Bergin, David and Reeves, Emer P. and Renwick, Julie and Wientjes, Frans B. and Kavanagh, Kevin
Superoxide Production in Galleria mellonella Hemocytes: Identification of Proteins Homologous to the NADPH Oxidase Complex of Human Neutrophils.
Infection and Immunity, 73 (7).
The insect immune response has a number of structural and functional similarities to the innate immune
response of mammals. The objective of the work presented here was to establish the mechanism by which insect
hemocytes produce superoxide and to ascertain whether the proteins involved in superoxide production are
similar to those involved in the NADPH oxidase-induced superoxide production in human neutrophils.
Hemocytes of the greater wax moth (Galleria mellonella) were shown to be capable of phagocytosing bacterial
and fungal cells. The kinetics of phagocytosis and microbial killing were similar in the insect hemocytes and
human neutrophils. Superoxide production and microbial killing by both cell types were inhibited in the
presence of the NADPH oxidase inhibitor diphenyleneiodonium chloride. Immunoblotting of G. mellonella
hemocytes with antibodies raised against human neutrophil phox proteins revealed the presence of proteins
homologous to gp91phox, p67phox, p47phox, and the GTP-binding protein rac 2. A protein equivalent to p40phox
was not detected in insect hemocytes. Immunofluorescence analysis localized insect 47-kDa and 67-kDa
proteins throughout the cytosol and in the perinuclear region. Hemocyte 67-kDa and 47-kDa proteins were
immunoprecipitated and analyzed by matrix-assisted laser desorption ionization-time of flight analysis. The
results revealed that the hemocyte 67-kDa and 47-kDa proteins contained peptides matching those of p67phox
and p47phox of human neutrophils. The results presented here indicate that insect hemocytes phagocytose and
kill bacterial and fungal cells by a mechanism similar to the mechanism used by human neutrophils via the
production of superoxide. We identified proteins homologous to a number of proteins essential for superoxide
production in human neutrophils and demonstrated that significant regions of the 67-kDa and 47-kDa insect
proteins are identical to regions of the p67phox and p47phox proteins of neutrophils.
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