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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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The percentage of fimbriated cells in the population is increased in crp and cya strains.(A) The percentage of fimA-expressing cells in presence (white bar) or absence (black bars) of 5 mM cAMP was determined by the indicator plate assay (see Materials and Methods) using mid-log phase cultures of the strains CBP198 (wt), CBP199 (Δcrp) and CMM198 (Δcya). Mean values and standard deviations from three independent experiments are shown. (B) Quantification of the percentage of ON-cells in bacterial populations by a PCR-based assay. Cultures of wt and cya derivatives of strains CBP198 and MG1655 were grown to mid-log phase in presence (white bars) or absence (black bars) of 5 mM cAMP. Mean values and standard deviations of three independent experiments are shown. (C) ON-OFF diagnostic of mid-log phase cultures of the J96 strain and its crp derivative; the arrowhead highlights the fragment corresponding to ON-cells detected in the J96crp samples. (D) Northern hybridization of total RNA extracted from mid-log cultures from strains J96 (wt), J96crp (Δcrp), VL751 (Δfim), and AAG42 (Δlrp) with specific probes for fimA, fimB, lrp, and 16S rRNA as indicated. (E) ON-OFF diagnostic of duplicated cultures of the phase variation deficient strains CBP374 (wt), CBP375 (Δcrp), and CMM374 (Δcya). A control showing the band pattern of an OFF population was included for comparison. The pictures in panels C and E are electronically inverted images of ethidium bromide stained acrylamide gels.
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ppat-1000303-g002: The percentage of fimbriated cells in the population is increased in crp and cya strains.(A) The percentage of fimA-expressing cells in presence (white bar) or absence (black bars) of 5 mM cAMP was determined by the indicator plate assay (see Materials and Methods) using mid-log phase cultures of the strains CBP198 (wt), CBP199 (Δcrp) and CMM198 (Δcya). Mean values and standard deviations from three independent experiments are shown. (B) Quantification of the percentage of ON-cells in bacterial populations by a PCR-based assay. Cultures of wt and cya derivatives of strains CBP198 and MG1655 were grown to mid-log phase in presence (white bars) or absence (black bars) of 5 mM cAMP. Mean values and standard deviations of three independent experiments are shown. (C) ON-OFF diagnostic of mid-log phase cultures of the J96 strain and its crp derivative; the arrowhead highlights the fragment corresponding to ON-cells detected in the J96crp samples. (D) Northern hybridization of total RNA extracted from mid-log cultures from strains J96 (wt), J96crp (Δcrp), VL751 (Δfim), and AAG42 (Δlrp) with specific probes for fimA, fimB, lrp, and 16S rRNA as indicated. (E) ON-OFF diagnostic of duplicated cultures of the phase variation deficient strains CBP374 (wt), CBP375 (Δcrp), and CMM374 (Δcya). A control showing the band pattern of an OFF population was included for comparison. The pictures in panels C and E are electronically inverted images of ethidium bromide stained acrylamide gels.

Mentions: So far, we have described that CRP-cAMP deficiency causes: i) a higher degree of type 1 fimbriation, ii) an increase in the expression of type 1 fimbriae in phase variation proficient strains, and iii) a reduction in fimA promoter activity in phase variation deficient strains. These results suggest that the percentage of fimbriated cells (ON-cells) in both crp and cya mutant strains is elevated when compared to wt, causing the overall increase in fimA expression. To test this prediction, the percentage of ON-cells in the population was monitored by plating cultures of the phase variable fimA-lacZ reporter strains on indicator plates containing X-gal. As predicted, a significant increase in the percentage of ON-cells in both the crp and cya mutant strains was observed (Fig. 2A). A quantitative PCR based method was validated (see Materials and Methods and Fig. S1) and used to detect and quantify the subpopulations having the invertible element either in the ON or in the OFF orientation [40]. Consistent with the results obtained from indicator plates, a significantly higher percentage of ON-cells was found for CMM198 (Δcya) as compared to CBP198 (wt) (Fig. 2B). Moreover, the cya deficiency was chemically complemented by addition of exogenous cAMP in the culture medium. The effect of CRP-cAMP deficiency in the switching between the ON and OFF orientation was further corroborated when in vivo switching frequencies were measured (see below). Similar results were obtained when using the reporterless and type 1 fimbriation-proficient strain MG1655 and its cya mutant derivative (Fig. 2B), thereby excluding the possibility that the lacZYA DNA sequence present in the reporter strains might affect the CRP-mediated effect on phase variation. Moreover, when derivatives of the uropathogenic isolate J96 were used, a significant increase in the percentage of ON-cells was detected in crp derivatives. While most of the cells in the wt population were in the OFF orientation under the culture conditions used, a subpopulation of cells with the invertible element in the ON orientation was clearly detected in the mutant derivative (Fig. 2C). To confirm these results, the level of fimA transcript in cultures of J96 and its derivative J96crp were quantified by Northern blot analysis (Fig. 2D). A 2.4-fold increase in fimA transcript was detected in J96crp as compared with wt, corroborating the results obtained when using fimA-lacZ reporter strains (Fig. 1A).


