title: Dendrimer-enabled transformation of Anaplasma phagocytophilum.
abstract: Anaplasma phagocytophilum is an obligate intracellular bacterium that causes the emerging infection, granulocytic anaplasmosis. While electroporation can transform A. phagocytophilum isolated from host cells, no method has been developed to transform it while growing inside the ApV (A. phagocytophilum-occupied vacuole). Polyamidoamine (PAMAM) dendrimers, well-defined tree-branched macromolecules used for gene therapy and nucleic acid delivery into mammalian cells, were recently shown to be effective in transforming Chlamydia spp. actively growing in host cells. We determined if we could adapt a similar system to transform A. phagocytophilum. Incubating fluorescently labeled PAMAM dendrimers with infected host cells resulted in fluorescein-positive ApVs. Incubating infected host cells or host cell-free A. phagocytophilum organisms with dendrimers complexed with pCis GFPuv-SS Himar A7 plasmid, which carries a Himar1 transposon cassette encoding GFPuv and spectinomycin/streptomycin resistance plus the Himar1 transposase itself, resulted in GFP-positive, antibiotic resistant bacteria. Yet, transformation efficiencies were low. The transformed bacterial populations could only be maintained for a few passages, likely due to random Himar1 cassette-mediated disruption of A. phagocytophilum genes required for fitness. Nonetheless, these results provide proof of principle that dendrimers can deliver exogenous DNA into A. phagocytophilum, both inside and outside of host cells.
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, PO Box 980678, Richmond, VA, 23298-0678, United States.
authors.forename: Aminat T
authors.initials: AT
authors.lastname: Oki
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, PO Box 980678, Richmond, VA, 23298-0678, United States.
authors.forename: David
authors.initials: D
authors.lastname: Seidman
authors.affiliation: Department of Biomedical Engineering, Virginia Commonwealth University School of Engineering, P.O. Box 843067, Richmond, VA 23284-3067, United States.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Lancina
authors.affiliation: Center for Nanomedicine, Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21235, United States.
authors.forename: Manoj K
authors.initials: MK
authors.lastname: Mishra
authors.affiliation: Center for Nanomedicine, Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, MD 21235, United States.
authors.forename: Rangaramanujam M
authors.initials: RM
authors.lastname: Kannan
authors.affiliation: Department of Biomedical Engineering, Virginia Commonwealth University School of Engineering, P.O. Box 843067, Richmond, VA 23284-3067, United States.
authors.forename: Hu
authors.initials: H
authors.lastname: Yang
authors.affiliation: Department of Microbiology and Immunology, Virginia Commonwealth University School of Medicine, PO Box 980678, Richmond, VA, 23298-0678, United States. Electronic address: jason.carlyon@vcuhealth.org.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Carlyon
mesh_terms: D041081:Anaplasma phagocytophilum / Q000235:genetics* / Q000472:pathogenicity; D000712:Anaplasmosis / Q000382:microbiology; D000818:Animals; D050091:Dendrimers / Q000494:pharmacology*; D018014:Gene Transfer Techniques; D006801:Humans; D010957:Plasmids / Q000235:genetics; D011073:Polyamines / Q000494:pharmacology*; D014170:Transformation, Genetic / Q000235:genetics*
chemical_list: D050091:Dendrimers; C531249:Poly(amidoamine); D011073:Polyamines
keywords: Anaplasmataceae; Dendrimers; Intracellular bacteria; Pathogen-occupied vacuole; Rickettsiales