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Infection and Immunity, December 2005, p. 8322-8333, Vol. 73, No. 12
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.12.8322-8333.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Leishmania pifanoi Amastigotes Avoid Macrophage Production of Superoxide by Inducing Heme Degradation

Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida

Received 18 June 2005/ Returned for modification 11 July 2005/ Accepted 23 August 2005

Whereas infections of macrophages by promastigote forms of Leishmania mexicana pifanoi induce the production of superoxide, infections by amastigotes barely induce superoxide production. Several approaches were employed to gain insight into the mechanism by which amastigotes avoid eliciting superoxide production. First, in experiments with nitroblue tetrazolium, we found that 25% of parasitophorous vacuoles (PVs) that harbor promastigotes are positive for the NADPH oxidase complex, in contrast to only 2% of PVs that harbor amastigotes. Second, confocal microscope analyses of infected cells labeled with antibodies to gp91^phox revealed that this enzyme subunit is found in PVs that harbor amastigotes. Third, in immunoblots of subcellular fractions enriched with PVs from amastigote-infected cells and probed with antibodies to gp91^phox, only the 65-kDa premature form of gp91^phox was found. In contrast, subcellular fractions from macrophages that ingested zymosan particles contained both the 91- and 65-kDa forms of gp91^phox. This suggested that only the immature form of gp91^phox is recruited to PVs that harbor amastigotes. Given that gp91^phox maturation is dependent on the availability of heme, we found that infections by Leishmania parasites induce an increase in heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation. Infections by amastigotes performed in the presence of metalloporphyrins, which are inhibitors of HO-1, resulted in superoxide production by infected macrophages. Taken together, we propose that Leishmania amastigotes avoid superoxide production by inducing an increase in heme degradation, which results in blockage of the maturation of gp91^phox, which prevents assembly of the NADPH oxidase enzyme complex.

Editor: F. C. Fang