Date Published: June 12, 2014
Publisher: Public Library of Science
Author(s): Halie K. Miller, Laura Kwuan, Leah Schwiesow, David L. Bernick, Erin Mettert, Hector A. Ramirez, James M. Ragle, Patricia P. Chan, Patricia J. Kiley, Todd M. Lowe, Victoria Auerbuch, Partho Ghosh.
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. coliiron-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.
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, . The T3SS functions as an injectisome that delivers bacterial effector proteins directly into the host cell cytoplasm . While the T3SS apparatus itself is structurally conserved, the repertoire of T3SS effector proteins used by each group of pathogens is distinct . Thus, the effect of the T3SS on the host is unique to the needs of the pathogen . While the T3SS is generally essential for a T3SS-expressing pathogen to cause disease, several aspects of the T3SS may be detrimental to bacterial growth . For example, T3SS components are recognized by the host immune system , . In addition, expression of the T3SS is energetically costly and, in some organisms, T3SS induction correlates with growth arrest . Therefore, regulation is essential for proper T3SS function in order to ensure that it occurs only during host cell contact in the appropriate host tissue , .
In this study, we present the first characterization of the iron-sulfur cluster regulator, IscR, of Yersinia. Initially identified through a genetic screen for modulators of Ysc T3SS function, iscR-deficient Y. pseudotuberculosis had a dramatic defect in secretion of T3SS effector proteins and in targeting macrophages through their T3SS, yet displayed normal growth in broth culture and wild type flagellar motility. Bioinformatic and DNA binding analysis revealed an IscR binding site upstream of the operon encoding the T3SS master regulator LcrF, indicating that IscR controls expression of the Ysc T3SS. Collectively, these findings indicated that IscR is a central component of the Y. pseudotuberculosis T3SS regulatory cascade.
All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the UCSC Institutional Animal Care and Use Committee.