Superoxide Production in Galleria mellonella Hemocytes: Identification of Proteins Homologous to the NADPH Oxidase Complex of Human Neutrophils

Abstract

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 gp91 phox , p67 phox , p47 phox , and the GTP-binding protein rac 2. A protein equivalent to p40

Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella

“…These cytosolic proteins interact with each other [21,22], with rac [23,24] and with the flavocytochrome [25][26][27] through a number of Src homology 3 (SH3), proline-rich, tetratricopeptide repeat, and PC motifs. Using immunological and matrixassisted laser desorption ionisation-time of flight analysis (MALDI-TOF), the presence of homologous proteins to p67 phox and p47 phox were found in insect hemocytes [6] further strengthening the similarities between the oxidative burst pathways in the two cell types.…”

“…Given the role of the innate immune response in protecting mammals from microbial infection and the high degree of similarity that exists between the mammalian and insect innate immune response, insect models have been developed for the study of microbial virulence [5,6]. Advantages of the use of insects include low cost, ease of rearing in the laboratory, genetic manipulability and fewer ethical considerations than the use of mammalian models [4].…”

“…Advantages of the use of insects include low cost, ease of rearing in the laboratory, genetic manipulability and fewer ethical considerations than the use of mammalian models [4]. We are interested in developing the use of larvae of the Greater Wax Moth Galleria mellonella (G. mellonella) which is attracting ever-increasing attention as a model organism for the study of a range of pathogenic bacteria (Pseudomonas aeruginosa [5], Proteus mirabilis [7], Escherichia coli, Bacillus cereus, [8] and Staphylococcus aureus [6]) and fungi (Cryptococcus neoformans [9], Aspergillus and Candida species [6,10]). The demonstration of a positive correlation between the virulence of Candida mutants in BalbC mice and G. mellonella larvae augments the use of G. mellonella as a model for evaluating microbial pathogenicity [11].…”

“…The burst in oxidative metabolism associated with activation of either human neutrophils or insect hemocytes results in the manufacture of reactive oxygen species (ROS) as detected by electron spin resonance spectroscopy [17] and more recently by cytochrome c reduction, with evidence of increased oxygen consumption resulting in superoxide (O 2 À ) production (0.25 mM/min/10 6 ) by hemocytes of G. mellonella [6].…”

“…We have previously demonstrated at the cellular level the respiratory burst of insect hemocytes and the presence of proteins (47 and 67 kDa) homologous to neutrophil p47 phox and p67 phox [6]. However, it remained unresolved whether activation of the NADPH oxidase of hemocytes resulted in translocation of these homologous phox components to the plasma membrane of activated hemocytes.…”