Cefoperazone-
sulbactam (SCF)-resistant Pseudomonas aeruginosa poses a big challenge in the use of SCF to treat
infection caused by the pathogen. We have recently shown exogenous
nitrite-enabled killing of naturally and artificially evolved Pseudomonas aeruginosa strains (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) by SCF. However, the underlying mechanism is unknown. Here, reprogramming metabolomics was adopted to investigate how
nitrite enhanced the SCF-mediated killing efficacy.
Nitrite-reprogrammed metabolome displayed an activated
pyruvate cycle (P cycle), which was confirmed by elevated activity of
pyruvate dehydrogenase (PDH), α-ketoglutarate
dehydrogenase, succinate dehydrogenase, and
malate dehydrogenase. The activated P cycle provided
NADH for the electron transport chain and thereby increased
reactive oxygen species (ROS), which potentiated SCF to kill AP-RCLIN-EVO and AP-RLAB-EVO. The
nitrite-enabled killing of AP-RCLIN-EVO and AP-RLAB-EVO by SCF was inhibited by PDH inhibitor
furfural and ROS scavenger
N-Acetyl-L-cysteine but promoted by ROS promoter Fe3+. SCF alone could not induce ROS, but SCF-mediated killing efficacy was enhanced by ROS. In addition, the present study demonstrated that
nitrite repressed
antioxidants, which were partly responsible for the elevated ROS. These results reveal a
nitrite-reprogrammed metabolome mechanism by which AP-RCLIN-EVO and AP-RLAB-EVO sensitivity to SCF is elevated. IMPORTANCE
Antibiotic-resistant Pseudomonas aeruginosa has become a real concern in hospital-acquired
infections, especially in
critically ill and immunocompromised patients. Understanding antibiotic resistance mechanisms and developing novel control measures are highly appreciated. We have recently shown that a reduced
nitrite-dependent NO biosynthesis contributes to
cefoperazone-
sulbactam (SCF) resistance, which is reverted by exogenous
nitrite, in both naturally and artificially evolved P. aeruginosa strains (AP-RCLIN-EVO and AP-RLAB-EVO, respectively). However, the mechanism is unknown. The present study reports that the
nitrite-enabled killing of AP-RCLIN-EVO and AP-RLAB-EVO by SCF is attributed to the promoted production of
reactive oxygen species (ROS).
Nitrite activates the
pyruvate cycle to generate
NADH for the electron transport chain, which in turn promotes ROS generation.
Nitrite-potentiated SCF-mediated killing is decreased by
pyruvate dehydrogenase inhibitor
furfural and ROS scavenger
N-Acetyl-L-cysteine but increased by ROS promoter Fe3+. Furthermore, SCF-mediated killing is promoted by H2O2 in a dose-dependent manner. In addition, the combination of
nitrite and H2O2 greatly enhances SCF-mediated killing. These results not only disclose a
nitrite-ROS-potentiated SCF-mediated killing, but also show SCF-mediated killing is dependent upon ROS.