NagPIBAF upregulation and ompO downregulation compromise oxidative stress tolerance of Stenotrophomonas maltophilia.

Background: Outer membrane protein OmpA is composed of two domains, an N-terminal β-barrel structure embedded in the outer membrane and a C-terminal globular domain noncovalently associated with the peptidoglycan layer in periplasm. Stenotrophomonas maltophilia KJ is a clinical isolate. In our recent study, we disclosed that KJ∆OmpA an OmpA C-terminal deletion mutant, compromised menadione tolerance. Furthermore, the involvement of σ, σ, and ompO in the ∆ompA-mediated phenotype was proposed. In that study, we hypothesized that there was an unidentified σ-regulated candidate responsible for ∆ompA-mediated menadione tolerance decrease, and the candidate was disclosed in this study.

Methods And Results: Transcriptome analysis of wild-type KJ and KJ∆OmpA revealed that a five-gene cluster, smlt4023-smlt4019 (annotated as nagPIBAF), was upregulated in KJ∆OmpA. Reverse transcription-PCR (RT-PCR) confirmed the presence of the nagPIBAF operon. The expression of the nagPIBAF operon was negatively regulated by NagI and σ, and triggered by N-acetylglucosamine. In-frame deletion mutant construction and menadione tolerance assay demonstrated that nagP, nagB, and nagA upregulation in KJ∆OmpA connected with ∆ompA-mediated menadione tolerance decrease. The intracellular reactive oxygen species (ROS) level assay further verified that in the presence of external oxidative stress such as menadione treatment, nagPIBAF operon upregulation and ompO inactivation synergistically increased intracellular ROS levels, which exceeded the capacity of bacterial oxidative stress alleviation systems and resulted in a decrease of menadione tolerance.

Conclusions: Loss of interaction between OmpA C-terminus and peptidoglycan causes envelope stress and activates σ regulon. ompO and rpoN are downregulated in response to σ activation. rpoN downregulation further derepresses nagPIBAF operon, which can favor the metabolism route of glycolysis, TCA cycle, and electron transport chain. nagPIBAF upregulation and OmpO downregulation synergistically increase intracellular ROS levels and result in menadione tolerance decrease.

Clinical Trial Number: Not applicable.