Abstract
BackgroundTick-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.ResultsTo 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.ConclusionThese 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.
Highlights
Tick-borne pathogens cause emerging zoonoses, and include fastidious organisms such as Anaplasma phagocytophilum
Southern Blot, confirmation PCR and rescue cloning To detect the presence of the GFPuv – Specr DNA in the fluorescent bacteria, a set of PCR primers not used in the plasmid construction (Forward UV-SS confirmation PCR, Reverse UV-SS confirmation PCR) was used to amplify a Transposon insertion points map to the following positions on the A. phagocytophilum (Ap) HZ genome[20]: 992097, 987221, 843456, 528403, 571958 (Figure 1A)
The tr promoter is one of the few characterized in Anaplasma and we have demonstrated it to be expressed in bacteria grown in both mammalian and tick cells
Summary
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. Anaplasma phagocytophilum (Ap, formerly the Human Granulocytic Ehrlichiosis agent) is a common tick borne obligate intracellular pathogen with an uncommon tropism for host granulocytes. Obstacles to transformation of obligate intracellular pathogens include: DNA delivery while retaining viability of extracellular bacteria, efficient reintroduction of the transformed bacterial population into host cells, selection (given the limited number of antibiotics ethically applicable to a pathogen), and the limited efficiency of homologous recombination and transposition systems.
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