A Study Of Metabolic Factors Involved In The Intracellular Germination Of Clostridium Botulinum Spores After Phagocytosis

Abstract

SUMMARY The environment within the metabolically active polymorphonuclear leucocyte during phagocytosis is conducive to the germination of phagocytically ingested spores of Clostridium botulinum type A. Specific metabolic inhibitors of leucocyte function—glyceraldehyde, hydrocortisone, colchicine, methimazole and azide—were used to study metabolic pathways during phagocytosis that may be responsible for inducing spore germination within the leucocyte. Leakage of 45Ca from labelled Cl. botulinum spores was taken as evidence of spore germination. Release of free 45Ca from phagocytosed spores in sporeleucocyte mixtures in in-vitro systems was normal in the presence of hydrocortisone, methimazole, azide and methimazole+azide. These findings indicate that H2O2 production, iodination and myeloperoxidase activities during phagocytosis are not required for germination. In the presence of glyceraldehyde or colchicine, release of 45Ca from spore-leucocyte mixtures was profoundly inhibited and this indicates that glycolysis and lysosome degranulation are essential for intraleucocytic spore germination. In leucocytes from patients with a genetic abnormality of phagocytosis, i.e., chronic granulomatous disease of childhood (CGD), germination of spores was significantly depressed. Since normal mechanisms of H2O2 production and possibly of lysosomal degranulation are blocked in CGD leucocytes, the results suggest that H2O2 and lysosomal enzymes are involved in triggering spore germination in this model. Nevertheless, a limited release of 45Ca was observed and this seems to indicate that small amounts of lysosomal enzymes are available for reaction with engulfed spores in CGD leucocytes. The results of these studies with metabolic inhibitors and genetic blocks lead to the view that germination of spores of Cl. botulinum within leucocytes is triggered by the combined action of lowered pH resulting from glycolysis together with lytic enzymes resulting from degranulation of lysosomes.