The OxyR-Responsive TacAT Module Promotes Oxidative Stress Adaptation and In Vivo Fitness in K. pneumoniae

Abstract

During infection, Klebsiella pneumoniae must withstand host-derived oxidative stress not only by detoxifying reactive oxygen species (ROS) but also by repairing oxidative damage to cellular physiology. How these detoxification and stress-adaptive responses are coordinated remains unclear. Here, we identify a plasmid-encoded tacAT locus as an OxyR-responsive regulatory module that supports peroxide tolerance and in vivo fitness in K. pneumoniae. Guided by the conservation of the OxyR DNA-binding domain and its consensus binding motif, we surveyed promoter-proximal regions of the K. pneumoniae CRK3022 chromosome and plasmids for candidate OxyR-responsive loci. This analysis identified tacAT, a putative type II toxin–antitoxin (TA) module, as the only plasmid-associated candidate. OxyR directly bound the tacAT promoter and contributed to tacAT induction during hydrogen peroxide (HoO2) stress. The TacAT complex exhibited DNA-binding and transcriptional regulatory activity, promoting the expression of genes linked to protein quality control and membrane/envelope homeostasis, including clpB, htpG, cadC, bhsA, and marA. Deletion of tacAT compromised survival under lethal peroxide challenge and attenuated bacterial dissemination, tissue pathology, and inflammatory responses in a murine bacteremia model. These findings define an OxyR–TacAT regulatory branch that connects peroxide sensing with stress-adaptive gene expression, revealing how a TA-associated module can be integrated into stress-adaptive programs during host-associated oxidative stress. Highlight Promoter motif screening identifies tacAT as a candidate OxyR-responsive TA-associated module OxyR directly binds the tacAT promoter and contributes to peroxide-induced tacAT expression TacAT promotes stress-response genes linked to protein quality control and envelope homeostasis TacAT supports oxidative stress survival and in vivo fitness of Klebsiella pneumoniae Graphical Abstract Proposed model of the OxyR-TacAT regulatory branch in K. pneumoniae. During infection, K. pneumoniae encounters host-derived oxidative stress. Oxidized OxyR activates classical antioxidant responses and contributes to tacAT induction by binding an OxyR-like motif in the tacAT promoter region. The induced TacAT complex binds OP1 within its own promoter and promotes the expression of stress-response genes, including clpB and htpG, which are associated with protein quality control, and cadC, bhsA, and marA, which are associated with membrane/envelope homeostasis. This regulatory branch supports bacterial survival under severe oxidative stress and contributes to in vivo fitness during systemic infection. Dashed arrows indicate proposed or indirect regulatory effects.