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.

Department(s)

Chemical and Biochemical Engineering

Comments

This study was supported by the National Institutes of Health (NIH) grant 2R01 AI072683.

Keywords and Phrases

Anaplasmataceae; Dendrimers; Intracellular bacteria; Pathogen-occupied vacuole; Rickettsiales

International Standard Serial Number (ISSN)

1286-4579

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2015 Institut Pasteur, All rights reserved.

Publication Date

01 Nov 2015

PubMed ID

26369714

Link to Full Text

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