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

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.

Figure 1. 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 OD_600nm 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.

Figure 2. 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.

Figure 3. Type 1 fimbriae expression profile in different growth conditions.

(A) fimA expression was determined by measuring ß-galactosidase activity at various optical densities from cultures of the fimA-lacZ reporter strains CBP198 (wt, black bars) and CBP199 (Δcrp, white bars) in LB medium at 37°C. (B) Quantification of the percentage of fimA-expressing cells in the population of strain CBP198 (wt) on indicator plates. Cultures were grown to mid-log phase at 37°C in M9 minimal medium containing either glycerol (glyc.) or glucose (gluc.) as a carbon source. Mean values and standard deviations from three independent experiments are shown.

Figure 4. The CRP-cAMP regulatory complex affects the FimB-mediated OFF to ON switch.

(A) Determination of the percentage of ON-cells in strains expressing either FimB (AAEC370A, fimB ⁺ fimE) or FimE (AAEC261A, fimB fimE ⁺) and their cya counterparts. The upper half of a representative gel used for ON-OFF diagnostic is shown. The estimated percentage of ON-cells in the cultures (% ON) is indicated as mean values and standard deviations in brackets of three independent experiments. (B) In vitro OFF to ON recombination assay in bacterial extracts either containing FimB or being recombinase free. Bacterial extracts were obtained from strain NEC026 (fim, cya ⁺) and its isogenic cya mutant CMM026, transformed with either an inducible fimB expression plasmid (pIB378, fimB+) or the vector control (pET11, fimB−). Extracts were mixed with the template plasmid pJL-2 (fim invertible element in the OFF orientation). The orientation of the plasmid-encoded fim invertible element was determined after 3 h incubation by using the PCR-based assay (see Materials and Methods). Results are provided in the bar diagram as percentage of invertible elements in the ON orientation. The picture in the right part of the Figure illustrates an ethidium bromide stained gel from one of the experiments used to obtain the data shown. (C) In vitro ON to OFF recombination assay in bacterial extracts either containing FimE or being recombinase free. A similar assay as presented in B was performed. In this case, bacterial extracts were obtained from strain NEC026 (wt) or its isogenic cya mutant CMM026, transformed with either an inducible fimE expression plasmid (pIB382, fimE+) or the vector control (pET11, fimE−). Extracts were mixed with the template plasmid pMM36 (fim invertible element in the ON orientation) and analyzed as in B. In both B and C, mean values and standard deviations from four independent experiments are shown. (D) Effect of CRP-cAMP deficiency in the percentage of ON-cells in cultures of strain J96 and its cya derivative carrying either the plasmid pPKL9 (constitutive fimB expression, fimB OE+) or the vector control (pBR322, fimB OE−). The percentage of ON-cells was quantified from cultures of two independent clones. In A and D, the image corresponds to the upper half of an ethidium bromide stained gel.

Figure 5. 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.

Figure 6. Induction of Lrp in a CRP-cAMP deficient background.

(A) Lrp levels in different genetic backgrounds. Whole bacterial cell lysates of the strains CBP198 (wt), CBP199 (Δcrp), CBP199/pLG339 (Δcrp/v.c.), CBP199/pLG339-CRP (Δcrp/crp), and AAG42 (Δlrp) were subjected to immunoblot analyses using Lrp-specific antiserum. Numbers below each lane represent the average of the signal intensity from three independent experiments relative to the corresponding wt value (set as one). (B) Analysis of Lrp levels in whole cell lysates of strain CBP198 (wt) and CMM198 (Δcya) grown in the presence of novobiocin at the indicated concentrations (same bacterial cultures as in Fig. 5B). (C) Effect of overexpression of lrp on the percentage of ON-cells. Cultures of strain CBP198 transformed with a plasmid that carries the lrp gene under the inducible P_ara-promoter (pAAG6) were grown to mid-log phase in presence of the indicated concentrations of arabinose. The induced levels of lrp were assessed by immunoblotting using Lrp-specific antiserum. Simultaneously, quantification of the percentage of ON-cells in the populations was performed by using the PCR-based method. The lower image corresponds to the upper half of a representative gel used for ON-OFF diagnostic; the results are given as numbers below each lane.

Figure 7. Schematic model of action of the CRP-cAMP complex on the regulation of type 1 fimbriation.

The integration of different environmental signals modify the levels of the CRP-cAMP complex which affects the phase variation of type 1 fimbriae by altering the FimB-mediated directionality of the OFF to ON recombination event. Based on our results, a model is proposed where a stimulation of the DNA gyrase activity by CRP-cAMP would repress the FimB-mediated recombination from the OFF to the ON orientation by a mechanism that requires the presence of Lrp . In this work, a repressing effect of CRP-cAMP on the expression of Lrp and a stimulatory effect of CRP-cAMP on fimA promoter activity have also been described. Green arrows indicate stimulatory effects, whereas red lines indicate repressing effects.