In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene

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Abstract
The Yersinia outer protein YopE belongs to the translocated effector proteins of pathogenic yersiniae. We constructed various truncated yopE genes fused to gfp (encoding the green fluorescent protein) to study yopE gene expression and YopE–GFP translocation of Y. enterocolitica in cell culture and mouse infection models. The hybrid gene fusions were co‐expressed in Y. enterocolitica (i) on a low‐copy plasmid in the presence of the virulence plasmid pYV08 (in trans configuration) and (ii) after co‐integration by homologous recombination of a yopE–gfp‐carrying suicide plasmid into pYV08 (co‐integrate configuration). After 30 min of infection of HEp‐2 cell monolayers, extracellularly located yersiniae began to emit green fluorescence after excitation. In contrast, internalized bacteria were weakly fluorescent. Translocation of YopE–GFP into HEp‐2 cells by attached yersiniae was visualized by optical sectioning of fluorescent HEp‐2 cells using confocal laser scanning microscopy and was confirmed by immunoprecipitation of cytosolic YopE–GFP from selectively solubilized HEp‐2 cells. The co‐translocation of other Yops was not significantly impaired by YopE–GFP as shown by YopH/YopE‐mediated suppression of the oxidative burst of infected neutrophils. The time course of yopE–gfp expression (in trans as well as in the co‐integrate configuration) in the HEp‐2 cell infection model as well as after in vitro induction was studied using a highly sensitive CCD camera and a flow cytometer. Similar results were obtained with a YopE–LUC (firefly luciferase) protein fusion as reporter. After intraperitoneal, intravenous and orogastrical infection of Balb/c mice with the recombinant yersiniae strains, green fluorescing bacteria could be visualized microscopically in the peritoneum, the spleen, the liver and in the Peyer's patches. However, only weakly fluorescent yersiniae were observed in the intestinal lumen. These results were quantified by flow cytometric measurements. The application of gfp as a reporter gene turned out to be promising for the study of protein translocation by protein type III secretion systems and differential virulence gene expression in vivo.