title: Identification of a novel Anaplasma marginale appendage-associated protein that localizes with actin filaments during intraerythrocytic infection.
abstract: The rickettsial pathogen Anaplasma marginale assembles an actin filament bundle during intracellular infection. Unlike other bacterial pathogens that generate actin filament tails, A. marginale infects mature erythrocytes, and the F-actin appendages are assembled on the cytoplasmic surface of a vacuole containing several organisms. To identify A. marginale molecules associated with these filaments, two complementary approaches were used: matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and tandem mass spectrometry of A. marginale proteins identified with an appendage-specific monoclonal antibody and expression screening of an A. marginale phage library. Amino acid and nucleotide sequences were mapped to a full-length gene in the genome of the St. Maries strain of A. marginale; the correct identification was confirmed by expression of full-length recombinant protein and its reactivity with appendage-specific antibodies. Interestingly, there is marked variation in the abilities of diverse A. marginale strains to assemble the F-actin appendages. Comparison of four strains, the Florida, Illinois, St. Maries, and Virginia strains, revealed substantial polymorphism in the gene encoding the appendage-associated protein, with amino acid sequence identity of as low as 34% among strains. However, this variation does not underlie the differences in expression, as there is no specific polymorphism associated with loss of ability to assemble actin appendages. In contrast, the ability to assemble an actin filament bundle reflected dramatic strain-specific differences in the expression level of the appendage-associated protein. Understanding how this protein influences the cycle of invasion, replication, and egress in the host cell may provide new insights into pathogen-host interactions.
authors.affiliation: Department of Veterinary Preventive Medicine, The Ohio State University, 1900 Coffey Rd., Columbus, OH 43210, USA. stich.2@osu.edu
authors.forename: Roger W
authors.initials: RW
authors.lastname: Stich
authors.forename: Glenn A
authors.initials: GA
authors.lastname: Olah
authors.forename: Kelly A
authors.initials: KA
authors.lastname: Brayton
authors.forename: Wendy C
authors.initials: WC
authors.lastname: Brown
authors.forename: Marcus
authors.initials: M
authors.lastname: Fechheimer
authors.forename: Kari
authors.initials: K
authors.lastname: Green-Church
authors.forename: Sathaporn
authors.initials: S
authors.lastname: Jittapalapong
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Kocan
authors.forename: Travis C
authors.initials: TC
authors.lastname: McGuire
authors.forename: Fred R
authors.initials: FR
authors.lastname: Rurangirwa
authors.forename: Guy H
authors.initials: GH
authors.lastname: Palmer
mesh_terms: D000199:Actins / Q000032:analysis*; D000595:Amino Acid Sequence; D042322:Anaplasma marginale / Q000737:chemistry* / Q000472:pathogenicity; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001426:Bacterial Proteins / Q000032:analysis*; D002417:Cattle; D004912:Erythrocytes / Q000382:microbiology*; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013045:Species Specificity
chemical_list: D000199:Actins; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins