Abstract
It is crucial to understand the in vitro and in vivo regulation of the virulence factor genes of bacterial pathogens. In this study, we describe the construction of a versatile reporter system for Yersinia enterocolitica serotype O:3 (YeO3) based on the luxCDABE operon. In strain YeO3-luxCDE we integrated the luciferase substrate biosynthetic genes, luxCDE, into the genome of the bacterium so that the substrate is constitutively produced. The luxAB genes that encode the luciferase enzyme were cloned into a suicide vector to allow cloning of any promoter-containing fragment upstream the genes. When the obtained suicide-construct is mobilized into YeO3-luxCDE bacteria, it integrates into the recipient genome via homologous recombination between the cloned promoter fragment and the genomic promoter sequence and thereby generates a single-copy and stable promoter reporter. Lipopolysaccharide (LPS) O-antigen (O-ag) and outer core hexasaccharide (OC) of YeO3 are virulence factors necessary to colonization of the intestine and establishment of infection. To monitor the activities of the OC and O-ag gene cluster promoters we constructed the reporter strains YeO3-Poc::luxAB and YeO3-Pop1::luxAB, respectively. In vitro, at 37°C both promoter activities were highest during logarithmic growth and decreased when the bacteria entered stationary growth phase. At 22°C the OC gene cluster promoter activity increased during the late logarithmic phase. Both promoters were more active in late stationary phase. To monitor the promoter activities in vivo, mice were infected intragastrically and the reporter activities monitored by the IVIS technology. The mouse experiments revealed that both LPS promoters were well expressed in vivo and could be detected by IVIS, mainly from the intestinal region of orally infected mice.
Highlights
The bacterial genus Yersinia has three species known to cause human infections: Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis
We wanted to incorporate both the luxCDE and luxAB genes into the bacterial genome to avoid possible copy-number effects associated in plasmid-borne reporter systems
As a proof of principle, we introduced the LPS O-ag and outer core hexasaccharide (OC) gene cluster promoters into the luxCDE-strain and demonstrated that the reporter system is functional both in vitro and in vivo
Summary
The bacterial genus Yersinia has three species known to cause human infections: Yersinia enterocolitica, Y. pseudotuberculosis and Y. pestis. The biotype 1B to 5 strains usually carry a 70 kb Yersinia virulence plasmid (pYV) that encodes several well-characterized virulence determinants [1]. The chromosomally encoded virulence factors include Ail (attachment and invasion locus), invasin, Yersinia stable toxin and lipopolysaccharide (LPS) [2,3,4]. Most pathogenic Y. enterocolitica strains that cause human yersiniosis belong to bioserotypes 1B/O:8, 2/O:5,27, 2/O:9, 3/O:3, 4/O:3 [5]. Yersiniosis is generally a self-limiting food-borne enteric infection with diarrhea, and occurs mostly in young children. The infection may be followed by sequelae such as reactive arthritis or erythema nodosum [6]. Y. enterocolitica mainly colonizes the Peyer’s patches (PPs) in the small intestine (INT), the mesenteric lymph nodes (MLNs), and sometimes disseminates to deeper tissues such as spleen (SP) and liver (LI) in mice [7, 8]
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