Respiratory burst facilitates the digestion of Escherichia coli killed by polymorphonuclear leukocytes

Abstract

We examined factors that may limit degradation of bacterial protein of Escherichia coli S15 killed by polymorphonuclear leukocytes (PMN). Both human and rabbit PMN degraded up to 40% of [14C]amino acid-labeled protein of ingested and killed E. coli in 2 h as determined by loss of acid-precipitable radioactivity. In contrast, equally bactericidal broken-PMN preparations or isolated granules degraded only about 10% of bacterial protein regardless of pH. To determine whether activation of the respiratory burst contributes to digestion, we compared degradation by intact PMN in room air and under N2. Depletion of O2 by N2 flushing had no effect on the bactericidal activity of either human or rabbit PMN but reduced degradation by approximately 50%. Protein degradation during phagocytosis was also reduced in the presence of cyanide or azide, inhibitors of myeloperoxidase (MPO). PMN of two patients with chronic granulomatous disease ingested and killed E. coli S15 as well as did normal PMN but degraded bacterial protein as did normal PMN incubated under N2. The low degradative activity of PMN disrupted by sonication could be raised to nearly the level of intact PMN incubated in room air by preincubation of the PMN with 10(-7) M formyl-methionyl-leucyl-phenylalanine (fMLP) before sonication and by pretreatment of E. coli with MPO. Depletion of O2 or chloride during these preincubations with formyl-methionyl-leucyl-phenylalanine respectively, virtually abolished and markedly diminished stimulation of bacterial protein degradation. We conclude that enhanced MPO-mediated O2 metabolism of intact PMN plays a role in the digestion of killed E. coli.

How human neutrophils kill and degrade microbes: an integrated view

“…Consequently, PMNs defective in oxidase activation, as seen in CGD, show profound abnormalities in their ability to trigger signaling cascades (74, 75), to kill a broad range of microbes, or to initiate productively the apoptotic program (76, 77) and subsequently be removed by macrophages (78). Although the killing of some organisms, such as catalase‐negative streptococci (79), E. coli (80–83), and Pseudomonas aeruginosa (84), is normal, overall anti‐microbial activity of CGD PMNs is profoundly depressed (26). Targeted deletions of components of the NADPH oxidase in murine models (85, 86) recapitulate the infectious complications seen in CGD (2, 87, 88), suggesting that the NADPH oxidase has a prominent role in the normal function of murine as well as human PMNs.…”

Glycine induces enhancement of bactericidal activity of neutrophils

“…1D ), indicating that GlyR is not involved in the basal phagocytosis-associated respiratory burst but only in the enhancement of it induced by supplemental glycine. Impaired NADPH oxidase activity is known to be accompanied by impairment in release of azurophil granules that contain various proteases, and protease activity; both chronic granulomatous disease neutrophils and DPI-treated neutrophils show impairment in azurophil granule release [ 42 - 44 ] and in the digestion of killed E. coli [ 36 , 45 , 46 ]. We found that ROS was essential in glycine-induced azurophil granule-phagosome fusion via the Ca 2+ /p38 MAPK/fluid-phase pinocytosis/azurophil granule-phagosome fusion pathway.…”

Oxygen‐Independent Microbicidal Mechanisms of Phagocytes

“…For others (e.g .• Staphylococcus aureus), the microbicidal defect was kinetic (8) and could be overcome by examining longer exposures of the microbes to the phagosomal environment. Many of the most severe manifestations of CGD are now attributed to a defect in the phagocytic degradation of microbial macromolecules (9), leading to the formation of reactive granulomas that impair organ function. This interpretation is favored by the severe granulomatous lung injury caused by dead fungal spores in transgenic CGD mice, but not in normal mice (10).…”