Activation of Adenylate Cyclase by Heat-Labile Escherichia Coli Enterotoxin

126

A genetically engineered Vibrio cholerae strain from which the cholera toxin genes had previously been deleted was used as a host in which to study the expression and secretion of related toxins and their subunits. Recombinant plasmids encoding heat-labile enterotoxins (LTs) from Escherichia coli of human and porcine origin were expressed in the V. cholerae host, and this resulted in the secretion of the LTs into the extracellular milieu. The secreted LTs were isolated and it was found that the A subunits of human and porcine LT were "unnicked" polypeptides, which indicates that nicking is not obligatory for toxin secretion. V. cholerae strains were also constructed that harbored plasmids encoding either the A or the B subunits of human LT (A+B-, or A-B+). Approximately 90% of the B subunits were secreted from the A-B+ strain, while all of the A subunits expressed by the A+B-strain remained cell associated. This implies that strains synthesizing both subunits assemble the A and B subunits prior to their secretion. We propose that the entry of the toxin into the secretory step of the export pathway is mediated by a secretory apparatus that recognizes structural domains within the B subunit of LT.Cholera is a severe and at times fatal diarrheal disease of man (1). It is caused by Vibrio cholerae, which secrete a potent cholera enterotoxin (2, 3), consisting of five identical B subunits (4) that bind to GM1 ganglioside receptors (5, 6) and a single A subunit that subsequently activates adenylate cyclase (7,8). Many Escherichia coli strains that cause diarrhea in man and domestic animals, such as pigs, produce heat-labile enterotoxins (LTs) that are similar to cholera toxin in immunological (9, 10), structural (11,12), and functional (13, 14) properties. However, several studies have revealed distinctive features between cholera toxin and the LTs. First, the A subunit of cholera toxin is activated by a proteolytic "nick," which gives rise to two polypeptides (A1 and A2) that are joined by a cystine bridge (15, 16). The A subunits of the LTs are "unnicked" (17, 18). Second, the B subunits of LTs bind to glycoprotein receptors as well as to GM1 ganglioside (19). Third, cholera toxin is secreted from V. cholerae (2,20), while the LTs are cell associated (21,22) and are located within the periplasmic space of E. coli (23,24).Our present investigations concern the export and secretion of cholera toxin and the LTs. Many of the steps in the export of these toxins are presumbly common in both V.cholerae and E. coli, including, for example, synthesis of subunit precursors, translocation across the cytoplasmic membrane, maturation and subunit assembly (25-27). The capacity of V. cholerae to secrete cholera toxin into the extracellular milieu is, however, an additional step not found in the export of LT by E. coli. This is unlikely to be due solely to differences between cholera toxin and LT, because the cloning of cholera toxin genes in E. coli resulted in the accumulation of cell-associated cholera toxin (28, 29). Th...

Section: Unit Of Ltmentioning

confidence: 99%

Mechanism of toxin secretion by Vibrio cholerae investigated in strains harboring plasmids that encode heat-labile enterotoxins of Escherichia coli.

Hirst¹,

Sánchez²,

Kaper³

et al. 1984

126

“…Cholera toxin (Ctx) is the prototype and best-characterized member of this family of enterotoxins; it consists of an A subunit (CtxA, 28 kDa) that has ADP-ribosyltransferase and NADhydrolase activities and five identical B subunits (CtxB, 12 kDa each) that bind to GM1 ganglioside (5). Certain diarrheagenic strains of Escherichia coli synthesize an enterotoxin (Etx) that is structurally and functionally similar to Ctx (10)(11)(12). The individual subunits of both toxins are synthesized as precursors that have amino-terminal hydrophobic leader (signal) sequences that specify transfer of the subunits across the bacterial cytoplasmic membrane (13)(14)(15).…”

mentioning

confidence: 99%

Coordinated assembly of multisubunit proteins: oligomerization of bacterial enterotoxins in vivo and in vitro.

