An Enterobacter cloacae toxin able to generate oxidative stress and to provoke dose-dependent lysis of leukocytes

We investigated an Enterobacter cloacae strain exhibiting high hemolytic and leukotoxic activity. Monomeric and polymeric forms of the toxin showed similar effects on blood cells, although the polymer was more active than the monomer. Fluorescence microscopy revealed that both forms of the FITC-labeled toxin interacted with leukocytes, principally with neutrophils. Prelytic concentrations of polymeric and monomeric toxin significantly increased the production of reactive oxygen species (ROS) in neutrophils. Conversely, lytic concentrations of both toxin forms showed an increase followed by a decrease of ROS due to neutrophil damage. Monocytes did not show oxidative stress at all the toxin concentrations assayed. The toxin-neutrophil interaction at prelytic concentrations of toxin-stimulated ROS production and led to oxidative stress with subsequent cell death by apoptosis. However, high concentrations of E. cloacae toxin damaged leukocytes, producing lysis before the trigger of apoptosis, which suggests that the toxic effect is concentration dependent. The inhibition of oxidative stress observed with genistein and chloroquine suggests a potential involvement of the tyrosine kinase and nitric oxide synthesis pathways in E. cloacae toxin-mediated elevation of ROS.