Abstract
Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.
Highlights
Type III secretion systems (T3SS) are important components in the progression of disease for a number of clinically relevant human pathogens, including those in the genera Shigella, Salmonella, Escherichia, Chlamydia, Vibrio, Pseudomonas, and Yersinia [1,2]
We identify a transcriptional regulator in the human gut pathogen, Yersinia pseudotuberculosis, which controls a specialized secretion system essential for bacterial growth in mammalian tissues
Y. pseudotuberculosis experiences large changes in iron bioavailability upon transit from the gut to deeper tissues and iron is a critical component in Yersinia virulence, as individuals with iron overload disorders have enhanced susceptibility to systemic Yersinia infections
Summary
Type III secretion systems (T3SS) are important components in the progression of disease for a number of clinically relevant human pathogens, including those in the genera Shigella, Salmonella, Escherichia, Chlamydia, Vibrio, Pseudomonas, and Yersinia [1,2]. The T3SS functions as an injectisome that delivers bacterial effector proteins directly into the host cell cytoplasm [2]. The iscR complement construct was generated by insertion of a fragment containing the iscR coding region as well as 530 bp of 59 upstream sequence. This was PCR amplified using primer pair FiscRC and RiscRC, and cloned into the vector pACYC184 via BamHI/SalI restriction sites [73,74]. Cells were allowed to recover in 1 mL SOC media for 1 h at 26uC followed by plating on LB containing carbenicillin to select for Yersinia bearing the plasmid of interest. Clones were confirmed by PCR analysis, using a combination of gene- and vector-specific primers, to construct both the DiscR complemented strain (DiscR pIscR) and the apo-IscR complemented strain (apo-IscR pIscR)
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