Hardy

Holmgren

Johansson

et al. 1988

133

121

In this paper we study the assembly, in vivo and in vitro, of a family of hexameric, heat-labile enterotoxins produced by diarrheagenic bacteria. The toxins, which consist of an A subunit and five B subunits, are assembled by a highly coordinated process that ensures secretion of the holotoxin complex. We show that (i) oxidation of cysteine residues in the B subunits is a prerequisite step for in vivo formation of B-subunit pentamers, (ii) reduction of dissociated B subunits in vitro abolishes their ability to reassemble, (iii) the kinetics of B-pentamer assembly inl vivo can be mimicked under defined conditions in vitro, (iv) A subunits cannot associate with fully assembled B pentamers in vitro, and (v) A subunits cause an 3-fold acceleration in the rate of B-subunit pentamerization in vivo, implying that A subunits play a coordinating role in the pathway of holotoxin assembly. The last finding is likely to be of general significance, since it provides a mechanism for preferentially excluding or favoring certain intermediates in the assembly of multisubunit proteins.

“…We therefore chose to use heat-labile enterotoxin, which is a well-defined diarrheagenic protein that consists of an A subunit (28 kDa) and five identical B subunits (12 kDa each) (14)(15)(16)(17)(18)(19)(20)(21). The toxin, which is structurally and functionally related to cholera toxin, is normally produced by enterotoxinogenic strains of E. coli (19,20,22), but it has also been studied experimentally in Vibrio cholerae where it exhibits the property of being efficiently secreted into the extracellular milieu (23)(24)(25).…”

mentioning

confidence: 99%

Conformation of protein secreted across bacterial outer membranes: a study of enterotoxin translocation from Vibrio cholerae.

Hirst

Holmgren

1987

148

The secretion of enterotoxin by Vibrio cholerae is'punctuated by the transient entry of the toxin subunits into the periplasm. In this paper, we show that the subunits oligomerize into an assembled holotoxin within the periplasm prior to their secretion across the outer membrane. The rate of toxin assembly was studied by pulse-labeling cells with [355]-methionine and then monitoring the turnover of radiolabeled subunits as they assembled withi the periplasm. The subunits entered the periplasm as monomers and assembled into oligomers with a half-time of -1 min. Since assembly was a rapid event compared to the rate of toxin efflux from the periplasm, which had a half-time of 13 nin, we conclude that all of the subunits that pass through the periplasm assemble before they traverse the outer membrane. The average concentration of subunit monomers and assembled holotoxin within the periplasm was calculated to be -20 and =260 ,ug/ml, respectively. This indicates that the periplasm is a suitably concentrated milieu where spontaneous toxin assembly can occur. Our findings suggest that protein movement across bacterial outer membranes, in apparent contrast to export across other biological membranes, involves translocation of polypeptides that have already folded into tertiary and even quaternary conformations.The movement of proteins across membranes is a fundamental characteristic of living cells. It plays an obligatory role in the replenishment and growth of membrane systems and cell envelopes, in organelle biogenesis, and in the secretion of, for example, protein hormones, immunoglobulins, and extracellular enzymes (for reviews, see refs. 1-4). A number of recent reports on protein import into mitochondria, and export of proteins across the cytoplasmic membrane of Escherichia coli have implied that nonnative, disordered, or unfolded protein states are needed to ensure successful translocation (5,6). In addition, several research groups, studying the secretion ofproteins into the endoplasmic reticulum (ER) and from the ER to the Golgi complex in eukaryotic cells, have found that protein folding, and in particular subunit oligomerization, are intraluminal processes occurring after translocation across the ER membrane (7-10). Thus, proteins that are translocated across ER and mitochondrial and bacterial cytoplasmic membranes would seem to share a common feature of initiating their export as unfolded polypeptides that develop native tertiary or quaternary conformations only after trAnslocation has occurred (5-12).