Association of Protease Activity in Vibrio cholerae Vaccine Strains with Decreases in Transcellular Epithelial Resistance of Polarized T84 Intestinal Epithelial Cells

Abstract

Culture supernatants prepared from reactogenic strains of Vibrio cholerae cause a decrease in the transcellular epithelial resistance of T84 intestinal cells. This decrease correlates with the presence of hemagglutinin/ protease but not with the presence of other potential accessory toxins or proteases. These data suggest a possible role for hemagglutinin/protease in reactogenicity, although other factors may also contribute.The potentially life-threatening disease cholera is caused by toxigenic strains of the gram-negative organism Vibrio cholerae. The hallmark symptom of cholera, profuse, watery diarrhea, is caused primarily by cholera toxin (CT). The genes encoding CT, ctxAB, are carried on a transducing phage, CTX⌽, that integrates into the V. cholerae genome (29). The "core" element of the phage genome carries four genes in addition to ctxAB : cep, orfU, ace, and zot (29). Both zot and orfU are known to be essential for phage morphogenesis (29). In addition, the recombinant products of the zot and ace genes have also been associated with changes in intestinal tissue conductance, suggesting that these genes encode accessory toxins of V. cholerae (6,27,28).The development of a safe and effective vaccine to protect against cholera is a multifaceted problem. An ideal cholera vaccine must confer long-lasting protective immunity, be inexpensive, and be easy to use. A vaccine strategy employing live attenuated V. cholerae strains should meet each of these requirements (15, 17). However, production of a safe, attenuated strain has been problematic. Shortly after the discovery of the genes zot and ace, one would have predicted that a core deletion, resulting in mutants unable to produce CT, zonula occludens toxin (Zot), and accessory cholera enterotoxin (Ace), would produce ideal vaccine candidate strains. However, even with the core element deleted, strains CVD110, CVD111, and CVD112 were still "reactogenic," causing residual side effects in volunteer recipients, including mild diarrhea, nausea, vomiting, abdominal cramps, and fever (21, 23). Similarly, vaccine strains with deletions of the entire integrated CTX⌽ element (attRS deletions) are also mildly reactogenic (5, 24). These data indicate that these strains encode additional reactogenic factors encoded at loci other that the integrated CTX⌽ prophage. Curiously, the reactogenic effect of these undefined factors is absent from vaccine candidate strains that have additional defects in motility (5,11,24). This paper describes experiments using transcellular epithelial resistance (TER) across polarized T84 epithelial cells to monitor potential reactogenic factors in various vaccine strains. We show that an activity associated with the zot gene is not detected in this system. However, we find that a decrease in TER correlates with the presence of the genes for production of hemagglutinin/protease (HA/protease), indicating that HA/protease may be a significant contributor to the reactogenicity of some V. cholerae vaccine strains.Addition of supernatant fluids to pol...

Growth Phase Regulation of Vibrio cholerae RTX Toxin Export

“…Strain E7946mpc, which was generated by Tn5 mutagenesis, has a multiple protease control (mpc) defect (28,40) and reduced protease activity both in log and stationary-phase growth (Table 1). E7946mpc has transposon insertions in toxS and dcuA, although these mutations are not linked to the protease control defect, which is due to a spontaneous mutation in the regulatory gene luxO (40).…”

Differential Infection of Polarized Epithelial Cell Lines by Sialic Acid-Dependent and Sialic Acid-Independent Rotavirus Strains

“…Paracellular permeability changes occur in response to a number of bacteria and contribute to diarrheal disease by altering intestinal secretion and absorption (3,16,17,27,40,43,51,54,56,58,64). Similar to some enteric bacteria, rotavirus produces an enterotoxin (1) and stimulates the enteric nervous system (37), and rotavirus or its proteins exert numerous physiological effects on the intestinal epithelium and host functions, including alteration of ion transport (25), disruption of the tight junction barrier (14,25,31,42,57,59), production of cytokine and chemokine responses (4,49,53), and stimulation of cell signaling pathways (35), all of which play a role in pathogenesis and/or immune responses.…”

Transcriptional Regulation of Vibrio cholerae Hemagglutinin/Protease by the Cyclic AMP Receptor Protein and RpoS

“…HA/protease perturbs the paracellular barrier of cultured intestinal epithelial cells (31,50) by acting on tight-junction-associated proteins (51) and promotes the detachment of vibrios from monolayers and mucin (4,15). Importantly, HA/protease contributes to the reactogenicity of live attenuated cholera vaccine strains (3,40).…”