vivo conditions and bacterial responses towards phage infection. In this work, RNA-seq of phage-infected bacteria grown under different media conditions and adhered to human lung epithelium was performed to capture a global view of the transcriptional events that occur under phage therapy. Thus, it was possible to prove that P. aeruginosa was significantly more virulent when cultured in media used to grow human lung epithelial cells, as well as when adhered to a monolayer of human lung epithelium, than when cultured in lysogeny broth. During phage infection, it was observed that global transcriptomes are substantially different at early infection, an important phase during which phage takeover of the bacterial cell occurs, with particular changes in metabolic pathways associated with energy acquisition. Curiously, in all growth conditions, there is a set of common mechanisms that are targeted by the phage, which influence the phage transcriptional landscape. These mechanisms include prophage induction, bacterial receptors shutdown, and motility inhibition. In addition, specific transcriptional events were captured for specific growth conditions, indicating that phage can adapt the bacterial cell to reallocate resources for infection and surpass the limitations imposed by bacterial defences. The results put into evidence the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, and the obviously phage versatility on bacterial cell invasion under different environments. Besides providing a better understanding on phage/host interactions, transcriptomics did not disclose the phage´s genes that are essential for phage replication. Therefore, a genomic approach based on the direct manipulation of phage´s genome was made to obtain phages with a reduced genome. This approach consists in exposing the phage to a mutagenic agent and heat treatments in the presence of a chelating agent. The surviving phages had large deletions (up to 3kb) in their early genes and revealed similar ability in the killing of the bacterial host. However, major changes occur in terms of the host range and in their long-term competitiveness, which might indicate that early genes are not essential for phage replication in lab conditions but confer competitive advantages to the phage. This approach enables the fast generation of mutants that can provide clues towards a better understanding of the role of unknown genes and potentially drive the discovery of novel phage sequences and phage core infection mechanisms at the molecular level. |
|---|---|
| Autores principais: | Brandão, Ana Catarina Ribeiro |
| Assunto: | Phage RNA-seq P. aeruginosa Fago |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Phages are bacterial viruses with high interest for science and technology due to their applicability in human health, biotechnology, agriculture, and veterinary, as well as in synthetic biology. As therapeutic agents, phages can be used to treat life-threatening bacterial infections, such as Pseudomonas aeruginosa infections, although its approval in human medicine is mainly limited by the lack of knowledge about phage mode of action under in vivo conditions and bacterial responses towards phage infection. In this work, RNA-seq of phage-infected bacteria grown under different media conditions and adhered to human lung epithelium was performed to capture a global view of the transcriptional events that occur under phage therapy. Thus, it was possible to prove that P. aeruginosa was significantly more virulent when cultured in media used to grow human lung epithelial cells, as well as when adhered to a monolayer of human lung epithelium, than when cultured in lysogeny broth. During phage infection, it was observed that global transcriptomes are substantially different at early infection, an important phase during which phage takeover of the bacterial cell occurs, with particular changes in metabolic pathways associated with energy acquisition. Curiously, in all growth conditions, there is a set of common mechanisms that are targeted by the phage, which influence the phage transcriptional landscape. These mechanisms include prophage induction, bacterial receptors shutdown, and motility inhibition. In addition, specific transcriptional events were captured for specific growth conditions, indicating that phage can adapt the bacterial cell to reallocate resources for infection and surpass the limitations imposed by bacterial defences. The results put into evidence the relevance of using complex settings that mimics in vivo conditions to study phage-bacteria interplay, and the obviously phage versatility on bacterial cell invasion under different environments. Besides providing a better understanding on phage/host interactions, transcriptomics did not disclose the phage´s genes that are essential for phage replication. Therefore, a genomic approach based on the direct manipulation of phage´s genome was made to obtain phages with a reduced genome. This approach consists in exposing the phage to a mutagenic agent and heat treatments in the presence of a chelating agent. The surviving phages had large deletions (up to 3kb) in their early genes and revealed similar ability in the killing of the bacterial host. However, major changes occur in terms of the host range and in their long-term competitiveness, which might indicate that early genes are not essential for phage replication in lab conditions but confer competitive advantages to the phage. This approach enables the fast generation of mutants that can provide clues towards a better understanding of the role of unknown genes and potentially drive the discovery of novel phage sequences and phage core infection mechanisms at the molecular level. |
|---|