Phagocytosis of Leishmania donovani amastigotes is Rac1 dependent and occurs in the absence of NADPH oxidase activation.
Macrophages produce little superoxide during phagocytosis of Leishmania donovani amastigotes. In this study, we characterized molecular events associated with L. donovani amastigotes uptake by mouse macrophages, to further define the mechanisms by which they are internalized without triggering superoxide production. Using transient transfections, we first showed that internalization of L. donovani amastigotes is mediated by the GTPases Rac1 and Arf6, of which Rac1 is recruited and retained on parasite-containing phagosomes. Next, we showed that, whereas internalization of amastigotes induced no superoxide release, co-internalization of serum-opsonized zymozan and amastigotes resulted in superoxide production. Furthermore, in co-internalization experiments, we detected superoxide production in over 95% of phagosomes containing IgG-opsonized SRBC compared to 5% of amastigote-harboring phagosomes. These results suggest that amastigotes evade the ability of macrophages to produce superoxide during phagocytosis. Consistently, we observed that amastigotes induced barely detectable phosphorylation of the NADPH oxidase component p47phox, leading to a defective phagosomal recruitment of p67phox and p47phox. Finally, we showed that amastigotes disrupt phagosomal lipid raft integrity, potentially interfering with NADPH oxidase assembly. Collectively, our results indicate that internalization of L. donovani amastigotes is a Rac1- and Arf6-dependent process that occurs in the absence of significant NADPH oxidase activation.
Lodge
Robert
R
INRS-Institut Armand Frappier and Centre for host-parasite interactions, Laval, QC, Canada.
Descoteaux
Albert
A
D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000276:immunology / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D004789:Enzyme Activation / Q000502:physiology*; D005260:Female; D005455:Fluorescent Antibody Technique; D007893:Leishmania donovani / Q000276:immunology*; D008264:Macrophages / Q000276:immunology / Q000378:metabolism* / Q000382:microbiology; D021962:Membrane Microdomains / Q000378:metabolism / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D019255:NADPH Oxidases / Q000276:immunology / Q000378:metabolism*; D010587:Phagocytosis / Q000276:immunology*; D010588:Phagosomes / Q000378:metabolism / Q000382:microbiology; D010766:Phosphorylation; D020830:rac1 GTP-Binding Protein / Q000276:immunology / Q000378:metabolism*
D000089982:ADP-Ribosylation Factor 6; D019255:NADPH Oxidases; D020727:ADP-Ribosylation Factors; C000715727:Arf6 protein, mouse; D020830:rac1 GTP-Binding Protein