Dendrimer-Enabled Transformation of Anaplasma Phagocytophilum
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.
A. Oki et al., "Dendrimer-Enabled Transformation of Anaplasma Phagocytophilum," Microbes and Infection, vol. 17, no. 11-12, pp. 817-822, Elsevier Masson SAS, Nov 2015.
The definitive version is available at https://doi.org/10.1016/j.micinf.2015.09.001
Chemical and Biochemical Engineering
Keywords and Phrases
Anaplasmataceae; Dendrimers; Intracellular bacteria; Pathogen-occupied vacuole; Rickettsiales
International Standard Serial Number (ISSN)
Article - Journal
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01 Nov 2015