Abstract
Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens.
Highlights
Rickettsia prowazekii causes the serious and historically significant human disease epidemic typhus
GFPUV is expressed well in several rickettsial species and the gene encoding this fluorescent protein has been used in both transposon and plasmid constructs designed for rickettsial transformations [23, 28]
In this paper our goal was to establish fluorescence parameters for sorting R. prowazekii infected cells into distinct populations based on the number of rickettsiae per cell and to demonstrate the early detection of R. prowazekii fluorescent transformants
Summary
Rickettsia prowazekii causes the serious and historically significant human disease epidemic typhus. This malady is transmitted by the human body louse and is associated with crowded populations living in unhygienic environments [1,2,3]. Due to a low infectious dose and the fact that R. prowazekii is stable for months in louse feces, there is the potential for aerosol spread and R. prowazekii was previously weaponized for use as a biological warfare agent [8, 9] It is currently classified as a Category B Select Agent
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