The interactions of hydrogen peroxide produced by Streptococcus pneumoniae with myeloperoxidase and lactoperoxidase

Streptococcus pneumoniae is a gram positive diplococci that can cause pneumonia, meningitis, and sepsis. S. pneumoniae produces its own hydrogen peroxide. Myeloperoxidase and lactoperoxidase are mammalian haem peroxidases capable of catalysing the formation of bactericidal oxidants in the presence of a halide or pseudohalide, and hydrogen peroxide. NADPH oxidase is an enzyme that reduces oxygen to superoxide which subsequently dismutates to oxygen and hydrogen peroxide. In the neutrophil, myeloperoxidase is present in the primary granules and in conjunction with NADPH oxidase, produces the antimicrobial oxidant, hypochlorous acid. Lactoperoxidase is present in saliva, milk and other secretory fluids and produces hypothiocyanous acid in the presence of thiocyanate and hydrogen peroxide. The purpose of this study was to determine the interactions between hydrogen peroxide produced by S. pneumoniae with myeloperoxidase and lactoperoxidase, and to assess whether neutrophils use oxidative mechanisms to kill S. pneumoniae. This study investigated if myeloperoxidase in the neutrophil phagosome could utilise hydrogen peroxide produced by S. pneumoniae to produce hypochlorous acid and kill this bacteria. This study also investigated whether S. pneumoniae could contribute its hydrogen peroxide to the lactoperoxidase system to kill other bacteria. Myeloperoxidase produced hypochlorous acid in the presence of S. pneumoniae and chloride and the bacteria were killed in the presence of the peroxidase. Treating S. pneumoniae with hypochlorous acid also killed the bacteria. Neutrophil killing assays demonstrated that the killing of S. pneumoniae was rapid and inhibition of NADPH oxidase reduced killing indicating oxidative mechanisms play a role in neutrophil killing of the bacteria. However, inhibition of myeloperoxidase had no effect on killing. Experiments with the hypochlorous acid specific fluorescent probe R19-S demonstrated that neutrophils were able to generate hypochlorous acid in response to S. pneumoniae. Hydrogen peroxide produced by S. pneumoniae did not contribute to the hypochlorous acid detected by this probe as inhibition of the NADPH oxidase abolished fluorescence increase. S. pneumoniae were resistant to lactoperoxidase and thiocyanate, and also the presence of 200 nmol hypothiocyanous acid at pH 7.4. Lactoperoxidase killing assays showed that when P. aeruginosa were incubated in the presence of S. pneumoniae, lactoperoxidase, and thiocyanate, the viability of P. aeruginosa was not affected, whereas large amounts of hypothiocyanous acid (200 nmol) had a toxic effect. Collectively, my findings give insight into the nature of S. pneumoniae and how neutrophils interact with the bacteria. My study shows that neutrophils produce hypochlorous acid in response to S. pneumoniae. The hydrogen peroxide utilised by myeloperoxidase appears to be from the neutrophil rather than the bacteria, and myeloperoxidase does not appear to be essential for killing. My findings also demonstrate the survival of S. pneumoniae in the presence of the lactoperoxidase system and its product, hypothiocyanous acid. This allows for a potential mutualistic relationship between humans and S. pneumoniae to be further investigated as other bacteria are known to be susceptible to hypothiocyanous acid