A new role for Zinc limitation in bacterial pathogenicity: modulation of α-hemolysin from uropathogenic Escherichia coli

Abstract

Metal limitation is a common situation during infection and can have profound effects on the pathogen's success. In this report, we examine the role of zinc limitation in the expression of a virulence factor in uropathogenic Escherichia coli. The pyelonephritis isolate J96 carries two hlyCABD operons that encode the RTX toxin α-hemolysin. While the coding regions of both operons are largely conserved, the upstream sequences, including the promoters, are unrelated. We show here that the two hlyCABD operons are differently regulated. The hly _II operon is efficiently silenced in the presence of zinc and highly expressed when zinc is limited. In contrast, the hly _I operon does not respond to zinc limitation. Genetic studies reveal that zinc-responsive regulation of the hly _II operon is controlled by the Zur metalloregulatory protein. A Zur binding site was identified in the promoter sequence of the hly _II operon, and we observe direct binding of Zur to this promoter region. Moreover, we find that Zur regulation of the hly _II operon modulates the ability of E. coli J96 to induce a cytotoxic response in host cell lines in culture. Our report constitutes the first description of the involvement of the zinc-sensing protein Zur in directly modulating the expression of a virulence factor in bacteria.

Figure 1

Differential expression of the two hlyCABD operons present in the J96 strain. (a) Percentage of identity at the level of the nucleotide sequence between the operon hly_I and hly_II of J96 and 536. n.d.: not determined. (b) Hemolytic phenotype of J96, JFV16 (J96ΔII) and JFV21 (J96ΔI) strains on Columbia Blood agar plates. (c) Coomassie blue stained SDS-PAGE (10%) of secreted protein extracts from cultures grown in LB₀ at 37 °C up to late-log phase (OD_600 nm of 1.0) of the indicated strains. Lane M: molecular mass markers (size in kDa indicated along the left side). The band corresponding to α-hemolysin (HlyA) is indicated with an arrowhead. Full-length gel image is shown in Fig. S6.

Figure 2

Expression of the hly_II operon of J96 is derepressed by a mutation in the zur gene. (a) Lac-phenotype on LB Xgal agar plates of the JFV3 (J96 hly_II::lacZ) strain and its Gm^R derivative (clone #13) obtained by random mutagenesis. Below is represented the location of the Gm^R transposon insertion in clone #13. (b) Transcriptional expression of the hly_II operon from cultures of the JFV3 strain and the two derivatives zur::Gm^R (clone #13) and zur::Cm^R (EV46) grown in LB at 37 °C up to late-log phase (OD_600 nm of 1.0). β-Galactosidase activity (Miller units) was determined in three independent cultures. Mean values with standard deviations are plotted. *P < 0.05, ANOVA with Tukey’s multiple comparisons test. (c) Electrophoretic analysis of secreted protein extracts from cultures grown in LB at 37 °C up to late-log phase of J96, JFV16 (J96ΔII) and JFV21 (J96ΔI) strains and their otherwise isogenic zur::Cm^R mutants (EV27, EV34 and EV38, respectively). Upper panels are Coomassie blue stained 10% SDS-PAGE and lower panels immunodetection using monoclonal anti-HlyA antibody. Samples for immunodetection from J96ΔII were concentrated 20x with respect to rest of samples for better visualization. Full-length gel and blot images are shown in Fig. S7. (d) Hemolytic phenotype of JFV16 (J96ΔII) and JFV21 (J96ΔI) strains and its zur mutant derivatives on Columbia Blood agar plates.

