title: In vitro intracellular trafficking of virulence antigen during infection by Yersinia pestis.
abstract: Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MPhis) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MPhis were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MPhis were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10-45 min of infection, (2) lysosomal protein(s) between 1-2 h of infection, (3) mitochondrial proteins between 2.5-3 h infection, and (4) Golgi protein(s) between 4-6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V.
authors.affiliation: Bacteriology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, United States of America.
authors.forename: Tracy L
authors.initials: TL
authors.lastname: DiMezzo
authors.forename: Gordon
authors.initials: G
authors.lastname: Ruthel
authors.forename: Ernst E
authors.initials: EE
authors.lastname: Brueggemann
authors.forename: Harry B
authors.initials: HB
authors.lastname: Hines
authors.forename: Wilson J
authors.initials: WJ
authors.lastname: Ribot
authors.forename: Carol E
authors.initials: CE
authors.lastname: Chapman
authors.forename: Bradford S
authors.initials: BS
authors.lastname: Powell
authors.forename: Susan L
authors.initials: SL
authors.lastname: Welkos
mesh_terms: D000942:Antigens, Bacterial / Q000378:metabolism*; D001692:Biological Transport; D015153:Blotting, Western; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008264:Macrophages / Q000276:immunology; D010930:Plague / Q000276:immunology*; D014774:Virulence; D015010:Yersinia pestis / Q000276:immunology* / Q000472:pathogenicity
chemical_list: D000942:Antigens, Bacterial