Novel antibacterial interventions against Pseudomonas aeruginosa infections by targeting c-di-GMP and unravelling DSF signalling pathway.

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterial pathogen causing acute and chronic infections in immunocompromised and hospitalized patients. Infections due to P. aeruginosa and other drug-resistant bacteria pose a major threat to healthcare systems worldwide and the development of new effective antibacterial agents is urgently needed. P. aeruginosa can also form biofilms that promote antibiotic tolerance and chronic infection, thereby posing additional major challenges for treatment. P. aeruginosa regulates the production of virulence factors through N-acyl homoserine lactone and quinoline PQS signal-mediated quorum sensing and communicate between species through cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. This thesis first focused on the DSF signalling mechanism of P. aeruginosa infection. In order to gain a deeper understanding of the communication regulatory mechanism of DSF signalling in P. aeruginosa infection, this work has focused on the role of the sensor kinase PA1396 in DSF perception. Further genomic analysis identified a gene encoding a response regulator, PA1397, upstream of PA1396, with a receptor domain and a DNA-binding domain. In order to systematically study the functions of DSF-related genes in P. aeruginosa, this study used a gene mutation and complementation strategy to mutate and complement the signalling orphan Hpt domains hptA, hptB, and hptC, and the results showed that these genes are highly active in P. aeruginosa and plays an important regulatory role in toxicity and motility, especially hptB. The functional role of the PA1397 gene and its interaction with DNA were further studied, and experiments proved that it may be involved in transcriptional regulation, suggesting its importance in DSF perception. Meanwhile, a comprehensive understanding of the signalling mechanisms of DSF perception in P. aeruginosa could provide valuable insights into the regulation of its virulence and facilitate the development of new therapeutic strategies. These findings lay the foundation for the development of antibacterial drugs against drug-resistant bacteria using signalling molecules as new pathways. The second focus of this thesis was to determine the role of c-di-GMP, a signalling molecule that regulates its fitness, antibiotic resistance, and biofilm formation, on the ability of P. aeruginosa to cause infection in a co-culture model of infection using human bronchial epithelial cells. P. aeruginosa uses c-di-GMP to regulate the expression of a series of genes to adapt to different environmental conditions, including antibiotic stress. Therefore, reducing intracellular c-di-GMP levels is considered a promising strategy to combat antibiotic-resistant P. aeruginosa infection. This paper also constructed and optimized the co-culture screening model to provide a physiologically realistic environment for the formation of bacterial biofilms, thereby improving the transferability of in vitro drug screening, and identified a promising compound that modulates c-di-MP levels and reduced virulence in the co-culture model. Based on the above research contents, this thesis provides an important theoretical and experimental basis for the molecular regulation of P. aeruginosa infection and the development of antibacterial drugs by exploring the regulatory mechanisms of DSF and c-di-GMP signalling in P. aeruginosa infection. At the same time, research into new potential therapeutic drugs has also been carried out. These findings are of great significance for combating the challenge of drug-resistant bacteria and improving clinical treatment effects.

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ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

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