Figure 3

Effect of zur mutation in UPEC virulence-associated phenotypes. (A) Hemolysis after 1 hour infection of sheep blood cell suspensions with J96, JFV21 (J96ΔI), JFV16 (J96ΔII) and EV64 (J96ΔIΔII) strains and their otherwise isogenic zur mutants (EV27, EV34, EV38 and EV65). The release of haemoglobin was monitored by OD_545 nm. (B) T24 cell monolayer detachment activity. Percentage of remaining attached T24 cells, measured as OD_590 nm, after 3.5 h post-infection with the same strains as in (A) Mean values with standard deviation from three independent experiments (A) and three biological replicates (B) are plotted. *P < 0.05 **P < 0.0001 n.s.: non-significant, t test with p-values adjusted by Bonferroni’s method (C) Phase contrast microscopy of T24 bladder epithelial cell monolayers infected for 2 hours with the indicated strains. Scale bar, 0.1 mm.

Figure 4

The expression of the hly_II operon of J96 responds to the external levels of zinc. (a) Transcriptional expression from the hly_II promoter in cultures of the strains JFV3 and its zur::Cm^R mutant derivative (EV46) grown in M-LB and M-LB replenished with either 1 or 10 μM ZnCl₂. Culture samples were taken at late-log growth phase (OD_600 nm of 1.0). β-galactosidase activity (expressed in Miller units) was determined from three independent cultures; mean values with standard deviation are plotted. *P < 0.05, ANOVA with Tukey’s multiple comparisons test (b) Growth curves of JFV3 strain cultured in M-LB and in M-LB replenished with 10 μM ZnCl₂. Growth was monitored by measuring OD_600 nm from two independent cultures; mean values are plotted.(c) Detection of the α-hemolysin in secreted protein extracts from cultures of J96, JFV21 (J96ΔI) and JFV16 (J96ΔII) strains and their otherwise isogenic zur mutants (EV27, EV34 and EV38) grown in M-LB and M-LB replenished with 10 μM ZnCl₂. Full-length gel images are shown in Fig. S8.

Figure 5

Rapid response of hly_II transcriptional expression to zinc addition. (a) Growth curves of the strain JFV3 and EV46 (zur::Cm^R) in the indicated media by measuring OD_600 nm from two independent cultures; mean values are plotted. (b) Transcriptional expression of hly_II in cultures of JFV3 and EV46 after zinc addition. Cultures were grown in M-LB up to an OD_600 nm of 0.3 when media was either replenished or not with 10 μM ZnCl₂. Culture samples were taken right before ZnCl₂ addition (0 minutes), and after 15, 30 and 60 minutes. As a control, JFV3 was cultured in M-LB replenished with 10 μM ZnCl₂ from the beginning. β-galactosidase activity (expressed in Miller units) was determined from three independent cultures; mean values with standard deviation are plotted.

Figure 6

Zur protein binds to the hly_II promoter. (a) Sequence of the hly_II operon upstream of hlyC. In bold are indicated the first codon (ATG) of hlyC and the putative −10 and −35 boxes of the promoter. Within the blue box is indicated the putative binding site of Zur. The red box indicates the transcriptional start found by 5′RACE assays. (b) Sequence of the putative Zur binding site found in the hly_II operon promoter. Bases recognized by Zur dimers are colored: green for conserved bases and red for the only base that is not conserved. A dashed line represents the symmetry axis. Below are depicted the positions of Zur binding sites in promoters of znuABC, zinT, rpmE2 and hly_II. Zur boxes were drawn to scale in respect of transcription start site. (c) Sequence of the 5′RACE assay using J96 total RNA samples grown in LB at 37 °C up to late log phase. The two bases identified as a transcriptional start are indicated with arrowheads. The N residue corresponds to a mix of A and C bases. (d) Electrophoretic mobility shift assay. Titration of a Cy5 labeled 50-bp hly_II promoter fragment carrying the putative Zur binding site was carried out in the presence of excess salmon sperm DNA which serves as a non-specific competitor. Samples contains 60 pM DNA, plus 0, 15, 30, 50, 100, 150, 200, 300, 350, 900 pM Zur (calculated as dimer), respectively. Samples were resolved on a 10% polyacrylamide gel. Both gel and electrophoresis buffers contain 50 µM ZnSO₄. Full-length gel image is shown in Fig. S9.