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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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Insights on the mechanism of action of the CRP-cAMP complex in vivo and in vitro.(A) Effect of the addition of cAMP during in vitro OFF to ON recombination. Bacterial extracts were obtained from strains NEC026 (wt) or CMM026 (Δcya) transformed with the inducible fimB expression plasmid (pIB378). Extracts were mixed with the template plasmid pJL-2 in absence or presence of increasing amounts of cAMP (1 to 50 mM final concentration). Mean values and standard deviations of three independent experiments are shown. (B) Effect of increasing amounts of the gyrase inhibitor novobiocin on fimA expression. ß-galactosidase activity was measured from strains CBP198 (wt, black bars) and CMM198 (Δcya, white bars) grown to mid-log phase in LB medium supplemented with 0, 6.25, 12.5, and 25 µg ml−1 novobiocin. Mean values and standard deviations from two independent experiments are shown. (C) Effect of DNA gyrase inhibition on the orientation of the fim invertible element in vivo. Upper panel: ON-OFF diagnostic of the samples used in Fig. 5B, representing the strain CBP198 (fimB+ fimE+) and its cya derivative CMM198 grown in presence of novobiocin (concentrations as indicated). Lower panel: ON-OFF diagnostic of the strain AAEC370A (fimB+ fimE) and its Δcya derivative CMM370A subject to the same growth conditions as in the upper panel. Both panels depict electronically inverted images of the upper half of acrylamide gels after ethidium bromide staining. (D) Effect of DNA gyrase inhibition on FimB-mediated OFF to ON switching in vitro. Increasing amounts of novobiocin (0, 50, 200 µg ml−1) were added to the in vitro recombination reactions. Bacterial extracts from strains NEC026 (wt, left panel) or CMM026 (Δcya, right panel) transformed with the inducible fimB expression plasmid (pIB378) were used together with the template plasmid pJL-2. Mean values and standard deviations in brackets of the estimated percentage of invertible elements in the ON orientation from four independent experiments are given as numbers below each lane. The images correspond to ethidium bromide stained gels from a representative experiment used to obtain the data shown.
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ppat-1000303-g005: Insights on the mechanism of action of the CRP-cAMP complex in vivo and in vitro.(A) Effect of the addition of cAMP during in vitro OFF to ON recombination. Bacterial extracts were obtained from strains NEC026 (wt) or CMM026 (Δcya) transformed with the inducible fimB expression plasmid (pIB378). Extracts were mixed with the template plasmid pJL-2 in absence or presence of increasing amounts of cAMP (1 to 50 mM final concentration). Mean values and standard deviations of three independent experiments are shown. (B) Effect of increasing amounts of the gyrase inhibitor novobiocin on fimA expression. ß-galactosidase activity was measured from strains CBP198 (wt, black bars) and CMM198 (Δcya, white bars) grown to mid-log phase in LB medium supplemented with 0, 6.25, 12.5, and 25 µg ml−1 novobiocin. Mean values and standard deviations from two independent experiments are shown. (C) Effect of DNA gyrase inhibition on the orientation of the fim invertible element in vivo. Upper panel: ON-OFF diagnostic of the samples used in Fig. 5B, representing the strain CBP198 (fimB+ fimE+) and its cya derivative CMM198 grown in presence of novobiocin (concentrations as indicated). Lower panel: ON-OFF diagnostic of the strain AAEC370A (fimB+ fimE) and its Δcya derivative CMM370A subject to the same growth conditions as in the upper panel. Both panels depict electronically inverted images of the upper half of acrylamide gels after ethidium bromide staining. (D) Effect of DNA gyrase inhibition on FimB-mediated OFF to ON switching in vitro. Increasing amounts of novobiocin (0, 50, 200 µg ml−1) were added to the in vitro recombination reactions. Bacterial extracts from strains NEC026 (wt, left panel) or CMM026 (Δcya, right panel) transformed with the inducible fimB expression plasmid (pIB378) were used together with the template plasmid pJL-2. Mean values and standard deviations in brackets of the estimated percentage of invertible elements in the ON orientation from four independent experiments are given as numbers below each lane. The images correspond to ethidium bromide stained gels from a representative experiment used to obtain the data shown.

Mentions: The slow response when adding exogenous cAMP to CMM198 cultures (Fig. 1C) suggested that the role of CRP-cAMP in the regulation of the phase variation occurs by an indirect mechanism. Nevertheless, to establish whether CRP-cAMP might also be directly involved in the switching process, in vitro recombination assays were performed using extracts of the cya strain while restoring CRP-cAMP activity by addition of increasing amounts of cAMP (Fig. 5A). No obvious alteration in the FimB-mediated switch was detected, strongly suggesting that CRP-cAMP does not directly interact with the nucleoprotein complex that is the substrate for the FimB recombinase. Accordingly, no effect was observed in the outcome of in vitro recombination assays when purified CRP was added to extracts obtained from a crp strain (data not shown).


