title: Bioinformatic and mass spectrometry identification of Anaplasma phagocytophilum proteins translocated into host cell nuclei.
abstract: Obligate intracellular bacteria have an arsenal of proteins that alter host cells to establish and maintain a hospitable environment for replication. Anaplasma phagocytophilum secrets Ankyrin A (AnkA), via a type IV secretion system, which translocates to the nucleus of its host cell, human neutrophils. A. phagocytophilum-infected neutrophils have dramatically altered phenotypes in part explained by AnkA-induced transcriptional alterations. However, it is unlikely that AnkA is the sole effector to account for infection-induced transcriptional changes. We developed a simple method combining bioinformatics and iTRAQ protein profiling to identify potential bacterial-derived nuclear-translocated proteins that could impact transcriptional programming in host cells. This approach identified 50 A. phagocytophilum candidate genes or proteins. The encoding genes were cloned to create GFP fusion protein-expressing clones that were transfected into HEK-293T cells. We confirmed nuclear translocation of six proteins: APH_0062, RplE, Hup, APH_0382, APH_0385, and APH_0455. Of the six, APH_0455 was identified as a type IV secretion substrate and is now under investigation as a potential nucleomodulin. Additionally, application of this approach to other intracellular bacteria such as Mycobacterium tuberculosis, Chlamydia trachomatis and other intracellular bacteria identified multiple candidate genes to be investigated.
authors.affiliation: Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Department of Pathology, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Department of Pathology, University of Maryland School of Medicine Baltimore, MD, USA ; Department of Microbiology and Immunology, University of Maryland School of Medicine Baltimore, MD, USA.
authors.forename: Sara H G
authors.initials: SH
authors.lastname: Sinclair
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Procter and Gamble Co. Cincinnati, OH, USA.
authors.forename: Jose C
authors.initials: JC
authors.lastname: Garcia-Garcia
authors.affiliation: Graduate Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Department of Pathology, The Johns Hopkins University School of Medicine Baltimore, MD, USA ; Department of Pathology, University of Maryland School of Medicine Baltimore, MD, USA ; Department of Microbiology and Immunology, University of Maryland School of Medicine Baltimore, MD, USA.
authors.forename: J Stephen
authors.initials: JS
authors.lastname: Dumler
keywords: Anaplasma phagocytophilum; iTRAQ; nuclear translocation; nucleomodulin; oxidative burst