The anaerobic pathogen Clostridium perfringens can escape the phagosome ofmacrophages under aerobic conditions

Clostridium perfringens is the most common cause of gas gangrene (clostridi al myonecrosis), a disease that begins when ischaemic tissues become contam inated with C. perfringens vegetative cells or spores. An aerotolerant anae robe, C, perfringens quickly multiplies in ischaemic tissues and spreads to healthy areas, leading to a high level of morbidity and mortality. As a sp ecies, the bacterium can synthesize 13 different toxins, and these are thou ght to be the major virulence factors of the disease. However, we present e vidence here that C, perfringens can also persist inside macrophages, under aerobic conditions, by escaping the phagosome into the cytoplasm. C, perfr ingens was not killed by the cells of a clone (J774-33) of the macrophage-l ike murine cell line J774A.1 under aerobic or anaerobic conditions, whereas the non-pathogenic bacterium Bacillus subtilis was killed by J774-33 cells under both conditions. Electron microscopy images showed that C. perfringe ns cells were intact and resided mostly in the cytoplasm of J774-33 cells, whereas B. subtilis was in the phagosome, Immunofluorescence microscopy sho wed that intracellular C, perfringens bacteria failed to co-localize with t he late endosome-lysosomal marker glycoprotein LAMP-1, whereas B. subtilis did co-localize with LAMP-1. C. perfringens also appeared to escape the pha gosome of both activated and unactivated mouse peritoneal macrophages, but not as efficiently as was seen with the J774-33 cell line. In addition, cyt ochalasin D was used to show that phagocytosis of C, perfringens was depend ent on actin polymerization and that the bacteria attach to J774-33 cells a t distinct areas of the cell membrane. We propose that the ability to escap e the phagosome and persist inside macrophages is an important factor in th e early stages of a gangrene infection, when bacterial numbers are low and phagocytic cells are present.