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)

Insights on the mechanism of action of the CRP-cAMP complex in vivo and in vitro.(A) Effect of the addition of cAMP during in vitro OFF to ON recombination. Bacterial extracts were obtained from strains NEC026 (wt) or CMM026 (Δcya) transformed with the inducible fimB expression plasmid (pIB378). Extracts were mixed with the template plasmid pJL-2 in absence or presence of increasing amounts of cAMP (1 to 50 mM final concentration). Mean values and standard deviations of three independent experiments are shown. (B) Effect of increasing amounts of the gyrase inhibitor novobiocin on fimA expression. ß-galactosidase activity was measured from strains CBP198 (wt, black bars) and CMM198 (Δcya, white bars) grown to mid-log phase in LB medium supplemented with 0, 6.25, 12.5, and 25 µg ml−1 novobiocin. Mean values and standard deviations from two independent experiments are shown. (C) Effect of DNA gyrase inhibition on the orientation of the fim invertible element in vivo. Upper panel: ON-OFF diagnostic of the samples used in Fig. 5B, representing the strain CBP198 (fimB+ fimE+) and its cya derivative CMM198 grown in presence of novobiocin (concentrations as indicated). Lower panel: ON-OFF diagnostic of the strain AAEC370A (fimB+ fimE) and its Δcya derivative CMM370A subject to the same growth conditions as in the upper panel. Both panels depict electronically inverted images of the upper half of acrylamide gels after ethidium bromide staining. (D) Effect of DNA gyrase inhibition on FimB-mediated OFF to ON switching in vitro. Increasing amounts of novobiocin (0, 50, 200 µg ml−1) were added to the in vitro recombination reactions. Bacterial extracts from strains NEC026 (wt, left panel) or CMM026 (Δcya, right panel) transformed with the inducible fimB expression plasmid (pIB378) were used together with the template plasmid pJL-2. Mean values and standard deviations in brackets of the estimated percentage of invertible elements in the ON orientation from four independent experiments are given as numbers below each lane. The images correspond to ethidium bromide stained gels from a representative experiment used to obtain the data shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2636892&req=5

ppat-1000303-g005: Insights on the mechanism of action of the CRP-cAMP complex in vivo and in vitro.(A) Effect of the addition of cAMP during in vitro OFF to ON recombination. Bacterial extracts were obtained from strains NEC026 (wt) or CMM026 (Δcya) transformed with the inducible fimB expression plasmid (pIB378). Extracts were mixed with the template plasmid pJL-2 in absence or presence of increasing amounts of cAMP (1 to 50 mM final concentration). Mean values and standard deviations of three independent experiments are shown. (B) Effect of increasing amounts of the gyrase inhibitor novobiocin on fimA expression. ß-galactosidase activity was measured from strains CBP198 (wt, black bars) and CMM198 (Δcya, white bars) grown to mid-log phase in LB medium supplemented with 0, 6.25, 12.5, and 25 µg ml−1 novobiocin. Mean values and standard deviations from two independent experiments are shown. (C) Effect of DNA gyrase inhibition on the orientation of the fim invertible element in vivo. Upper panel: ON-OFF diagnostic of the samples used in Fig. 5B, representing the strain CBP198 (fimB+ fimE+) and its cya derivative CMM198 grown in presence of novobiocin (concentrations as indicated). Lower panel: ON-OFF diagnostic of the strain AAEC370A (fimB+ fimE) and its Δcya derivative CMM370A subject to the same growth conditions as in the upper panel. Both panels depict electronically inverted images of the upper half of acrylamide gels after ethidium bromide staining. (D) Effect of DNA gyrase inhibition on FimB-mediated OFF to ON switching in vitro. Increasing amounts of novobiocin (0, 50, 200 µg ml−1) were added to the in vitro recombination reactions. Bacterial extracts from strains NEC026 (wt, left panel) or CMM026 (Δcya, right panel) transformed with the inducible fimB expression plasmid (pIB378) were used together with the template plasmid pJL-2. Mean values and standard deviations in brackets of the estimated percentage of invertible elements in the ON orientation from four independent experiments are given as numbers below each lane. The images correspond to ethidium bromide stained gels from a representative experiment used to obtain the data shown.
Mentions: The slow response when adding exogenous cAMP to CMM198 cultures (Fig. 1C) suggested that the role of CRP-cAMP in the regulation of the phase variation occurs by an indirect mechanism. Nevertheless, to establish whether CRP-cAMP might also be directly involved in the switching process, in vitro recombination assays were performed using extracts of the cya strain while restoring CRP-cAMP activity by addition of increasing amounts of cAMP (Fig. 5A). No obvious alteration in the FimB-mediated switch was detected, strongly suggesting that CRP-cAMP does not directly interact with the nucleoprotein complex that is the substrate for the FimB recombinase. Accordingly, no effect was observed in the outcome of in vitro recombination assays when purified CRP was added to extracts obtained from a crp strain (data not shown).

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