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Two Nucleotide Second Messengers Regulate the Production of the Vibrio Cholerae Colonization Factor GbpA

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Two Nucleotide Second Messengers Regulate the Production of the Vibrio Cholerae Colonization Factor GbpA

Ankunda T Kariisa et al. BMC Microbiol.

Abstract

Background: The nucleotide second messengers cAMP and c-di-GMP allow many bacteria, including the human intestinal pathogen Vibrio cholerae, to respond to environmental stimuli with appropriate physiological adaptations. In response to limitation of specific carbohydrates, cAMP and its receptor CRP control the transcription of genes important for nutrient acquisition and utilization; c-di-GMP controls the transition between motile and sessile lifestyles often, but not exclusively, through transcriptional mechanisms. In this study, we investigated the convergence of cAMP and c-di-GMP signaling pathways in regulating the expression of gbpA. GbpA is a colonization factor that participates in the attachment of V. cholerae to N-acetylglucosamine-containing surfaces in its native aquatic environment and the host intestinal tract.

Results: We show that c-di-GMP inhibits gbpA activation in a fashion independent of the known transcription factors that directly sense c-di-GMP. Interestingly, inhibition of gbpA activation by c-di-GMP only occurs during growth on non-PTS dependent nutrient sources. Consistent with this result, we show that CRP binds to the gbpA promoter in a cAMP-dependent manner in vitro and drives transcription of gbpA in vivo. The interplay between cAMP and c-di-GMP does not broadly impact the CRP-cAMP regulon, but occurs more specifically at the gbpA promoter.

Conclusions: These findings suggest that c-di-GMP directly interferes with the interaction of CRP-cAMP and the gbpA promoter via an unidentified regulator. The use of two distinct second messenger signaling mechanisms to regulate gbpA transcription may allow V. cholerae to finely modulate GbpA production, and therefore colonization of aquatic and host surfaces, in response to discrete environmental stimuli.

