Cyclic di-GMP (c-di-GMP) is one of the most important molecules in P. aeruginosa that promote biofilm formation, which is governed by a highly complex gene and protein network. It is produced and hydrolysed by two types of proteins, diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. Disrupting the activities of DGCs or stimulating the activities of PDEs will inhibit biofilms. It was unclear which of the 40 proteins involved in the metabolism of c-di-GMP in P. aeruginosa plays the most important role in biofilm formation in CF lungs. As such, this project first analysed the gene sequences and expression profiles of 40 genes in 32 non-familial P. aeruginosa clinical isolates grown as biofilms in microaerophilic synthetic sputum, which recapitulated the habitat of P. aeruginosa in CF airways. Such large-scale analysis and subsequent gene deletion studies identified the essential role of SiaD, a DGC protein, in the formation of suspended biofilms that resemble those observed in expectorated sputum from CF patients. The computer-aided virtual screening technique was next employed to search for novel compounds that can inhibit the activity of SiaD within a comprehensive library consisting of 21495 compounds with known bioactivities to increase the potential of clinical usage. Screening results predicted a strong binding of two compounds, named echinacoside and bimosiamose, with the active site of SiaD. After c-di-GMP quantification and microscopic evaluation, it was found that the water-soluble echinacoside could decrease the c-di-GMP level in P. aeruginosa and inhibit biofilm formation in synthetic sputum. Echinacoside was then tested in combination with four antibiotics (tobramycin, ciprofloxacin, meropenem, and ceftazidime) frequently prescribed to CF patients. Results showed that after 18 hours of treatments, echinacoside successfully increased the efficacy of tobramycin against pre-established aggregates in synthetic sputum formed by >80% of tested P. aeruginosa clinical strains by 2.7 to 48-fold, including those that are intrinsically resistant to tobramycin. As P. aeruginosa residing in the thick mucus can also directly attach to airway epithelial cells, the effect of echinacoside and tobramycin combination treatment against biofilms formed on 3-D differentiated alveolar cells was assessed. Echinacoside was proven not toxic to human cells even at high concentrations. After co-culturing P. aeruginosa and alveolar cells for 4 hours, substantial biofilms could be found on the cell surface, and infected cells were given different treatments for another 18 hours. Without any treatment, only around 40% of human cells remained alive, and tobramycin treatment alone failed to prevent cell death. In contrast, the 18-hr combinatorial therapy of both echinacoside and tobramycin resulted in a 20% increase in cell survival rate compared to tobramycin monotherapy, demonstrating its potential for future clinical applications.
Although echinacoside was shown in this study to be a tobramycin potentiator against P. aeruginosa aggregates in different in vitro models, whether it maintains the same function in P. aeruginosa-infected CF lung needs extensive further research, which hospitals, academic researchers, and pharmaceutical companies may exploit in the future. As inhalation has been adopted as the most efficient route for the delivery of tobramycin directly into CF lungs, formulations for echinacoside that are compatible with tobramycin inhalation solution may be needed. If echinacoside can also be prepared as a formulation suitable for inhalation, it may increase the retention for prolonged exposure and maximize the therapeutic index. The results from this project will be disseminated at upcoming conferences, including ESCMID/ASM Joint Conference (2024) and Microbiology Society annual conference (2025), which academic colleagues, medical doctors, and industrial partners will attend. As the potential users of my results could be researchers, pharmaceutical companies, doctors, and eventually patients, potential collaboration may be set up during these conferences.
For more information about the methodology and data, please see the open-access manuscript at
https://www.biorxiv.org/content/10.1101/2024.02.09.579617v1.full(se abrirá en una nueva ventana).
