Mechanism of inducible nitric oxide synthase exclusion from mycobacterial phagosomes.
Mycobacterium tuberculosis is sensitive to nitric oxide generated by inducible nitric oxide synthase (iNOS). Consequently, to ensure its survival in macrophages, M. tuberculosis inhibits iNOS recruitment to its phagosome by an unknown mechanism. Here we report the mechanism underlying this process, whereby mycobacteria affect the scaffolding protein EBP50, which normally binds to iNOS and links it to the actin cytoskeleton. Phagosomes harboring live mycobacteria showed reduced capacity to retain EBP50, consistent with lower iNOS recruitment. EBP50 was found on purified phagosomes, and its expression increased upon macrophage activation, paralleling expression changes seen with iNOS. Overexpression of EBP50 increased while EBP50 knockdown decreased iNOS recruitment to phagosomes. Knockdown of EBP50 enhanced mycobacterial survival in activated macrophages. We tested another actin organizer, coronin-1, implicated in mycobacterium-macrophage interaction for contribution to iNOS exclusion. A knockdown of coronin-1 resulted in increased iNOS recruitment to model latex bead phagosomes but did not increase iNOS recruitment to phagosomes with live mycobacteria and did not affect mycobacterial survival. Our findings are consistent with a model for the block in iNOS association with mycobacterial phagosomes as a mechanism dependent primarily on reduced EBP50 recruitment.
Davis
Alexander S
AS
Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, New Mexico, United States of America.
Vergne
Isabelle
I
Master
Sharon S
SS
Kyei
George B
GB
Chua
Jennifer
J
Deretic
Vojo
V
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D020868:Gene Silencing; D054884:Host-Pathogen Interactions / Q000187:drug effects / Q000502:physiology*; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000382:microbiology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D050296:Microbial Viability; D009161:Mycobacterium / Q000502:physiology*; D009163:Mycobacterium bovis / Q000378:metabolism / Q000472:pathogenicity; D009169:Mycobacterium tuberculosis / Q000378:metabolism / Q000472:pathogenicity*; D052247:Nitric Oxide Synthase Type II / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010588:Phagosomes / Q000201:enzymology* / Q000276:immunology; D010750:Phosphoproteins; D034741:RNA, Small Interfering / Q000235:genetics; D017923:Sodium-Hydrogen Exchangers
D000199:Actins; D008070:Lipopolysaccharides; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D007371:Interferon-gamma; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse