Transformation of Anaplasma phagocytophilum.
BACKGROUND
Tick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum. Because of their obligate intracellular nature, methods for mutagenesis and transformation have not been available.


RESULTS
To facilitate genetic manipulation, we transformed A. phagocytophilum (Ap) to express a green fluorescent protein (GFP) with the Himar1 transposase system and selection with the clinically irrelevant antibiotic spectinomycin.


CONCLUSIONS
These transformed bacteria (GFP/Ap) grow at normal rates and are brightly fluorescent in human, monkey, and tick cell culture. Molecular characterization of the GFP/Ap genomic DNA confirmed transposition and the flanking genomic insertion locations were sequenced. Three mice inoculated with GFP/Ap by intraperitoneal injection became infected as demonstrated by the appearance of morulae in a peripheral blood neutrophil and re-isolation of the bacteria in culture.
Felsheim
Roderick F
RF
Department of Entomology, University of Minnesota, St. Paul, MN 55108, USA. felsh001@umn.edu
Herron
Michael J
MJ
Nelson
Curtis M
CM
Burkhardt
Nicole Y
NY
Barbet
Anthony F
AF
Kurtti
Timothy J
TJ
Munderloh
Ulrike G
UG
D041081:Anaplasma phagocytophilum / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D013987:Ticks; D014169:Transformation, Bacterial / Q000502:physiology*
D049452:Green Fluorescent Proteins