Vibrio Cholerae:Function Of Cysteine Residues In The Intracellular Domain Of TcpP Protein

Vibrio cholerae is a pathogen of cholera in acute intestinal diarrhea in humans.The rapid loss of body fluids in people with severe cholera can lead to dehydration,shock and even death.In the Vibrio cholerae toxin regulatory network system,there is an important toxin regulatory protein TcpP,a transcellular inner membrane protein with three distinct domains:the N-terminal intracellular DNA binding domain,a transmembrane region and a C-terminal periplasmic domain.Previous studies have found that Vibrio cholerae DsbA forms the intermolecular disulfide bond in the periplasmic space C207 and C218 in the presence of taurobilinate,and the regulatory protein TcpP forms more dimers.The TcpP protein is The form of the dimer activates the expression of the downstream toxin gene toxT.Our study found that the mutation of the cysteine residue at position 58 of TcpP to serine(TcpPC58S)is not able to form a dimer or activate the expression of the toxin gene,and the function of four cysteine residues in the intracellular domain of TcpP still not clear.Therefore,this study mainly studies the function of cysteine residues in the intracellular domain of TcpP and its role in the formation of TcpP dimers.By mutating the four cysteine residues to a serine residue by site-directed mutagenesis,it was found that TcpPC58S could not activate the expression of the downstream toxin gene after mutation of the cysteine residue at position 58.Western blot analysis showed that TcpPC58S could still bind to the inner membrane of the cell,and its protein stability was similar to that of wild type.This indicates that the mutation of C58 does not have an important effect on the folding of the TcpP protein.However,the application of E.coli two-hybrid technique and membrane protein purification analysis revealed that TcpPC58S could not form a dimer even in the presence of a bile salt.Previous studies have shown that TcpPC218S can activate the expression of toxT without the induction of bile salts.Our study found that TcpPC58/218S did not form a dimer and could not activate the expression of the toxin gene,indicating that the 218th cysteine of TcpP could not restore the activity of TcpPC58S and the formation of dimer.At the same time,the fusion of the intracellular domain of TcpPC58S with the periplasmic transmembrane region of ToxR could not form a dimer.This indicates that the cysteine residue at position 58 plays an important role in the formation of TcpP dimer,and further proves that TcpP binds to the promoter of the downstream regulatory gene in the form of a dimer,thereby activating the expression of the downstream gene.The formation of disulfide bonds between cysteine residues is important for protein folding and function.In order to further study whether the expression of TcpP activated toxin gene requires the formation of disulfide bonds between C58,and in order to avoid the influence of cysteine in the periplasmic space of TcpP,TcpPC207/218S was used to analyze the intracellular domain disulfide bond,and it was found that there was no disulfide bond formation in the TcpPC207/218S monomer molecule.The TcpP intracellular domain was fused to the periplasmic transmembrane domain of ToxR to form a chimeric protein(Cp-TR-PRR C236/294S),which indicated that the chimeric protein was able to form a dimer and activate the expression of the toxin gene.Using this inlay protein study,it was found that the CP-TR-PR C236/294s mutant has no disulfide bond formation,indicating that the formation of the TcpP dimer does not require the intracellular domain cysteine to form an intermolecular disulfide bond.In order to better study the function of the cysteine residue at position 58 of TcpP,we constructed TcpPC58A,TcpPC58T,TcpPC58L and TcpPC58G mutants and found that TcpPc58L and TcpPC58G lost their activity,While TcpPC58A and TcpPc58T are still capable of activating the expression of downstream virulence genes.Thus,although the formation of a TcpP dimer does not require the intracellular domain to form an intramolecular or intermolecular disulfide bond,the 58th cysteine residue is important for the promoter of TcpP binding to a downstream regulatory gene.To investigate whether only the 58th cysteine is sufficient for TcpP to activate downstream toxin gene expression,we constructed the TcpPC19/51/124S mutant and found that TcpPC19/51/124S activates downstream virulence genes with similar expression to TcpPWT.the colonization experiment of Vibrio cholerae showed that TcpPC19/51/124S had similar colonization ability to wild type,while TcpPC58S could hardly colonize the intestine of suckling mice.However,in the colonization model of adult mouse,the colonization ability of TcpPC19/51/124S was weaker than that of wild type,while TcpPC58S had stronger colonization ability than wild type.These results indicate that among the four cysteine residues of the TcpP intracellular domain,only the cysteine residue at position 58 is sufficient to allow the TcpP protein to have activity to activate downstream virulence gene expression,however,the other three cysteine residues play different physiological functions in Vibrio cholerae in different growth environments.Taken together,this study demonstrates that the 58th cysteine residue of TcpP is essential for the formation of TcpP dimer and activation of downstream toxin gene expression,and only the 58th cysteine in the intracellular domain of TcpP is sufficient to activate the expression of virulence genes;the formation of the TcpP dimer does not require the intracellular domain cysteine residue to form an intermolecular or intramolecular disulfide bond.The cysteine residue of the TcpP intracellular domain plays an important role in the expression of downstream virulence genes of TcpP activation and the regulation of gene expression in adapting to different living environments.This study provides a theoretical basis for the study of the regulation mechanism of Vibrio cholerae toxin gene.