Periodic Reporting for period 1 - DRIPBEAT (Drug repurposing for inhibiting pathogenic biofilms and potentiating the activity of antibiotics)
Reporting period: 2022-03-07 to 2024-03-06
Cystic fibrosis (CF) is a genetic disease for which the major symptom is abnormally thick, sticky mucus produced by the human body, especially the lungs. It makes patients vulnerable to a toxic bacterium, Pseudomonas aeruginosa, that can be found almost everywhere. P. aeruginosa invading patients’ lungs is trapped by the mucus and grows well in this environment. As P. aeruginosa cells grow, they gather and build themselves a ‘house’ called biofilm for their own protection, making each embedded bacterium almost impossible to kill using antibiotics, and living P. aeruginosa cells constantly damage lung tissue and function. Therefore, disrupting biofilms to revert bacteria to free-floating individuals will significantly enhance their susceptibility to antibiotics. As chronic infections with P. aeruginosa are the main perpetrators of morbidity and mortality in CF patients, this project aims to discover novel anti-biofilm agents as adjunctive therapy that potentiate the efficacy of frequently prescribed antibiotics against P. aeruginosa, thus improving the therapeutic outcomes.
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(opens in new window).
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(opens in new window).
This project, for the first time, systematically investigated the expression profiles of c-di-GMP-related genes among various CF P. aeruginosa isolates in a patient-mimetic model, which led to the identification of the essential role of SiaD in auto-aggregation in sputum. More importantly, we discovered the novel anti-biofilm and antibiotic-potentiating functions of echinacoside originally investigated as a neuroprotective agent. The outputs from this project will be of great interest to other researchers where a useful and comprehensive methodology to search for novel anti-biofilm agents was introduced, and several genes with unknown functions were highlighted from the gene expression dataset, which may open new avenues for future therapeutic designs. Moreover, as echinacoside was already proven safe in healthy individuals in a recent clinical trial, and showed an outstanding tobramycin-potentiating effect, it is highly possible that echinacoside can be re-purposed as an anti-biofilm adjunctive therapy for CF patients. With a mean prevalence of CF disease being 0.737 per 10,000 individuals in the EU and 1/3 of them suffering from chronic P. aeruginosa infection, our data may provide novel solutions to improve the life quality of CF patients, alleviate the burden on the health system, and create new market opportunities for pharmaceutical companies.