The plant-derived naphthoquinone lapachol causes an oxidative stress response in Staphylococcus aureus

Nico Linzner,Verena Nadin Fritsch,Tobias Busche,Quach Ngoc Tung,Vu Van Loi,Jörg Bernhardt,Jörn Kalinowski,Haike Antelmann

doi:10.1016/j.freeradbiomed.2020.07.025

Abstract

Staphylococcus aureus is a major human pathogen, which causes life-threatening systemic and chronic infections and rapidly acquires resistance to multiple antibiotics. Thus, new antimicrobial compounds are required to combat infections with drug resistant S. aureus isolates. The 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone lapachol was previously shown to exert antimicrobial effects. In this study, we investigated the antimicrobial mode of action of lapachol in S. aureus using RNAseq transcriptomics, redox biosensor measurements, S-bacillithiolation assays and phenotype analyses of mutants. In the RNA-seq transcriptome, lapachol caused an oxidative and quinone stress response as well as protein damage as revealed by induction of the PerR, HypR, QsrR, MhqR, CtsR and HrcA regulons. Lapachol treatment further resulted in up-regulation of the SigB and GraRS regulons, which is indicative for cell wall and general stress responses. The redox-cycling mode of action of lapachol was supported by an elevated bacillithiol (BSH) redox potential (EBSH), higher endogenous ROS levels, a faster H2O2 detoxification capacity and increased thiol-oxidation of GapDH and the HypR repressor in vivo. The ROS scavenger N-acetyl cysteine and microaerophilic growth conditions improved the survival of lapachol-treated S. aureus cells. Phenotype analyses revealed an involvement of the catalase KatA and the Brx/BSH/YpdA pathway in protection against lapachol-induced ROS-formation in S. aureus. However, no evidence for irreversible protein alkylation and aggregation was found in lapachol-treated S. aureus cells. Thus, the antimicrobial mode of action of lapachol in S. aureus is mainly caused by ROS formation resulting in an oxidative stress response, an oxidative shift of the EBSH and increased protein thiol-oxidation. As ROS-generating compound, lapachol is an attractive alternative antimicrobial to combat multi-resistant S. aureus isolates.

Highlights

Staphylococcus aureus is an important human pathogen, which can cause acute skin and soft tissue infections, and life-threatening systemic and chronic diseases, such as sepsis, endocarditis, pneumonia and osteomyelitis [1,2,3,4]
The minimal inhibitory concentration (MIC) of lapachol was determined as 1.25 mM in S. aureus (Table S4), which was higher compared to the growth-inhibitory amount
We have analyzed the antimicrobial mode of action of the naphthoquinone lapachol in the major pathogen S. aureus

Summary

Introduction

Staphylococcus aureus is an important human pathogen, which can cause acute skin and soft tissue infections, and life-threatening systemic and chronic diseases, such as sepsis, endocarditis, pneumonia and osteomyelitis [1,2,3,4]. MHQ was shown to induce a strong thiol-specific oxidative and quinone stress response in the S. aureus transcriptome [26]. Using RNA-seq transcriptomics, lapachol induced an oxidative and quinone stress response as well as strong protein damage in S. aureus. This signature was revealed by the induction of the QsrR, MhqR, PerR, HypR, CtsR and HrcA regulons and of the enzymes of the Brx/BSH/YpdA redox pathway. The oxidative mode of action of lapachol was demonstrated by an oxidative shift of the BSH redox potential, elevated ROS formation and faster H2O2 detoxification capacity, increased protein S-bacillithiolation of GapDH and thiol-oxidation of the HypR repressor in vivo. Our results indicate that the antimicrobial effect of lapachol is mainly caused by ROS-formation, resulting in an impaired redox homeostasis and increased protein thioloxidation in S. aureus

Experimental procedures

Results

Discussion