Abstract

--- - To gain a better insight of how Cu ions toxify cells, metabolomic analyses were performed in S. aureus strains that lacks the described Cu ion detoxification systems (Delta copBL Delta copAZ; cop(-)). Exposure of the cop(-) strain to Cu (II) resulted in an increase in the concentrations of metabolites utilized to synthesize phosphoribosyl diphosphate (PRPP). PRPP is created using the enzyme phosphoribosylpyrophosphate synthetase (Prs) which catalyzes the interconversion of ATP and ribose 5-phosphate to PRPP and AMP. Supplementing growth medium with metabolites requiring PRPP for synthesis improved growth in the presence of Cu (II). A suppressor screen revealed that a strain with a lesion in the gene coding adenine phosphoribosyltransferase (apt) was more resistant to Cu. Apt catalyzes the conversion of adenine with PRPP to AMP. The apt mutant had an increased pool of adenine suggesting that the PRPP pool was being redirected. Over-production of apt, or alternate enzymes that utilize PRPP, increased sensitivity to Cu (II). Increasing or decreasing expression of prs resulted in decreased and increased sensitivity to growth in the presence of Cu (II), respectively. We demonstrate that Prs is inhibited by Cu ions in vivo and in vitro and that treatment of cells with Cu (II) results in decreased PRPP levels. Lastly, we establish that S. aureus that lacks the ability to remove Cu ions from the cytosol is defective in colonizing the airway in a murine model of acute pneumonia, as well as the skin. The data presented are consistent with a model wherein Cu ions inhibits pentose phosphate pathway function and are used by the immune system to prevent S. aureus infections. - Author summaryAntimicrobial resistance is a global health emergency. There is an increasing demand to develop new drugs and repurpose chemotherapeutics to prevent and treat infections. Metals and metal ions have been used as effective antimicrobials for millennia. This effectiveness is likely due to their ability to simultaneously inhibit multiple cellular processes. Copper (Cu) is a metal that has been used by humans as a prophylactic. Furthermore, the human immune system utilizes Cu to aid bacterial killing or growth inhibition. The human pathogen Staphylococcus aureus has two described Cu ion detoxification systems, underscoring the efficacy of Cu ions to negatively impact S. aureus growth and survival. Questions remain about how bacteria are toxified by Cu ions. Understanding how Cu kills and inhibits bacterial growth will provide useful information for designing and implementing antimicrobial chemotherapy to decrease bacteria burden. Herein, we demonstrate that the essential enzyme Prs is a target of Cu ions in S. aureus. Using mouse models of pneumonia and skin infections, we demonstrate that S. aureus that cannot effectively remove Cu ions from the inside of the cell are defective in colonizing tissues.