A new level in the Vibrio cholerae ToxR virulence cascade: AphA is required for transcriptional activation of the tcpPH operon

The expression of the ToxR virulence regulon is dependent upon the regulato ry proteins ToxR/ToxS, TcpP/TcpH and ToxT. We describe here a previously un identified gene in Vibrio cholerae, aphA ((a) under bar ctivator of tcpP an d tcpH expression), which is required for the transcription of the tcpPH op eron. Under conditions normally optimal for virulence gene expression, an i n frame aphA deletion decreased the expression of a cholera toxin promoter fusion (ctx-lacZ) and prevented the production of the toxin co-regulated pi lus (TCP). Plasmids producing ToxT or TcpP/H, but not ToxR, restored ctx-la cZ expression and TCP production in the Delta aphA strain, suggesting that the mutation interferes with toxT expression by influencing the transcripti on of tcpPH. Indeed, the expression of a chromosomal tcpP-lacZ fusion was r educed in the Delta aphA mutant and increased in both V. cholerae and Esche richia coli by introducing aphA expressed from an inducible promoter. These results support a model in which AphA functions at a previously unknown st ep in the ToxR virulence cascade to activate the transcription of tcpPH. Tc pP/TcpH, together with ToxR/ToxS, then activate the expression of toxT, res ulting ultimately in the production of virulence factors such as cholera to xin and TCP.