Lincomycin increases synthetic rate and periplasmic pool size for cholera toxin

Abstract

Increased enterotoxigenicity of Vibrio cholerae 569B grown with low concentrations of lincomycin, previously described in terms of increased extracellular biological activity (capillary permeability factor and fluid accumulation in ligated rabbit ileal loops), was further characterized. Polyacrylamide gel electrophoresis and single radial immunodiffusion showed that lincomycin-stimulated cells produced increased molar quantities of cholera toxin (CT) both extra-and intracellularly. The intracellular CT was released in comparable amounts by sonication, deoxycholate extraction, and polymyxin B treatment. Polymyxin B release of CT was nearly complete under conditions wherein only 6% of total cellular,tf-galactosidase was released, implying a periplasmic pool of CT in stimulated cells. No intracellular choleragenoid (CT subunit B) was found in stimulated cells by polymyxin B release. No proteolysis of '4C-labeled CT was detected after prolonged incubation with sonicated nonstimulated cultures or sonicated concentrated cells. These data support the conclusion that the stimulatory effect of lincomycin involves an increase in the rate of synthesis of the CT molecule, and argue against alternative models involving inhibition of putative normal degradation of CT, increased release of otherwise cell-bound CT, or activation of inactive, or less active, forms of CT.Cholera toxin (CT) is a protein enterotoxin consisting of subunits A and B. The A portion has been shown to catalyze the intracellular ADP ribosylation and activation of adenylate cyclase (8), and it contains two polypeptide chains, Al and A2, linked by a disulfide bond (6). It appears that subunit A is synthesized as a single polypeptide chain which is nicked extracellularly to produce Al and A2 (9). The B portion, which binds to receptors of CT target cells (7), is composed of four or five identical polypeptide chains (6, 13), here denoted Bm (for monomer). The molecular weights of Al, A2, and Bm have been reported to be 23,000, 6,000, and 11,000, respectively (6).Cellular mechanisms which regulate the synthesis of cholera toxin in Vibrio cholerae are of particular interest because of both the relevance of the toxin to human health and its apparent irrelevance to the producing organism. Largely because of this absence of any known function of CT in the growth of V. cholerae, little is known about the regulation of its synthesis. As a possible means of probing this regulation, we have been examining the increased enterotoxigenicity caused by lincomycin.We have previously described large increases in extracellular CT in V. cholerae 569B when cells are grown with low concentrations of lincomycin, an inhibitor of protein synthesis (14), as measured either by capillary permeability factor or by fluid accumulation in ligated rabbit ileal loops. Heat-labile Escherichia coli enterotoxin (LT) was also shown to be increased by lincomycin (14). Data are presented here which strongly support the conclusion that lincomycin stimulates an increase in the rate of synthesis of t...

Identification of Novel Components Influencing Colonization Factor Antigen I Expression in Enterotoxigenic Escherichia coli

“…Although lincomycin has been reported to induce LT secretion in the CFA/I expressing strain H10407 [ 46 ], it had no effect on LT secretion in the experimental conditions tested here. While the effect of lincomycin may be strain specific, leading to increased production of other virulence factors including CFA/I, as shown here for strain 258909–3, lincomycin has also been reported to induce LT production with no associated increase in secretion [ 56 ].…”

Shigella dysenteriae 1 cytotoxin: periplasmic protein releasable by polymyxin B and osmotic shock

“…The release of both cholera (15) and Escherichia coli LT (7) toxins by polymyxin B has been shown to occur at an antibiotic concentration of 2 mg/ml. However, it has been shown by Cerny and Teuber (1) that treatment of E. coli cells with as little as 100 ,g of polymyxin B per ml is sufficient to release periplasmic enzymes.…”

Cellular location of heat-labile enterotoxin in Escherichia coli

“…cells by methods known to release periplasmic proteins (6,21). A periplasmic pool of cholera toxin A subunit has also been detected (24).…”