Mycobacteria inhibit nitric oxide synthase recruitment to phagosomes during macrophage infection.
Inducible nitric oxide synthase (iNOS) is a cytoplasmic protein responsible for the generation of nitric oxide (NO. ) in macrophages. In this work, we hypothesized that the intracellular localization of iNOS is significant for effective delivery of NO. to phagosomes containing ingested microorganisms. Using immunofluorescence microscopy and Western blot analysis, iNOS was shown to localize in the vicinity of phagosomes containing latex beads in stimulated macrophages. iNOS also localized to phagosomes containing Escherichia coli. The colocalization of iNOS with ingested latex beads was an actin-dependent process, since treatment with the actin microfilament disrupter cytochalasin D prevented iNOS recruitment to latex bead phagosomes. In contrast to E. coli and inert particle phagosomes, mycobacterial phagosomes did not colocalize with iNOS. This study demonstrates that (i). iNOS can be recruited to phagosomes; (ii). this recruitment is dependent on a functional actin cytoskeleton; (iii). certain microorganisms have the ability to prevent or reduce colocalization with iNOS; and (iv). spatial exclusion of iNOS may play a role in Mycobacterium tuberculosis pathogenesis.
Miller
Barbara H
BH
Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, Michigan 48109-0620, USA.
Fratti
Rutilio A
RA
Poschet
Jens F
JF
Timmins
Graham S
GS
Master
Sharon S
SS
Burgos
Marcos
M
Marletta
Michael A
MA
Deretic
Vojo
V
D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D066298:In Vitro Techniques; D008262:Macrophage Activation; D008264:Macrophages / Q000201:enzymology* / Q000382:microbiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009163:Mycobacterium bovis / Q000472:pathogenicity; D009169:Mycobacterium tuberculosis / Q000472:pathogenicity*; D019001:Nitric Oxide Synthase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010588:Phagosomes / Q000201:enzymology* / Q000382:microbiology*
D000199:Actins; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse