Oxidative Burst and Necroptosis in Macrophages Infected with Carbapenem-Resistant Klebsiella Pneumoniae (CRKP)

Nova Southeastern University, Davie, Florida, USA

Michael Kling OMS-11,2, Danielle Ahn MD1, Alice Prince MD1 1Department of Pediatric Infectious Disease, Columbia University Medical Center, New York, NY, USA 2Dr. Kiran C. Patel College of Osteopathic Medicine, Fort Lauderdale, FL, USA Objective. To clarify how KP35, a representative clinical isolate of CRKP ST258, evades innate immune clearance. Background. CRKP ST258 are a multidrug-resistant Gram-negative bacteria responsible for numerous, often lethal, health care- and community-associated infections. The predominant species of CRKP ST258, e.g. KP35, have become resistant to phagocytic killing. Ahn et al. demonstrated that neutrophils failed to clear KP35 after 2 hours, while KPPR1, a well-studied reference strain, was cleared within 30 minutes. In similar studies, KP35 rapidly depleted alveolar macrophages. We hypothesized that KP35 does not activate the oxidative burst and that it promotes necroptosis in macrophages. Methods. Macrophage-induced THP-1 cells were infected with either KP35, KPPR1, or S. aureus USA300. qRT-PCR analyses were performed for expression of NOX2/gp91phox – the catalytic core of NADPH oxidase. Flow cytometry were performed for mitochondrial and intracellular reactive oxygen species (mROS and iROS). Immunoblots for p-MLKL were performed to investigate the induction of necroptosis. Results. KP35 does not upregulate NOX2 transcription. KP35 kills macrophages without causing an increase in iROS, and with or without causing an increase in mROS. KP35 does not induce phosphorylation of MLKL in macrophages. Conclusion. KP35 does not upregulate NOX2, but activation of the oxidative burst has not been ruled out as flow cytometry was confounded by significant cell death. Two populations of dead cells emerged during flow cytometry: the first, positive for mROS and the second, negative for mROS. This suggest that KP35 induces two forms of cell death. The lack of p-MLKL rules out necroptosis. Grants. NIH.