Figures

Fig. 1
Fig. 1
c-di-GMP inhibits gbpA expression independently of the Vc1 riboswitch. a GbpA production by V. cholerae wild type and ∆Vc1 strains, each with vector (pBAD33) or pPDE, was determined by western blot. PDE gene expression was induced as described in the Methods. The image shown is a representative of three independent experiments. Densitometry analyses were done by normalizing the intensities of the bands corresponding to GbpA to the intensities of a cross-reactive band in the same lane (indicated by an asterisk), then comparing the normalized value to that of wild type V. cholerae with vector only. The fold change relative to wild type is indicated below each lane. b qRT-PCR was used to measure gbpA transcript levels in the strains described in (A), in V. cholerae with vector (black bars) or with pPDE (grey bars). The data were normalized relative to the wild-type containing vector only, using rpoB as the reference gene. c PgbpA-Vc1-lacZ or PgbpA-∆Vc1-lacZ fusions were each introduced into V. cholerae with vector (black bars) or pPDE (grey bars), and the β-galactosidase activity in culture lysates of these strains was measured. d qRT-PCR was used to measure gbpA transcript levels in V. cholerae with vector, pPDE, pPDEmut or pVC1592. The data were normalized relative to the wild-type containing vector, using rpoB as the reference gene. b-d Shown are the mean values and standard deviations of at least three independent experiments. ***P < 0.001 by unpaired t-test comparing the indicated sets of data
Fig. 2
Fig. 2
Known c-di-GMP effectors FlrA, VpsT and VpsR do not regulate gbpA in response to c-di-GMP. a GbpA levels in the supernatants of wild type V. cholerae, ∆flrA,vpsT and ΔvpsR strains, each with wild type (vector) or reduced levels of c-di-GMP (pPDE), were measured by western blot. PDE gene expression was induced as described in the Methods. The image shown is a representative of three separate experiments. Densitometry analyses were done by comparing the intensities of the GbpA bands to the intensities of a cross-reactive band in the same lane (indicated by an asterisk), then normalizing the value to that of wild type V. cholerae with vector. The fold change relative to the wild type is indicated below each lane. b qRT-PCR was used to measure the gbpA transcript abundance in wild type, ∆flrA,vpsT and ΔvpsR strains of V. cholerae, each with wild type (vector, black bars) and reduced levels of c-di-GMP (pPDE, grey bars). The data were normalized relative to the wild-type containing vector only, using rpoB as the reference gene. Shown are the means and standard deviations from at least three independent samples. For the indicated comparisons, ***P < 0.001 by unpaired t-test
Fig. 3
Fig. 3
c-di-GMP inhibition of gbpA expression is influenced by carbon source availability. V. cholerae strains containing the PgbpA-Vc1-lacZ reporter fusion, with wild type c-di-GMP (vector, black bars) or reduced c-di-GMP (pPDE, grey bars), were grown in M9 minimal medium with 0.5 % (w/v) N-acetylglucosamine (GlcNAc), glucose, maltose, sucrose, fructose or casamino acids at 37 °C with aeration to mid-logarithmic phase. PDE gene expression was induced as described in the Methods. Transcription was measured using β-galactosidase assays. Shown are the means and standard deviations from at least three independent experiments. For the indicated comparisons, ***P < 0.001 by unpaired t-test
Fig. 4
Fig. 4
Reduction of c-di-GMP induces gbpA expression in a CRP dependent manner. a GbpA levels in the supernatants of wild type V. cholerae, ∆nagC and ∆crp strains, each with wild type (vector) and reduced levels of c-di-GMP (pPDE), were measured by western blot. PDE gene expression was induced as described in the Methods. The image shown is a representative of three separate experiments. Densitometry analyses were done by comparing the intensities of the GbpA bands to the intensities of a cross-reactive band in the same lane (indicated by an asterisk), then normalizing the value to that of wild type V. cholerae with vector. The fold change relative to the wild type is indicated below each lane. b qRT-PCR was used to measure the gbpA transcript abundance in wild type, ∆nagC and ∆crp strains of V. cholerae, each with wild type (vector, black bars) and reduced levels of c-di-GMP (pPDE, grey bars). The data were normalized relative to the wild-type containing vector only, using rpoB as the reference gene. Shown are the means and standard deviations from at least three independent samples. For the indicated comparisons, *P < 0.05, ***P < 0.001 by unpaired t-test
Fig. 5
Fig. 5
Inactivation of the cAMP-CRP signaling pathway prevents c-di-GMP inhibition of gbpA expression. a GbpA levels in the supernatants of wild type V. cholerae, ∆cpdA (constitutively active CRP) and ∆cya (constitutively inactive CRP) strains, each with wild type (vector) and reduced levels of c-di-GMP (pPDE), were measured by western blot. PDE gene expression was induced as described in the Methods. The image shown is a representative of three separate experiments. Densitometry analyses were done by comparing the intensities of the GbpA bands to the intensities of a cross-reactive band in the same lane (indicated by an asterisk), then normalizing the value to that of wild type V. cholerae with vector. The fold change relative to the wild type is indicated below each lane. b qRT-PCR was used to measure the gbpA transcript abundance in wild type, ∆cpdA and ∆cya strains of V. cholerae, each with wild type (vector, black bars) and reduced levels of c-di-GMP (pPDE, grey bars). The data were normalized relative to the wild-type containing vector only, using rpoB as the reference gene. Shown are the means and standard deviations from at least three independent samples. For the indicated comparisons, *P < 0.05, ***P < 0.001 by unpaired t-test
Fig. 6
Fig. 6
c-di-GMP does not regulate CRP gene transcription, protein stability or DNA binding. a Transcript levels for crp in V. cholerae with wild type (vector) or reduced c-di-GMP (pPDE) were measured by qRT-PCR. PDE gene expression was induced as described in the Methods. The data were normalized relative to the wild-type containing vector only, using rpoB as the reference gene. Shown are the means and standard deviations from at three independent samples. b CRP protein (23.6 kDa) levels in lysates of V. cholerae with wild type (vector) or reduced c-di-GMP (pPDE) were measured by western blot. V. cholerae Δcrp containing vector was included as a negative control. RNA Polymerase was detected on the same blot as a loading control. The images shown are from a representative of three independent experiments. Densitometry analyses were done by comparing the intensities of the GbpA bands to the intensities of the RNAP band in the same lane, then normalizing the value to that of wild type V. cholerae with vector. The fold change relative to the wild type is shown below each lane. c Using electrophoretic mobility shift assays, purified recombinant CRP was tested for the ability to bind and shift a DNA fragment encompassing the gbpA promoter in the presence or absence of cAMP and/or c-di-GMP. As a control, a non-specific DNA (indicated by “NS”) fragment was added to all binding reactions and was confirmed not to be shifted by CRP. *In the final lane, c-di-GMP was added in 10-fold excess of cAMP
Fig. 7
Fig. 7
cAMP-CRP specifically impacts gbpA expression in response to c-di-GMP. a Putative CRP-regulated genes were selected for analysis by qRT-PCR to assess transcript abundance in wild type (grey bars) and Δcrp (black bars) strains. The data were normalized relative to the wild-type, using rpoB and gyrA as the reference genes. b The transcript abundance for the genes analyzed in (a) was determined for V. cholerae with wild type (vector, grey bars) and reduced levels of c-di-GMP (pPDE, white bars). The data were normalized relative to the wild-type containing vector only, using rpoB and gyrA as the reference genes. a and (b) Shown are the means and standard deviations from at least three independent samples. For the indicated comparisons, ***P < 0.001 by unpaired t-test

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