Degradation of the membrane-localized virulence activator TcpP by the YaeL protease in Vibrio cholerae

Abstract

A common mechanism inhibiting the activity of transcription factors is their sequestration to the membrane until they are needed, at which point they are released from the membrane by proteolysis. Acting in contrast to this inhibition mechanism are virulence regulators of Vibrio cholerae, the ToxR and TcpP proteins, which are localized to the inner membrane of the cell, where they bind promoter DNA and activate gene expression. TcpP is rapidly degraded in the absence of another protein, TcpH. We used a genetic screen to identify regulators of TcpP stability and identified the YaeL membrane-localized zinc metalloprotease as responsible for degrading TcpP in the absence of TcpH. In Escherichia coli, DegS and YaeL cooperate to degrade RseA, an antisigma factor that sequesters sigma(E) to the inner membrane, thereby inhibiting the activity of sigma(E). When yaeL was disrupted in a V. cholerae tcpH mutant, we observed accumulation of a lower molecular weight species of TcpP. This observation is consistent with TcpP being partially degraded in the absence of YaeL. A mutant lacking both DegS and YaeL continued to accumulate the TcpP degradation product, indicating that protease other than DegS is acting before YaeL in degrading TcpP. The YaeL-dependent degradation pathway is active in TcpH(+) cells under conditions that are not favorable for virulence gene activation. This work expands the knowledge of YaeL-dependent processing in the bacterial cell and reveals an unexpected layer of virulence gene regulation in V. cholerae.

Fig. 1.

Effect of yaeL deletion in the presence and absence of TcpH. Overnight cultures of the following strains were subcultured 1:100 and grown in the presence of 0.1% arabinose: O395 containing pBAD18-Kan (lane 1); ΔtcpP (lane 2) and ΔtcpH containing pBAD18 (lane 3); ΔyaeL containing pBAD18-Kan (lane 4), and pBAD18-Kan-yaeL (lane 5); and ΔtcpHΔyaeL containing pBAD18-Kan (lane 6), pBAD18-Kan-yaeL (lane 7), pBAD18-tcpH (lane 8), and pBAD18-Kan-yaeLE23A (lane 9). Cells were harvested after growth to midlogarithmic phase in pH 6.5 LB at 30°C. Samples were separated by SDS/PAGE and analyzed by immunoblotting with antibodies against TcpP and TcpA.

Fig. 2.

Stability of TcpP truncations in the presence and absence of YaeL. The following plasmids were expressed in either O395 ΔtcpPH (lanes 1-5) or O395 ΔtcpPHΔyaeL (lanes 6-10): pBAD18-Kan (vector; lanes 1 and 6), pBAD18-Kan-P_cyt (amino acids 1-138; lanes 2 and 7), pBAD18-Kan-P_mem (amino acids 1-169; lanes 3 and 8), pBAD18-Kan-P_peri (amino acids 1-190; lanes 4 and 9), and pBAD18-Kan-P_full (amino acids 1-222; lanes 5 and 10). Overnight cultures were subcultured 1:100 and grown in the presence of 0.1% arabinose to midlogarithmic phase in pH 6.5 LB at 30°C. Samples were harvested, separated by SDS/PAGE, and analyzed by immunoblotting with antibodies against TcpP and TcpA.

Fig. 3.

TcpP degradation in the presence of TcpH. Overnight cultures of O395 (A) and ΔyaeL (B) V. cholerae were subcultured 1:100 and grown to midlogarithmic phase in pH 6.5 LB at 30°C. The cells were harvested by centrifugation, and equal portions of each sample were resuspended either in pH 6.5 LB and grown at 30°C or in pH 8.5 LB and grown at 37°C for an additional 4 h. Samples were harvested for Western blot analysis before resuspension in new media (time 0), and at each hour after resuspension for both conditions. Samples were separated by SDS/PAGE and analyzed by immunoblotting with antibodies against TcpP.