Investigating Nucleoside Analogues as Anti-bacterial Agents Targeting Pathogenic Escherichia coli

The global rise of antimicrobial resistance has created an urgent need for alternative therapeutic strategies that suppress pathogenicity without driving resistance. Purine nucleoside analogue CIF, originally developed as an anticancer agent, has been studied for its potential anti-virulence activity against enteropathogenic Escherichia coli (EPEC) and related enteric pathogens. CIF was identified in a 3-hour EPEC infection assay screening 1,120 FDA-approved drugs and found to inhibit the growth of EPEC, enterohaemorrhagic E. coli (EHEC), Salmonella and the commensal E. coli K-12 MG1655.

Transcriptomic profiling of EPEC revealed downregulation of ribosome genes, which was validated by experiments showing that CIF inhibited protein synthesis. Notably, inhibition occurred without DNA Damage, setting CIF apart from traditional genotoxic nucleoside analogues. Transcriptomics revealed the repression of virulence genes in the locus of enterocyte effacement (LEE), which encodes the EPEC Type 3 Secretion System (T3SS), a crucial component for infection. Biochemical experiments showed that CIF disabled the T3SS, explaining its identification in the screen.

CIF acutely suppressed bacterial growth for 6 hours, after which all resumed growth and reached the stationary phase by 24 hours. Repeated exposure did not produce resistant mutants, indicating that recovery reflected tolerance rather than stable resistance. Mass spectrometry demonstrated that EPEC metabolised CIF, which coincided with a reduced concentration of CIF extracellularly and intracellularly. Importantly, host cell assays showed that bacteria-exposed CIF had a reduced ability to induce DNA damage responses in mammalian cells, confirming that bacterial metabolism directly diminished its efficacy against cancer cells. This PhD thesis identifies CIF as a novel inhibitor of EPEC growth, protein synthesis, and the function of the T3SS, a key element in infection. The study also provides insight into how bacteria metabolise drugs to restart growth, which must be considered when designing treatment regimens for human diseases such as cancer.