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

Abstract

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...

Bacterial Surface Association of Heat-labile Enterotoxin through Lipopolysaccharide after Secretion via the General Secretory Pathway

“…When introduced on an exogenous plasmid, LT is secreted solubly from V. cholerae in the same manner as CT (18,29,30), whereas K-12 E. coli transformed with a CT-expressing plasmid does not secrete CT (30,33). These results suggest that E. coli does not possess or express the secretion apparatus present in V. cholerae.…”

“…LT Secretion via the GSP-Because LT and CT secretion depended on the native gsp gene cluster in V. cholerae (18,32), we wanted to investigate whether the GSP secretion machinery is responsible for LT secretion across the outer membrane and, consequently, the association of LT to the cell surface in E. coli. Wild type ETEC strains are not ideal for the dissection of this secretory pathway, because ETEC may possess multiple gsp loci in its chromosome and in its multiple uncharacterized virulence plasmids and the gsp and LT operons are repressed by HNS (36,37).…”

“…Extracellular CT secretion occurs through the GSP (32). An examination of many Gram-negative bacteria including those from genera Pseudomonas, Klebsiella, Erwinia, and Vibrio has shown that homologs of this pathway (Xcp, Pul, Out, and Eps, respectively) are responsible for the secretion of a large host of soluble extracellular proteases and toxins across the outer membrane (24).When introduced on an exogenous plasmid, LT is secreted solubly from V. cholerae in the same manner as CT (18,29,30), whereas K-12 E. coli transformed with a CT-expressing plasmid does not secrete CT (30,33). These results suggest that E. coli does not possess or express the secretion apparatus present in V. cholerae.…”

Genetic fusion of a non‐toxic heat‐stable enterotoxin‐related decapeptide antigen to cholera toxin B‐subunit

“…Furthermore, the STa-related decapeptide-CTB hybrid protein was able to efficiently bind anti-STa antibodies both in GMI-ELISA and in immunoblot analyses. The hybrid protein exhibited, in addition, a number of attributes characteristic of the CTB moiety such as the ability to pentamerize, to bind to the GM1 receptor and to be secreted by V. cholerae [23]. The latter two properties made possible purification of the decapeptide-CTB from V. cholerae culture supernatants by a single-step receptorspecific affinity chromatography procedure developed for the purification of CTB [17].…”

“…As mentioned above the decapeptide-CTB protein retained full capacity to bind to GM1 while reacting strongly with anti-STa antibodies. These properties allow the use of the hybrid protein as an immunoreagent for detection of STa by GMl-inhibition ELISA [23]. Indeed, evidence is now available indicating that not only the decapeptide-CTB hybrid can be used as a reagent to detect STa in culture supernatants of clinical isolates of enterotoxigenic E. co# but that this protein is superior to other genetically obtained ST-LT hybrids [10,11] in this test (J.S.…”