Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants

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.

Fluorescent Protein Expressing Rickettsia buchneri and Rickettsia peacockii for Tracking Symbiont-Tick Cell Interactions

“…Low penetration of plasmid DNA into the symbionts, massive killing due to electroporation conditions and excessive antibiotic selection are potential factors that need to be examined. Recently, Driskell et al [ 37 ] used fluorescence activated cell sorting to harvest cells infected with R. prowazekii expressing fluorescent reporter proteins. To reduce time and variability, frozen stocks of electrocompetent rickettsiae were used.…”

Rickettsia Aglow: A Fluorescence Assay and Machine Learning Model to Identify Inhibitors of Intracellular Infection

“…Once the cultures in the flasks exhibited either lytic activity or >90% GFP expression post-transformation (see the GFP measurement method below), isolation was performed according to published methods. 39,40 If isolated R. canadensis were to be used for passaging or assays, DNA was extracted following manufacturer's protocols for DNeasy Blood and Tissue Kit (Qiagen, 69504). To quantify R. canadensis in a stock, we subsequently performed quantitative PCR on the isolated DNA.…”

Engineering of obligate intracellular bacteria: progress, challenges and paradigms

“…Cells infected with labelled C. trachomatis and Rickettsia spp. that express a fluorescent protein have been sorted by FACS 33,54–56 . Similarly, single cells that are infected with labelled C. trachomatis have been successfully isolated through laser microdissection 57 .…”