Transformation of Anaplasma phagocytophilum

Abstract

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.

Conclusion: 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.

Figure 1

Transposition insertion diagram and Southern blot. (A) This diagram represents eight Ap genomic locations and direction of the transposon (arrow under the line) as determined by rescue cloning from GFP/Ap genomic DNA and sequence mapping onto the Ap HZ genome. Four insertions were found in putative coding sequences and four were in intergenic sites. The band sizes on the left of the diagram correspond to bands in the BglII digest lane of the Southern blot. (B) Southern blot of restriction digested GFP/Ap genomic DNA probed with a GFPuv coding sequence probe. The labels indicate restriction enzyme used and the numbers on the right indicate the sizes of visible bands in the BglII lane.

Figure 2

Images of GFP/Ap growing within various host cells. HL-60 (A) with partial bright field to illuminate the non fluorescent host cell. Tick cell ISE6 (B) expressing DSred and containing indistinct bacteria in morulae. Monkey RF/6A (C) and human HMEC-1 (D) endothelial cells expressing mCherry and containing GFP/Ap morulae with distinct bacteria. Bars 5 μm.

Figure 3

Physical maps of the Himar1 transposon and transposase plasmids. (A) pHIMAR1-UV-SS carries the A. marginale promoter tr driving expression of GFPuv and spectinomycin resistance between the left and right Himar transposon repeats. (B) pET28AMTR-A7-HIMAR contains the A7 hyperactive mutant of the Himar1 transposase also driven by the Am tr promoter.