LcrQ blocks the role of LcrF in regulating the Ysc-Yop type III secretion genes in Yersinia pseudotuberculosis

Abstract

Pathogenic Yersinia species employ the Ysc-Yop type III secretion system (T3SS) encoded by a highly conserved pYV virulence plasmid to export the virulence effectors into host cells. The Ysc-Yop T3SS is tightly regulated by multiple contributing proteins that function at different levels. However, systematic transcriptional regulation analysis of Ysc-Yop T3SS is lacking and the detailed mechanism under this regulation process is still elusive. Aimed at systematically characterizing transcriptional regulations of all T3SS genes in Y. pseudotuberculosis, we amplified 97 non-coding fragments from the pYV plasmid and analyzed transcriptional responses of the T3SS genes under different growth conditions. Transcriptions of T3SS genes were induced at 37°C and genes encoding T3SS effectors were highly induced by further depletion of Ca2+. The temperature induced gene transcription process is mediated by modules encoded on the chromosome, while the Ca2+ depletion-induced process is controlled by the positive regulatory protein LcrF as well as the negative regulatory protein LcrQ. In this process, LcrQ shares the same targets with LcrF and the effect of LcrQ is dependent on the presence of LcrF. Furthermore, over-expression of LcrF showed the same phenotype as that of the lcrQ mutant strain and intracellular amount balance of LcrQ and LcrF is important in T3SS regulation. When the expression level of LcrF exceeds LcrQ, expression of the Ysc-Yop T3SS genes is activated and vice versa. Together, these data support a model in which LcrQ blocks the activation role of LcrF in regulating the transcription of T3SS genes in Yersinia.

Figure 1. Promoter activity analyses of genes on the pYV plasmid in YPIII.

(A) The promoter activities of 99 non-coding fragments at 26°C, 37°C or 37°C with Ca²⁺ depletion. Fragments with relative promoter activities higher than the pZT100 plasmid (202±12, p<0.01) at one of these conditions were marked with red dot. (B) & (C) Relative promoter activities of selected fragments in YPIII and ΔpYV strains at 26°C and 37°C (B) or at 37°C with Ca²⁺ depletion versus at 37°C (C).

Figure 2. Role of LcrF in controlling the transcription of T3SS genes.

(A) Relative promoter activities of genes in lcrF mutant (ΔlcrF) compared with YPIII parent strain under T3SS inducible conditions. Promoters with decreased activities were marked with red dot. (B) & (C) Effect of LcrF over-expression (pOVR-LcrF) on the promoter activities of its targets in ΔlcrF strain at 37°C with or without Ca²⁺. ** p<0.01.

Figure 3. Role of LcrQ in regulating the transcription of T3SS genes.

(A) Comparison of relative promoter activities of T3SS genes under T3SS inducible conditions in LcrQ over-expressed strain with YPIII carrying the pOVR200 plasmid. Promoters repressed by LcrQ were marked with red dot. (B) Secretion (Supernatant) and expression (Pellet) of T3SS effectors in LcrQ over-expressed and ΔlcrF strains under T3SS inducible conditions. RpoA was used as a loading control. (C) Secretion and expression of T3SS effectors in ΔlcrQ and LcrF over-expressed strains at 37°C with or without Ca²⁺. (D) & (E) Effects of LcrQ over-expression on the promoter activities of three LcrQ targets (yopE, lcrG and yopH) in YPIII ΔpYV and ΔlcrF strains.

Figure 4. Effects of co-expression of LcrF and LcrQ on T3SS.

(A) Secretion and expression of Yops and LcrQ in strains with overexpression of LcrF or LcrQ or co-expression of these two proteins. (B) Effects of co-expression of GST-fused LcrQ (G-Q) with LcrF on Yops and LcrQ expression and secretion. (C) Influences of over-expressing LcrF in an yscV mutant (ΔyscV _618–644) on expression of Yops. (D) Promoter activities of LcrF targets in ΔyscV _618–644 strains carrying pOVR-LcrF or pOVR200.

Figure 5. Interaction between LcrQ and LcrF by a bacterial two-hybrid assay.

Model for bacterial adenylate cyclase two-hybrid assay is shown in the upper panel. Interaction between LcrQ and LcrF as detected by bacterial two-hybrid system is shown in the lower panel. ** p<0.01.

Figure 6. Proposed model for the transcriptional responses of the T3SS genes in Yersinia.