Isolation and Characterization of a Vibrio cholerae Strain Unable to Secrete Cholera Toxin, and Cloning and Characterization of Genes from Vibrio cholerae that Confer a Congo Red Dye Binding Phenotype on Escherichia coli.

摘要

Vibrio cholerae is the causative agent of cholera gravis. The main virulence determinant for this organism is the production of cholera toxin (CT) , a bipartite toxin enzyme which is responsible for causing the physiological changes in humans that result in massive diarrhea and subsequent fluid loss that is the hallmark of cholera disease. In order for CT to reach the epithelial target cells of the small intestine it must be synthesized and secreted from the bacterium afterinfection of the host organism by V. cholerae. The goal of this study was to isolate and characterize genes that encode proteins responsible for the translocation of CT across the outer membrane of V. cholera. An El Tor V. cholerae strain was isolated by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis that was defective in CT secretion and designated CC9453. CC9453 was found to exhibit a pleiotropic phenotype so far as it was also defective in the secretion of protease, DNAse, and hemolysin enzymes. Attempts to complement the genetic defect responsible for the secretion of these proteins failed. A cloned gene able to complement the CT secretion defect of classical biotype v. cholerae strain M14 was unable to complement the defect of CC9453. The nature of the mutation in CC9453 remains unknown. The second part of this study describes the isolation and characterization of genes from V. cholerae that confer a congo red binding phenotype on Escherichia coli. Five different alleles were obtained from V. cholerae strain U1 that enabled E. coli to bind congo red. One of these alleles (cbp) was extensively characterized and found to encode a lipoprotein.