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)

The percentage of fimbriated cells in the population is increased in crp and cya strains.(A) The percentage of fimA-expressing cells in presence (white bar) or absence (black bars) of 5 mM cAMP was determined by the indicator plate assay (see Materials and Methods) using mid-log phase cultures of the strains CBP198 (wt), CBP199 (Δcrp) and CMM198 (Δcya). Mean values and standard deviations from three independent experiments are shown. (B) Quantification of the percentage of ON-cells in bacterial populations by a PCR-based assay. Cultures of wt and cya derivatives of strains CBP198 and MG1655 were grown to mid-log phase in presence (white bars) or absence (black bars) of 5 mM cAMP. Mean values and standard deviations of three independent experiments are shown. (C) ON-OFF diagnostic of mid-log phase cultures of the J96 strain and its crp derivative; the arrowhead highlights the fragment corresponding to ON-cells detected in the J96crp samples. (D) Northern hybridization of total RNA extracted from mid-log cultures from strains J96 (wt), J96crp (Δcrp), VL751 (Δfim), and AAG42 (Δlrp) with specific probes for fimA, fimB, lrp, and 16S rRNA as indicated. (E) ON-OFF diagnostic of duplicated cultures of the phase variation deficient strains CBP374 (wt), CBP375 (Δcrp), and CMM374 (Δcya). A control showing the band pattern of an OFF population was included for comparison. The pictures in panels C and E are electronically inverted images of ethidium bromide stained acrylamide gels.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636892&req=5

ppat-1000303-g002: The percentage of fimbriated cells in the population is increased in crp and cya strains.(A) The percentage of fimA-expressing cells in presence (white bar) or absence (black bars) of 5 mM cAMP was determined by the indicator plate assay (see Materials and Methods) using mid-log phase cultures of the strains CBP198 (wt), CBP199 (Δcrp) and CMM198 (Δcya). Mean values and standard deviations from three independent experiments are shown. (B) Quantification of the percentage of ON-cells in bacterial populations by a PCR-based assay. Cultures of wt and cya derivatives of strains CBP198 and MG1655 were grown to mid-log phase in presence (white bars) or absence (black bars) of 5 mM cAMP. Mean values and standard deviations of three independent experiments are shown. (C) ON-OFF diagnostic of mid-log phase cultures of the J96 strain and its crp derivative; the arrowhead highlights the fragment corresponding to ON-cells detected in the J96crp samples. (D) Northern hybridization of total RNA extracted from mid-log cultures from strains J96 (wt), J96crp (Δcrp), VL751 (Δfim), and AAG42 (Δlrp) with specific probes for fimA, fimB, lrp, and 16S rRNA as indicated. (E) ON-OFF diagnostic of duplicated cultures of the phase variation deficient strains CBP374 (wt), CBP375 (Δcrp), and CMM374 (Δcya). A control showing the band pattern of an OFF population was included for comparison. The pictures in panels C and E are electronically inverted images of ethidium bromide stained acrylamide gels.
Mentions: So far, we have described that CRP-cAMP deficiency causes: i) a higher degree of type 1 fimbriation, ii) an increase in the expression of type 1 fimbriae in phase variation proficient strains, and iii) a reduction in fimA promoter activity in phase variation deficient strains. These results suggest that the percentage of fimbriated cells (ON-cells) in both crp and cya mutant strains is elevated when compared to wt, causing the overall increase in fimA expression. To test this prediction, the percentage of ON-cells in the population was monitored by plating cultures of the phase variable fimA-lacZ reporter strains on indicator plates containing X-gal. As predicted, a significant increase in the percentage of ON-cells in both the crp and cya mutant strains was observed (Fig. 2A). A quantitative PCR based method was validated (see Materials and Methods and Fig. S1) and used to detect and quantify the subpopulations having the invertible element either in the ON or in the OFF orientation [40]. Consistent with the results obtained from indicator plates, a significantly higher percentage of ON-cells was found for CMM198 (Δcya) as compared to CBP198 (wt) (Fig. 2B). Moreover, the cya deficiency was chemically complemented by addition of exogenous cAMP in the culture medium. The effect of CRP-cAMP deficiency in the switching between the ON and OFF orientation was further corroborated when in vivo switching frequencies were measured (see below). Similar results were obtained when using the reporterless and type 1 fimbriation-proficient strain MG1655 and its cya mutant derivative (Fig. 2B), thereby excluding the possibility that the lacZYA DNA sequence present in the reporter strains might affect the CRP-mediated effect on phase variation. Moreover, when derivatives of the uropathogenic isolate J96 were used, a significant increase in the percentage of ON-cells was detected in crp derivatives. While most of the cells in the wt population were in the OFF orientation under the culture conditions used, a subpopulation of cells with the invertible element in the ON orientation was clearly detected in the mutant derivative (Fig. 2C). To confirm these results, the level of fimA transcript in cultures of J96 and its derivative J96crp were quantified by Northern blot analysis (Fig. 2D). A 2.4-fold increase in fimA transcript was detected in J96crp as compared with wt, corroborating the results obtained when using fimA-lacZ reporter strains (Fig. 1A).

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