Cyclic AMP and its receptor protein negatively regulate the coordinate expression of cholera toxin and toxin-coregulated pilus in Vibrio cholerae

Abstract

Insertion mutations in two Vibrio cholerae genes, cya and crp, which encode adenylate cyclase and the cyclic AMP (cAMP) receptor protein (CRP), respectively, derepressed the expression of a chromosomal cholera toxin (CT) promoter-lacZ fusion at the nonpermissive temperature of 37 degrees C. In the classical biotype strain O395, the crp mutation increased the production of both CT and toxin-coregulated pilus (TCP) in vitro under a variety of growth conditions not normally permissive for their expression. The most dramatic increase in CT and TCP was observed with the crp mutant in Luria-Bertani (LB) medium pH 8.5, at 30 degrees C. El Tor biotype strains differ from classical strains in that they do not produce CT or TCP when grown in LB media. Incorporation of the crp mutation into El Tor strain C6706 permitted production of these proteins in LB medium pH 6.5, at 30 degrees C. In the infant mouse cholera model, the crp mutation decreased colonization in both biotypes at least 100-fold relative to the wild-type strains. The data presented here suggest a model whereby cAMP-CRP negatively regulates the expression of CT and TCP in both classical and El Tor biotypes under certain environmental conditions and also influences pathogenesis by regulating other processes necessary for optimal growth in vivo.

Figure 1

β-Galactosidase production in the ctx–lacZ fusion strains. Left half: toxR⁺ strains KSK369 (crp), black bars, and KSK218 (crp⁺), grey bars. Right half: toxR strains KSK374 (crp), black bars, and KSK236 (crp⁺), grey bars.

Figure 2

Autoagglutination pattern of KSK218 and its crp derivative KSK369. Cultures of KSK218 (A) and KSK369 (B) were grown in LB medium, pH 8.5, at 30°C. Autoagglutinated bacteria are shown by the arrow.

Figure 3

TcpA and CT production in O395 and its crp derivative KSK377. Strains were grown in LB medium pH 6.5, at 30°C (lanes 2 and 3), pH 8.5, at 30°C (lanes 4 and 5), pH 6.5, at 37°C (lanes 6 and 7), and pH 8.5, at 37°C (lanes 8 and 9). For each strain, approximately 6 μg of total protein was subjected to SDS/PAGE and stained with Coomassie blue. CS2-1 (tcpA), lane 1; O395, even-numbered lanes; KSK377, odd-numbered lanes. TcpA is shown by the arrow. The amount of CT in each culture supernatant is expressed at the bottom as ng/mg of protein per ml.

Figure 4

TcpA and CT production in C6706 and its crp derivative KSK394. Strains were grown at 30°C in LB medium, pH 6.5 (lanes 1, 2, and 4), or pH 8.5 (lanes 3 and 5). For each strain, approximately 2 μg of total protein was subjected to SDS/PAGE, electroblotted to nitrocellulose paper, and probed with anti-TCP antiserum (20). O395, lane 1; C6706, lanes 2 and 3; KSK394, lanes 4 and 5. TcpA is shown by the arrow. The amount of CT in each culture supernatant is expressed at the bottom as ng/mg of protein per ml.

Figure 5

Time course of colonization in the infant mouse cholera model. Twenty infant mice were orally inoculated with a mixture of strains O395, KSK377 (crp), and CS2-1 (tcpA). At various times over a 24-hr period, four mice were sacrificed and the total intestinal cfu per mouse for each strain was determined and averaged. ▪, O395; •, KSK377; ▴, CS2-1.