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Type 1 fimbriae, a colonization factor of uropathogenic Escherichia coli, are controlled by the metabolic sensor CRP-cAMP.

Müller CM, Aberg A, Straseviçiene J, Emody L, Uhlin BE, Balsalobre C - PLoS Pathog. (2009)

Bottom Line: Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation.The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity.Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.

ABSTRACT
Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.

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Effect of CRP-cAMP deficiency on the expression of the fimA gene.(A–B) fimA expression was monitored by measuring ß-galactosidase activity from a chromosomal transcriptional fimA-lacZ fusion in either phase variation proficient (A) or phase variation deficient (B) strain backgrounds. For complementation purposes, plasmid pCBP68 (crp+) carrying the crp gene in the vector pLG338 (v.c.) was used. Bacterial cultures were grown in LB medium at 37°C to mid-log phase. Black bars represent values derived from cultures grown without cAMP and white bars represent values obtained from cultures grown in presence of 5 mM cAMP. Strains used in A: CBP198 (wt), CBP199 (Δcrp), and CMM198 (Δcya); Strains used in B: CBP374 (wt), CBP375 (Δcrp), and CMM374 (Δcya). (C–D) Effect of addition of 5 mM cAMP (open symbols) on fimA expression in either phase variation proficient (C) or phase variation deficient (D) strain backgrounds. Bacterial cultures were grown in LB medium at 37°C to an OD600nm of 0.05 before the addition of cAMP. As controls, cultures with no addition of cAMP (filled symbols) were used. Strains used in C: CBP198 (wt, squares) and CMM198 (Δcya, triangles). Strains used in D: CBP374 (wt, squares) and CMM374 (Δcya, triangles). All results shown are the mean values and standard deviations from three independent experiments.
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ppat-1000303-g001: Effect of CRP-cAMP deficiency on the expression of the fimA gene.(A–B) fimA expression was monitored by measuring ß-galactosidase activity from a chromosomal transcriptional fimA-lacZ fusion in either phase variation proficient (A) or phase variation deficient (B) strain backgrounds. For complementation purposes, plasmid pCBP68 (crp+) carrying the crp gene in the vector pLG338 (v.c.) was used. Bacterial cultures were grown in LB medium at 37°C to mid-log phase. Black bars represent values derived from cultures grown without cAMP and white bars represent values obtained from cultures grown in presence of 5 mM cAMP. Strains used in A: CBP198 (wt), CBP199 (Δcrp), and CMM198 (Δcya); Strains used in B: CBP374 (wt), CBP375 (Δcrp), and CMM374 (Δcya). (C–D) Effect of addition of 5 mM cAMP (open symbols) on fimA expression in either phase variation proficient (C) or phase variation deficient (D) strain backgrounds. Bacterial cultures were grown in LB medium at 37°C to an OD600nm of 0.05 before the addition of cAMP. As controls, cultures with no addition of cAMP (filled symbols) were used. Strains used in C: CBP198 (wt, squares) and CMM198 (Δcya, triangles). Strains used in D: CBP374 (wt, squares) and CMM374 (Δcya, triangles). All results shown are the mean values and standard deviations from three independent experiments.

Mentions: Using strains CBP198 and CBP199, a clear fimA up-regulation was observed in the crp background as compared with wt (Fig. 1A), indicating that CRP-cAMP represses type 1 fimbriation at the transcriptional level, consistent with the agglutination data (Table 1). On the other hand, when using CBP374 and CBP375 strains (Fig. 1B), a significantly lower fimA promoter activity was detected in the crp mutant, thereby suggesting that CRP-cAMP stimulates fimA promoter activity itself. Having in consideration that CRP activity is strictly dependent on its co-factor cAMP, in-frame cya deletion mutant strains (cAMP-deficient strains) were created and compared with isogenic crp and wt strains. The effect of the cya mutation on fimA expression resembled the one observed in the crp strain (Fig. 1A and 1B). Moreover, restoration of CRP-cAMP activity in crp and cya derivatives by using either the low copy number plasmid pCBP68 (pLG338-crp) or external addition of cAMP, respectively, restored fimA expression to wt levels (Fig. 1A and 1B). Taken together, we may conclude that CRP-cAMP has a dual role in the transcriptional expression of type 1 fimbriae: i) to stimulate transcription of the fimA promoter in phase-ON-cells and, ii) to repress the overall type 1 fimbriae expression.


Type 1 fimbriae, a colonization factor of uropathogenic Escherichia coli, are controlled by the metabolic sensor CRP-cAMP.

