Repurposing clofarabine against enteric pathogens via metabolic starvation

Abstract

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) cause severe global diarrhoeal disease, exacerbated by rising antimicrobial resistance. Pathogenesis relies on "attaching and effacing" lesions, where bacteria generate actin pedestals to adhere, replicate, and destroy intestinal microvilli. To identify novel anti-virulence candidates targeting this process, we conducted a high-throughput phenotypic screen quantifying pedestal formation via high-content fluorescence microscopy. We identified the anticancer nucleotide analogue clofarabine as a potent inhibitor of EPEC growth and pathogenesis. Distinct from its oncology mechanism - inducing DNA chain termination and apoptosis - clofarabine competes with bacterial ATP-dependent processes to impede gene transcription, protein synthesis, and Type III Secretion System (T3SS) function. Remarkably, EPEC counteracts this inhibition by cleaving clofarabine, enabling restored protein synthesis and resumed growth. Furthermore, we report for the first time that clofarabine suppresses protein translation in human monocytes, suggesting an uncharacterised translational impact on mammalian cells. These findings reveal host and bacterial translation as interconnected vulnerabilities to purine antimetabolites, offering a novel strategy to disarm enteric pathogens while uncovering a bacterial enzymatic mechanism for neutralizing host-targeted therapeutics.