Expression Analysis of the Yersiniabactin Receptor Gene fyuA and the Heme Receptor hemR of Yersinia enterocolitica In Vitro and In Vivo Using the Reporter Genes for Green Fluorescent Protein and Luciferase

Abstract

The enteropathogenic Yersinia enterocolitica strains have several systems for scavenging iron from their environment. We have studied the expression of the fyuA gene, which encodes the outer membrane receptor for the siderophore yersiniabactin (Ybt), and the hemR gene, which encodes the receptor for heme, using the reporter genes gfp (encoding green fluorescent protein) and luc (encoding firefly luciferase). To study gene expression in vitro as well as in vivo, we have constructed several translational reporter gene fusions to monitor simultaneously expression of fyuA and hemR or expression of one gene by a gfp-luc tandem reporter. Results of the in vitro expression analysis (liquid media) indicated that fyuA and hemR are strongly derepressed under iron starvation conditions, resulting in strong fluorescence and/or luminescence at 27°C. In the in vivo BALB/C mouse infection model, tissue-specific expression of fyuA and hemR reporter fusions was observed. Surprisingly, fyuA and hemR reporter constructs were weakly expressed by yersiniae located in the liver and intestinal lumen, whereas strong expression was found for yersiniae in the peritoneal cavity and moderate expression was found in the spleen. Strikingly, yersiniae carrying fyuA or hemR reporter fusions exhibited threefold-stronger signals when grown in the peritoneal cavity of mice than those growing under iron derepression in vitro. This hyperexpression suggests that besides Fur derepression, additional activators may be involved in the enhanced expression of fyuA and hemR under peritoneal growth conditions. Differential expression of the fyuA and hemR reporter fusions could not be observed, suggesting similar regulation of fyuA and hemR in the mouse infection model.

Yersinia enterocolitica Infection of Mice Reveals Clonal Invasion and Abscess Formation

“…To determine if yersiniae no longer expressing RFP or GFP were present in microcolonies, we also immunostained cryosections of Y. enterocolitica-infected mouse organs 5 days p.i. with antiYadA rabbit antiserum followed by an anti-rabbit fluorescein isothiocyanate-or tetramethyl rhodamine isocyanate-conjugated antibody as previously described (20). This immunostaining revealed that all yersiniae and microcolonies in the tissue sections were detectable by RFP or GFP fluorescence (data not shown), indicating the reliable expression of the reporter genes and the maintenance of the reporter plasmids during the infection experiments.…”

“…For the detection of yersiniae by immunofluorescence, cryosections were fixed with 3.7% paraformaldehyde and incubated for 30 min in 2% bovine serum albumin to block nonspecific binding. Bacteria were stained with a primary monoclonal YadA antibody and a fluorescein isothiocyanate-or tetramethyl rhodamine isocyanatelabeled secondary antibody as previously described (20). Composite images of PPs, livers, and spleens were automatically assembled using a motorized Olympus BX61 fluorescence microscope with analysis software (Olympus Soft Imaging System).…”

The irp2 and fyuA genes in High Pathogenicity Islands are involved in the pathogenesis of infections caused by avian pathogenic Escherichia coli (APEC)

“…The HPI is a long chromosomal fragment containing several regulatory genes and the genes related to synthesis, uptake, and transport of siderophores such as yersiniabactin (Ybt), which is involved in the virulence and pathogenicity of Yersinia species (Rakin et al 1999, Jacobi et al 2001.…”

The High-Pathogenicity Island Is Absent in Human Pathogens of Salmonella enterica Subspecies I but Present in Isolates of Subspecies III and VI

“…The functionality of yersiniabactin biosynthesis was evaluated by detecting the presence of yersiniabactin in the cell-free supernatant of iron-starved bacteria by using a translational reporter gene fusion system with luciferase (20,28). Synthesis and release of yersiniabactin were observed in all HPI-positive strains tested except 1380, which carries a truncated island, and 50/90 and HPI-negative control strains (Table 1).…”