Intracellular trafficking of Pseudomonas ExoS, a type III cytotoxin.
Pseudomonas aeruginosa ExoS is a bifunctional type III cytotoxin that disrupts Ras- and Rho-signaling pathways in mammalian cells. A hydrophobic region (residues 51-77, termed the membrane localization domain) targets ExoS to the plasma membrane (PM) and late endosomes of host cells. In the current study, metabolic inhibitors and dominant-negative proteins that disrupt known vesicle-trafficking pathways were used to define the intracellular trafficking of ExoS. Release of ExoS from PM was independent of dynamin and ADP ribosylation factor 6 but inhibited by methyl-beta-cyclodextrin, a cholesterol-depleting reagent, and perinuclear localization of ExoS was disrupted by nocodazole. p50 dynamitin, a dynein inhibitor partially disrupted perinuclear localization of ExoS. Methyl-beta-cyclodextrin and nocodazole inhibited the ability of type-III-delivered ExoS to ADP-ribosylated Golgi/endoplasmic reticulum-resident Ras. Methyl-beta-cyclodextrin also relocated ExoS from the perinuclear region to the PM, indicating that ExoS can cycle through anterograde as well as through retrograde trafficking pathways. These findings show that ExoS endocytosis is cholesterol dependent, and it utilizes host microtubules, for intracellular trafficking. Understanding how type III cytotoxins enter and traffic within mammalian cells may identify new targets for therapeutic intervention of gram-negative bacterial pathogens.
Deng
Qing
Q
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Zhang
Yue
Y
Barbieri
Joseph T
JT
D036002:ADP Ribose Transferases / Q000378:metabolism* / Q000502:physiology; D000089982:ADP-Ribosylation Factor 6; D001427:Bacterial Toxins / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002784:Cholesterol / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology*; D007956:Leukocidins / Q000378:metabolism* / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D000089982:ADP-Ribosylation Factor 6; D001427:Bacterial Toxins; D007956:Leukocidins; C510238:Pseudomonas aeruginosa Cytotoxins; D002784:Cholesterol; D036002:ADP Ribose Transferases; C444820:exoenzyme S; C000715348:ARF6 protein, human