Initial studies of the structural signal for extracellular transport of cholera toxin and other proteins recognized by Vibrio cholerae.
نویسندگان
چکیده
The specificity of the pathway used by Vibrio cholerae for extracellular transport of cholera toxin (CT) and other proteins was examined in several different ways. First, V. cholerae was tested for the ability to secrete the B polypeptides of the type II heat-labile enterotoxins of Escherichia coli. Genes encoding the B polypeptide of LT-IIb in pBluescriptKS- phagemids were introduced into V. cholerae by electroporation. Culture supernatants and periplasmic extracts were collected from cultures of the V. cholerae transformants, and the enterotoxin B subunits were measured by an enzyme-linked immunosorbent assay. Results confirmed that the B polypeptides of both LT-IIa and LT-IIb were secreted by V. cholerae with efficiencies comparable to that measured for secretion of CT. Second, the plasmid clones were introduced into strain M14, an epsE mutant of V. cholerae. M14 failed to transport the B polypeptides of LT-IIa and LT-IIb to the extracellular medium, demonstrating that secretion of type II enterotoxins by V. cholerae proceeds by the same pathway used for extracellular transport of CT. These data suggest that an extracellular transport signal recognized by the secretory machinery of V. cholerae is present in LT-IIa and LT-IIb. Furthermore, since the B polypeptide of CT has little, if any, primary amino acid sequence homology with the B polypeptide of LT-IIa or LT-IIb, the transport signal is likely to be a conformation-dependent motif. Third, a mutant of the B subunit of CT (CT-B) with lysine substituted for glutamate at amino acid position 11 was shown to be secreted poorly by V. cholerae, although it exhibited immunoreactivity and ganglioside GM1-binding activity comparable to that of wild-type CT-B. These findings suggest that Glu-11 may be within or near the extracellular transport motif of CT-B. Finally, the genetic lesion in the epsE allele of V. cholerae M14 was determined by nucleotide sequence analysis.
منابع مشابه
Expression of Recombinant Protein B Subunit Pili from Vibrio Cholera
Background & Aims: Vibrio cholerae is a gram-negative bacterial pathogen that causes cholera disease. Following ingestion by a host and entry into the upper intestine, V. cholera colonizes and begins to emit enterotoxin. One of the most pathogenic factors of Vibrio cholera is toxin-coregulated pili (TCP). ToxinCoregulated pili is as the primary factor requiered for the colonization and insisten...
متن کاملDesigning and Expression of Recombinant Chimeric Protein Containing CtxB and OmpW from Vibrio Cholerae and Evaluation of Its Immunogenicity
Background: Cholera disease caused by Vibrio cholerae remains a major cause of morbidity and mortality throughout the world. Various strategies with different proteins as immunogens have been tried for vaccine development, none of which have been sufficiently effective to preclude cholera. Chimeric proteins, with their ability to present multiple antigens at the same time, can play important ro...
متن کاملRapid Screening of Toxigenic Vibrio cholerae O1 Strains from South Iran by PCR-ELISA
Background: The ability to sensitively detect Vibrio cholera with PCR-ELISA method represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen. The aim of this research is to evaluate the suitability of a PCR-enzyme-linked immunosorbent assay for sensitive and rapid detection of V. cholera O1. Methods: The 398-bp sequence of a gene that cod...
متن کاملPCR-MEDIATED CLONING A ND EXPRESSION OF THE GENE FOR THE B-SUBUNIT OF VIBRIO CHOLERAE TOXIN ISOLATED RECENTLY IN IRAN
Knowing the nucleotide sequence of the cholera toxin operon, we designed oligonucleotide primers for its-PCR amplification from local clinical isolates of V. cholerae. The resulting amplification product was cloned in a common pUC18 vector. Subsequently, a part of this operon encoding the cholera toxin Bsubunit (CTB) was reamplified and cloned between the BamH1 and EcoR1 sites of the same ...
متن کاملSimple and Accurate Detection of Vibrio Cholera Using Triplex Dot Blotting Assay
Cholera outbreak is more common in developing countries. The causative agent of the disease is Vibrio cholerae strains O1 and O139. Traditional diagnostic testing for Vibrio is not always reliable, because Vibrio can enter a viable but non cultivable state. Therefore, nucleic acid-based tests have emerged as a useful alternative to traditional enrichment testing. In this investigation, a...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Infection and immunity
دوره 63 10 شماره
صفحات -
تاریخ انتشار 1995