A fadD mutant of Vibrio cholerae is impaired in the production of virulence factors and membrane localization of the virulence regulatory protein TcpP

Abstract

In the enteric pathogen Vibrio cholerae, expression of the major virulence factors is controlled by the hierarchical expression of several regulatory proteins comprising the ToxR regulon. In this study, we demonstrate that disruption of the fadD gene encoding a long-chain fatty acyl coenzyme A ligase has marked effects on expression of the ToxR virulence regulon, motility, and in vivo lethality of V. cholerae. In the V. cholerae fadD mutant, expression of the major virulence genes ctxAB and tcpA, encoding cholera toxin (CT), and the major subunit of the toxin-coregulated pilus (TCP) was drastically repressed and a growth-phase-dependent reduction in the expression of toxT, encoding the transcriptional activator of ctxAB and tcpA, was observed. Expression of toxT from an inducible promoter completely restored CT to wild-type levels in the V. cholerae fadD mutant, suggesting that FadD probably acts upstream of toxT expression. Expression of toxT is activated by the synergistic effect of two transcriptional regulators, TcpP and ToxR. Reverse transcription-PCR and Western blot analysis indicated that although gene expression and production of both TcpP and ToxR are unaffected in the fadD mutant strain, membrane localization of TcpP, but not ToxR, is severely impaired in the fadD mutant strain from the mid-logarithmic phase of growth. Since the decrease in toxT expression occurred concomitantly with the reduction in membrane localization of TcpP, a direct correlation between the defect in membrane localization of TcpP and reduced toxT expression in the fadD mutant strain is suggested.

FIG. 1.

Growth curves of V. cholerae O395 and ΔfadD::cat. The strains were grown in LB medium (A) or M9 minimal medium containing 5 mM palmitic acid (B), and the numbers of CFU were determined at different times.

FIG. 2.

CT production in V. cholerae wild-type and fadD mutant strains. Strains O395, ΔfadD::cat, and ΔfadD::cat containing plasmid pfadD were grown in LB medium under conditions permissive for CT production (pH 6.6, 30°C), and at different times the level of CT in culture supernatants corresponding to 10⁹ CFU was measured. The data presented are averages of three independent experiments, and error bars represent standard deviations.

FIG. 3.

Virulence gene expression in V. cholerae fadD mutant. RNA was isolated from V. cholerae O395, ΔfadD::cat, and ΔfadD::cat containing plasmid pfadD; and the strains were grown in LB medium under permissive conditions to an OD₆₀₀ of 0.3 (A) or 0.7 (B), for estimation of ctxAB, tcpA, and 16S rRNA expression by qRT-PCR. (C) Semiquantitative RT-PCR was performed with RNA from cultures grown to an OD₆₀₀ of 0.7. Expression of each gene was normalized to that of 16S rRNA (C, lower panels). The level of ctxA or tcpA expression in strain O395 was arbitrarily taken to be 100. In panels A and B, the results of three independent experiments are represented as means ± standard deviations. A P value of <0.001 was considered significant.

FIG. 4.

Expression and stability of toxT mRNA. (A) Strains O395 and ΔfadD::cat were grown in LB medium under permissive conditions, and at different times RNA was isolated for estimation of toxT expression by qRT-PCR. 16S rRNA was taken as the internal control. The amount of toxT mRNA in strain O395 cultures in the early exponential phase (OD₆₀₀, 0.25) was arbitrarily taken to be 100. (B) RNA was isolated from strains O395 (lane a), ΔfadD::cat (lane b), and ΔfadD::cat/pfadD (lane c) grown to the late exponential phase (OD₆₀₀, 0.7), for estimation of toxT and 16S rRNA expression by semiquantitative RT-PCR. (C) Rifampin was added to O395 and ΔfadD::cat cultures (OD₆₀₀, 0.6) to inhibit transcription initiation, and aliquots were collected at different times. RNA was isolated, and toxT-specific transcripts were estimated by qRT-PCR. The amount of toxT mRNA in strain O395 at the time of addition of rifampin (0 min) was arbitrarily taken to be 100. Results of three independent experiments are represented as means ± standard deviations. A P value of <0.001 was considered significant.

FIG. 5.

Expression of virulence regulatory genes. Expression of aphA, aphB, tcpP, and toxR was examined in strains O395 and ΔfadD::cat grown to mid-exponential phase (OD₆₀₀, 0.5). 16S rRNA expression was used as an internal control. The level of gene expression in strain O395 was arbitrarily taken to be 100. The data presented are averages of three independent experiments, and error bars represent standard deviations. A P value of <0.001 was considered significant.

FIG. 6.

Effect of tcpP overexpression on virulence gene expression. Expression of tcpP, toxT, and ctxA was examined in strains O395, ΔfadD::cat, O395/ptcpPH, and ΔfadD::cat/ptcpPH grown under permissive conditions. 16S rRNA expression was used as an internal control. The level of gene expression in strain O395 was arbitrarily taken to be 1.

FIG. 7.

Western blot analysis. Cell lysate and membrane proteins of strains O395 (lane a) and ΔfadD::cat (lane b) were separated by SDS-PAGE and analyzed by Western blotting with anti-ToxR or anti-TcpP sera. Coomassie blue staining of parallel lanes is shown as a control for protein load (left). (A) Total cell lysates of strains grown to an OD₆₀₀ of 0.7. Membrane fractions of the strains were grown to an OD₆₀₀ of 0.2 (B) or an OD₆₀₀ of 0.7 (C).

FIG. 8.

Effect of fatty acid on ctxA expression. Strains O395 and ΔfadD::cat/pKEK162 were grown under permissive conditions in LB medium without or with linoleic acid (0.015%) to the late logarithmic phase, and ctxAB expression in the strains was estimated by qRT-PCR. The fold change in ctxA expression in each strain grown without and with linoleic acid is indicated.

FIG. 9.

Swarming of V. cholerae strains O395, ΔfadD::cat, and ΔfadD::cat/pfadD on motility plates.

FIG. 10.

Expression of fabB, fadB, and ctxA genes in the V. cholerae fadR mutant. RT-PCR analysis was performed to estimate expression of the genes and 16S rRNA in V. cholerae O395 (lanes a) and the fadR mutant (lanes b).