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.

Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle

“…There are no reports of genetic modification in Orientia tsutsugamushi or Wolbachia. Transformation has been achieved using electroporation [247][248][249][250][251]151 and polyamidoamine dendrimers 252 . Protein overexpression has been achieved in Rickettsia spp.…”

“…Where genetic tools are available, overexpression of fluorescence proteins has been used for imaging intracellular Rickettsiales in cultured cells and animals 5,252,256,[262][263][264][265][266] . Given the limited availability of genetic tools, however, alternative approaches for fluorescence microscopy are required.…”

Multiple Ehrlichia chaffeensis Genes Critical for Its Persistent Infection in a Vertebrate Host Are Identified by Random Mutagenesis Coupled with In Vivo Infection Assessment

“…Three different plasmid constructs encoding the Himar1 transposase, antibiotic resistance conferred by aadA , and a fluorescent protein (mCherry or GFP) driven by the Anaplasma marginale Am-tr promoter were used for mutagenesis of E. chaffeensis : (i) pCis mCherry-SS Himar A7 containing the mCherry and aadA genes ( 14 , 25 ), (ii) pHimar1 A7 loxP plasmid containing the mCherry and aadA genes flanked by loxP sites, and (iii) pCis GFPuv-SS Himar A7 containing the gfpuv and aadA genes ( 14 , 27 ). The pHimar1 A7 loxP plasmid was generated by inserting mismatched loxP sites ( 76 , 77 ) flanking the transposon segment into pCis mCherry-SS Himar A7.…”

Synthesis and Antimicrobial Properties of a Ciprofloxacin and PAMAM-dendrimer Conjugate

“…The lack of antimicrobial activity of the control dendrimer compared to the synergistic activity of the dendrimer Ciprofloxacin conjugate relative to Ciprofloxacin suggest that the synergism is related to Ciprofloxacin mode of action. Here, it is of interest that PAMAM dendrimers promote transfection of DNA in both mammalian cells [64] and in prokaryotic cells as demonstrated for Anaplasma phagocytophilum, an obligate intracellular bacterium [65]. Thus, the ability of PAMAM to bind DNA indicates that the dendrimer may interact with the association of Ciprofloxacin with bacterial topoisomerases e.g., by binding to DNA fragments generated by Ciprofloxacin activity.…”