Yersinia Type III Secretion System Master Regulator LcrF

Abstract

Many Gram-negative pathogens express a type III secretion (T3SS) system to enable growth and survival within a host. The three human-pathogenic Yersinia species, Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica, encode the Ysc T3SS, whose expression is controlled by an AraC-like master regulator called LcrF. In this review, we discuss LcrF structure and function as well as the environmental cues and pathways known to regulate LcrF expression. Similarities and differences in binding motifs and modes of action between LcrF and the Pseudomonas aeruginosa homolog ExsA are summarized. In addition, we present a new bioinformatics analysis that identifies putative LcrF binding sites within Yersinia target gene promoters.

FIG 1

Alignment of verified and putative LcrF and ExsA binding sites within target gene promoters. (A) Promoter regions of Yersinia and Pseudomonas genes controlled by LcrF and ExsA, respectively, were aligned using SeaView (81). The sequences are from Y. enterocolitica 8081 (NC 008791), Y. pestis CO92 (NC 003131), Y. pseudotuberculosis IP 32953 (NC 006153), P. aeruginosa (NC 002516), and P. aeruginosa UCBPP PA14 (NC 008463). Predicted −10 regions are highlighted in blue. Identified 5′-AAAA-N₆-GNCT-N₅-TGANA-3′ consensus sites containing three conserved regions are highlighted in red, and the conserved nucleotides at each position of the motif are denoted in bold above the alignment, with uppercase letters denoting highly conserved residues and lowercase letters denoting more-degenerate residues. LcrF binding sites 1 and 2 are indicated by arrows. The sequences of the virA, virB, and yopH promoters from all three Yersinia species are identical, and thus a single sequence is shown. virA and virB are proximal but are divergently encoded. Thus, we propose that what appears to be an inverted 5′-AAAA-N₆-GNCT-N₅-TGANA-3′ motif upstream of the virA and virB promoters (blue solid-line box) actually belongs to the divergent virB and virA promoters, respectively. yopH and sycE have two tandem 5′-AAAA-N₆-GNCT-N₅-TGANA-3′ motifs (both highlighted in red), which overlap the protected regions identified by Wattiau and Cornelis (17). (B) Sequences upstream of Yersinia yopE were aligned using SeaView. Two regions containing inverted 5′-AAAA-N₆-GNCT-N₅-TGANA-3′ motifs which may belong to sycE are outlined in blue. The putative translational yopE start site is denoted in bold. (A and B) YtxR binding sites upstream of yopH (A) and yopE (B) are underlined in green (38). Regions experimentally found to be strongly bound by LcrF/VirF or ExsA are underlined with black solid lines, and those found to be weakly bound are underlined with black dashed lines (17). Identified putative LcrF binding sites are highlighted in red and the conserved nucleotides denoted in bold above the alignment, with uppercase letters denoting highly conserved residues and lowercase letters denoting more-degenerate residues.

FIG 2

Regulatory elements encoded within the yscW-lcrF sequence. Nucleotide sequences of the yscW-lcrF promoter regions for Y. pestis CO92, Y. enterocolitica 8081, and Y. pseudotuberculosis YPIII were aligned using ClustalW2. Nucleotides whose sequences are not identical are marked in red, while the conserved nucleotides are indicated by asterisks. The identified binding sequence for IscR is marked in purple, and critical residues required for IscR binding within this motif are in bold (28, 64). The binding site for RcsB is marked in green (71). The region experimentally determined to be involved in YmoA/H-NS binding, marked in brown, is downstream of the transcriptional start site (49). The sequence encoding the RNA thermometer is marked in yellow and is encoded within the intergenic region between yscW and lcrF (49). The DNA bending region identified within the yscW-lcrF operon is denoted with a dotted line and is within the intergenic region between yscW and lcrF (59). The −10 and −35 boxes, the transcriptional start site, the ribosome binding sites (rbs) upstream of the yscW and lcrF coding sequence, and the fourU element in the intergenic region between the yscW and lcrF coding regions are marked in bold (49). All coding sequences are marked in blue.

FIG 3

Multiple environmental signals control lcrF expression and, subsequently, T3SS expression through several distinct transcriptional and translational regulatory mechanisms. The data summarized in this review suggest that the YmoA, RcsB, and IscR regulators control transcription of lcrF in response to temperature, extracytoplasmic stress, iron bioavailability, oxygen tension, and reactive oxygen species. In addition, the RNA thermometer found upstream of lcrF allows LcrF translation only at the mammalian host body temperature, 37°C. As Yersinia transits from the environment or the flea vector to the mammalian host and then from localized to disseminated sites of infection, changes in temperature, iron availability, and stresses such as ROS may direct the regulatory network controlling LcrF, optimizing T3SS deployment and virulence.