Abstract
Pseudomonas aeruginosa is an opportunistic pathogen responsible for a variety of infections. P. aeruginosa presents a challenge to conventional antibiotic therapeutics because of the emergence and rapid spread of antibiotic resistance. Antibiotic resistant P. aeruginosa poses a major threat to public health, with many nosocomial infections caused by this problematic pathogen. Similar to many pathogenic bacteria, there is an urgent need for novel therapeutics to treat P. aeruginosa infections. One approach to antibiotic discovery is to screen drugs approved for other purposes for antibiotic activity. Identified compounds can potentially be repurposed and fast-tracked into clinical use. This approach has identified the antimycotic prodrug 5-fluorocytosine (5-FC) as significantly reducing pathogenicity of P. aeruginosa without affecting growth in a murine infection model. 5-FC was shown to reduce expression of PvdS, an alternate sigma factor. As PvdS is important for regulation of multiple virulence factors of P. aeruginosa, 5-FC was hypothesised to reduce pathogenicity through reduction of PvdS. The aims of the presented research were to further explore the effects of 5-FC on a well-characterised reference strain and various isolates of P. aeruginosa, to determine the mechanism of action of 5-FC in P. aeruginosa, and identify how treatment with 5-FC decreases pathogenicity.
5-FC reduced expression of pvdS, identified using reporter gene assays. Production of the PvdS-regulated iron-chelator pyoverdine was also reduced. Using assays for motility and biofilm initiation, 5-FC reduced the three motilities of P. aeruginosa, but had little effect on adherence to an abiotic surface. Over 60 isolates from clinical and environmental sources were screened for effects on growth and pyoverdine production. While the majority of isolates responded similarly to 5-FC treatment, some isolates had diverse responses. Further phenotypic studies on a subset of 19 isolates highlighted the varied responses to 5-FC.
5-FC was previously shown to function in P. aeruginosa through uptake and subsequent conversion to 5-fluorouracil. Through the construction of mutant strains, the enzymes necessary for the full mechanism of action of 5-FC in P. aeruginosa were identified, indicating that 5-fluorouridine triphosphate, a metabolite of 5-FC, is incorporated into the RNA. This was conclusively demonstrated through the use of mass spectrometry, with fluorinated uracil comprising 5% of the total uracil nucleobases in the RNA.
To further understand how 5-FC reduced pathogenicity, RNA-seq was used. After 4 h of treatment, over 300 genes involved in a variety of different functions and pathways were found to be differentially expressed. A large portion of the genes were pathogenicity-related. In particular, 5-FC strongly increased the expression of genes required for the production of pyocyanin, a metabolite implicated in virulence and facilitating redox balance in P. aeruginosa. A proteomics approach also identified proteins involved in pyocyanin synthesis as upregulated in 5-FC-treated bacteria.
Overall, this study has provided a clearer understanding of the phenotypic effects and the mechanisms of action of 5-FC on P. aeruginosa and has highlighted important points for consideration before the clinical applicability of the drug is further explored.