title: Arf6-dependent intracellular trafficking of Pasteurella multocida toxin and pH-dependent translocation from late endosomes.
abstract: The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH(4)Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity.
authors: Department of Microbiology, University of Illinois at Urbana-Champaign, 601 South Goodwin Avenue, Urbana, IL 61801, USA. repella@illinois.edu
authors: Tana L
authors: TL
authors: Repella
authors: Mengfei
authors: M
authors: Ho
authors: Tracy P M
authors: TP
authors: Chong
authors: Yuka
authors: Y
authors: Bannai
authors: Brenda A
authors: BA
authors: Wilson
mesh_terms: D016475:3T3 Cells; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000493:pharmacokinetics* / Q000633:toxicity*; D001427:Bacterial Toxins / Q000493:pharmacokinetics* / Q000633:toxicity*; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002772:Cholera Toxin / Q000493:pharmacokinetics; D004705:Endocytosis; D011992:Endosomes / Q000187:drug effects* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016979:Pasteurella multocida / Q000378:metabolism*; D021381:Protein Transport; D014168:Transferrin / Q000493:pharmacokinetics
chemical_list: D000089982:ADP-Ribosylation Factor 6; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C062258:Pasteurella multocida toxin; D014168:Transferrin; D002772:Cholera Toxin; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; C000715727:Arf6 protein, mouse