Expression of Hemolysin Is Regulated Under the Collective Actions of HapR, Fur, and HlyU in Vibrio cholerae El Tor Serogroup O1

Abstract

The biotype El Tor of serogroup O1 and most of the non-O1/non-O139 strains of Vibrio cholerae can produce an extracellular pore-forming toxin known as cholera hemolysin (HlyA). Expression of HlyA has been previously reported to be regulated by the quorum sensing (QS) and the regulatory proteins HlyU and Fur, but lacks the direct evidence for their binding to the promoter of hlyA. In the present work, we showed that the QS regulator HapR, along with Fur and HlyU, regulates the transcription of hlyA in V. cholerae El Tor biotype. At the late mid-logarithmic growth phase, HapR binds to the three promoters of fur, hlyU, and hlyA to repress their transcription. At the early mid-logarithmic growth phase, Fur binds to the promoters of hlyU and hlyA to repress their transcription; meanwhile, HlyU binds to the promoter of hlyA to activate its transcription, but it manifests direct inhibition of its own gene. The highest transcriptional level of hlyA occurs at an OD600 value of around 0.6–0.7, which may be due to the subtle regulation of HapR, Fur, and HlyU. The complex regulation of HapR, Fur, and HlyU on hlyA would be beneficial to the invasion and pathogenesis of V. cholerae during the different infection stages.

Direct Binding and Regulation by Fur and HapR of the Intermediate Regulator and Virulence Factor Genes Within the ToxR Virulence Regulon in Vibrio cholerae

“…Fur coordinates with HapR to tightly regulate tcpP, toxT, and tcpA, leading to the cell density transcriptional manner of expression of these genes. HapR repression of Fur (Gao et al, 2018), AphA activation of tcpP (Kovacikova and Skorupski, 2001;Kovacikova et al, 2004), TcpP activation of toxT (Krukonis et al, 2000), autoregulation of Fur (Lee et al, 2007), and ToxT activation of tcpA (DiRita et al, 1991) were reported by other researchers. LCD, low cell density; MCD, medium cell density; HCD, high cell density.…”

“…Vibrio cholerae El Tor serogroup O1 strain C7258 (Peru, 1991) was used as the derivative (wild type, WT). Non-polar fur and hapR single-gene deletion mutants fur and hapR derived from the WT strain were constructed in our previous study (Gao et al, 2018). The deletion mutant of lacZ (designated as lacZ) was constructed from WT by homologous recombination using the suicide plasmid pWM91, which was similarly performed as previously described (Wu et al, 2015).…”

“…For the luminescence assay (Xu et al, 2010;Gao et al, 2018), the promoter DNA region of each target gene was cloned into the pBBRlux harboring a promoterless luxCDABE reporter gene and a chloramphenicol resistance gene. The recombinant plasmid was transferred into WT and each mutant strain, respectively.…”

“…The primer extension assay was essentially performed as previously described (Gao et al, 2018). Twelve micrograms of total RNA was annealed with 1 pmol of 5 -HEX-labeled reverse primer to generate cDNAs using the Primer Extension System (Promega), and the products of primer extension were then analyzed using the ABI 3500XL DNA Genetic analyzer.…”

“…In addition, the mechanisms of the Fur-dependent activation of TCP expression are also unclear. Moreover, transcription of fur was under the direct control of HapR, and HapR coordinates with Fur to regulate hlyA transcription (Gao et al, 2018), suggesting Fur integrated into QS to co-regulate gene expression in V. cholerae.…”