Müller CM, Aberg A, Straseviçiene J, Emody L, Uhlin BE, Balsalobre C - PLoS Pathog. (2009)

Effect of CRP-cAMP deficiency on the expression of the fimA gene.(A–B) fimA expression was monitored by measuring ß-galactosidase activity from a chromosomal transcriptional fimA-lacZ fusion in either phase variation proficient (A) or phase variation deficient (B) strain backgrounds. For complementation purposes, plasmid pCBP68 (crp+) carrying the crp gene in the vector pLG338 (v.c.) was used. Bacterial cultures were grown in LB medium at 37°C to mid-log phase. Black bars represent values derived from cultures grown without cAMP and white bars represent values obtained from cultures grown in presence of 5 mM cAMP. Strains used in A: CBP198 (wt), CBP199 (Δcrp), and CMM198 (Δcya); Strains used in B: CBP374 (wt), CBP375 (Δcrp), and CMM374 (Δcya). (C–D) Effect of addition of 5 mM cAMP (open symbols) on fimA expression in either phase variation proficient (C) or phase variation deficient (D) strain backgrounds. Bacterial cultures were grown in LB medium at 37°C to an OD600nm of 0.05 before the addition of cAMP. As controls, cultures with no addition of cAMP (filled symbols) were used. Strains used in C: CBP198 (wt, squares) and CMM198 (Δcya, triangles). Strains used in D: CBP374 (wt, squares) and CMM374 (Δcya, triangles). All results shown are the mean values and standard deviations from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636892&req=5

ppat-1000303-g001: Effect of CRP-cAMP deficiency on the expression of the fimA gene.(A–B) fimA expression was monitored by measuring ß-galactosidase activity from a chromosomal transcriptional fimA-lacZ fusion in either phase variation proficient (A) or phase variation deficient (B) strain backgrounds. For complementation purposes, plasmid pCBP68 (crp+) carrying the crp gene in the vector pLG338 (v.c.) was used. Bacterial cultures were grown in LB medium at 37°C to mid-log phase. Black bars represent values derived from cultures grown without cAMP and white bars represent values obtained from cultures grown in presence of 5 mM cAMP. Strains used in A: CBP198 (wt), CBP199 (Δcrp), and CMM198 (Δcya); Strains used in B: CBP374 (wt), CBP375 (Δcrp), and CMM374 (Δcya). (C–D) Effect of addition of 5 mM cAMP (open symbols) on fimA expression in either phase variation proficient (C) or phase variation deficient (D) strain backgrounds. Bacterial cultures were grown in LB medium at 37°C to an OD600nm of 0.05 before the addition of cAMP. As controls, cultures with no addition of cAMP (filled symbols) were used. Strains used in C: CBP198 (wt, squares) and CMM198 (Δcya, triangles). Strains used in D: CBP374 (wt, squares) and CMM374 (Δcya, triangles). All results shown are the mean values and standard deviations from three independent experiments.
Mentions: Using strains CBP198 and CBP199, a clear fimA up-regulation was observed in the crp background as compared with wt (Fig. 1A), indicating that CRP-cAMP represses type 1 fimbriation at the transcriptional level, consistent with the agglutination data (Table 1). On the other hand, when using CBP374 and CBP375 strains (Fig. 1B), a significantly lower fimA promoter activity was detected in the crp mutant, thereby suggesting that CRP-cAMP stimulates fimA promoter activity itself. Having in consideration that CRP activity is strictly dependent on its co-factor cAMP, in-frame cya deletion mutant strains (cAMP-deficient strains) were created and compared with isogenic crp and wt strains. The effect of the cya mutation on fimA expression resembled the one observed in the crp strain (Fig. 1A and 1B). Moreover, restoration of CRP-cAMP activity in crp and cya derivatives by using either the low copy number plasmid pCBP68 (pLG338-crp) or external addition of cAMP, respectively, restored fimA expression to wt levels (Fig. 1A and 1B). Taken together, we may conclude that CRP-cAMP has a dual role in the transcriptional expression of type 1 fimbriae: i) to stimulate transcription of the fimA promoter in phase-ON-cells and, ii) to repress the overall type 1 fimbriae expression.

Bottom Line: Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation.The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity.Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, Umeå, Sweden.

ABSTRACT
Type 1 fimbriae are a crucial factor for the virulence of uropathogenic Escherichia coli during the first steps of infection by mediating adhesion to epithelial cells. They are also required for the consequent colonization of the tissues and for invasion of the uroepithelium. Here, we studied the role of the specialized signal transduction system CRP-cAMP in the regulation of type 1 fimbriation. Although initially discovered by regulating carbohydrate metabolism, the CRP-cAMP complex controls a major regulatory network in Gram-negative bacteria, including a broad subset of genes spread into different functional categories of the cell. Our results indicate that CRP-cAMP plays a dual role in type 1 fimbriation, affecting both the phase variation process and fimA promoter activity, with an overall repressive outcome on fimbriation. The dissection of the regulatory pathway let us conclude that CRP-cAMP negatively affects FimB-mediated recombination by an indirect mechanism that requires DNA gyrase activity. Moreover, the underlying studies revealed that CRP-cAMP controls the expression of another global regulator in Gram-negative bacteria, the leucine-responsive protein Lrp. CRP-cAMP-mediated repression is limiting the switch from the non-fimbriated to the fimbriated state. Consistently, a drop in the intracellular concentration of cAMP due to altered physiological conditions (e.g. growth in presence of glucose) increases the percentage of fimbriated cells in the bacterial population. We also provide evidence that the repression of type 1 fimbriae by CRP-cAMP occurs during fast growth conditions (logarithmic phase) and is alleviated during slow growth (stationary phase), which is consistent with an involvement of type 1 fimbriae in the adaptation to stress conditions by promoting biofilm growth or entry into host cells. Our work suggests that the metabolic sensor CRP-cAMP plays a role in coupling the expression of type 1 fimbriae to environmental conditions, thereby also affecting subsequent attachment and colonization of host tissues.

Show MeSH
Related in: MedlinePlus