ClpC1-dependent iron homeostasis underlies mycobacterial defense against oxygen-driven Fenton reaction during reactivation

ABSTRACT The caseinolytic protease (Clp) system, which is highly conserved and essential for protein homeostasis in mycobacteria, comprises the ClpP1P2 proteolytic core and the essential ATPases ClpC1 and ClpX. While ClpX has a defined role in DNA replication, and some differential substrates have been identified, the physiological functions distinguishing ClpC1 from ClpX remain incompletely understood. Here, using Clustered Regularly Interspaced Short Palindromic Repeats-mediated knockdown in Mycolicibacterium smegmatis and the attenuated Mycobacterium tuberculosis H37Ra strain, we showed that ClpX impairment caused complete growth arrest, cell filamentation, and dramatically increased intracellular reactive oxygen species (ROS). In contrast, clpC1 knockdown resulted in only moderate growth defects but induced profound hypersensitivity to high oxygen and catalase. Mechanistically, ClpC1 maintained iron homeostasis; its loss elevated free Fe² + levels, driving Fenton chemistry, catastrophic ROS bursts, and redox collapse upon oxygen exposure. This iron-dependent survival defect was confirmed in a murine model. We thus establish ClpC1 as a central regulator of iron metabolism and oxidative defense, implicating its roles in bacterial reactivation from dormancy and persistence under fluctuating oxygen conditions. Furthermore, knockdown of either clpC1 or clpX increased mycobacterial susceptibility to multiple antibiotics, highlighting the Clp system as an attractive anti-tuberculosis drug target. Together, our findings reveal overlapping yet non-redundant roles for ClpC1 and ClpX, delineating their distinct regulatory networks. These results advance our understanding of the Clp system. Importantly, although these conclusions are derived from studies in surrogate mycobacterial species, they raise the possibility that similar mechanisms may underlie adaptation, dormancy, and resuscitation in pathogenic Mycobacterium tuberculosis . IMPORTANCE Tuberculosis remains a major global health burden. The ClpC1/ClpX-ClpP1P2 proteolytic complex has emerged as a highly attractive drug target. This study investigates the role of ClpX in maintaining fundamental bacterial biological processes and suggests a specific function in regulating iron homeostasis and oxidative defense. This proposed mechanism may contribute to the bacterium’s ability to survive environmental shifts, such as resuscitation from latency. By demonstrating an involvement of caseinolytic proteases (Clps) in iron metabolism, this research provides additional insights into mycobacterial adaptive strategies within the host. Importantly, the results highlight a putative vulnerability during latent infection and reactivation: mycobacteria appear to depend on ClpX and ClpC1 for dormancy regulation, while ClpC1 is likely important for mitigating reoxygenation-induced oxidative stress. This discovery may inform future therapeutic approaches specifically targeting the reactivation phase of latent tuberculosis.