The nucleobase analog 4-thiouracil hijacks the pyrimidine salvage pathway to inhibit <i>Staphylococcus aureus</i> growth

<i>Staphylococcus aureus</i> is associated with greater than one million global deaths annually and is capable of infecting every human tissue. The increasing emergence of antibiotic-resistant strains emphasizes the urgent need to develop new therapeutic strategies to treat infections. Nucleoside analogs that disrupt pyrimidine or purine nucleotide metabolism serve as a promising approach for treating drug-resistant infections, as these pathways differ between host and bacteria. Here, we demonstrate that the uracil derivative 4-thiouracil (4-TU) inhibits <i>S. aureus</i> growth by hijacking the pyrimidine salvage pathway, leading to incorporation of 4-TU into RNA. We found that mutations in uracil phosphoribosyltransferase (<i>upp</i>) confer resistance to 4-TU and prevent incorporation into RNA. Expression of a <u>t</u>hio<u>u</u>racil <u>d</u>e<u>s</u>ulfurase (<i>tudS</i>) from <i>Clostridioides difficile</i> is sufficient to detoxify 4-TU and diminish 4-TU levels in RNA. Taken together, these results suggest that 4-TU-mediated disruption of pyrimidine metabolism limits <i>S. aureus</i> growth, which may serve as a promising therapeutic target.