Characterisation of the E. coli and Salmonella qseC and qseE mutants reveals a metabolic rather than adrenergic receptor role

Abstract

Catecholamine stress hormones (norepinephrine, epinephrine, and dopamine) are signals that have been shown to be used as environmental cues, which affect the growth and virulence of normal microbiota as well as pathogenic bacteria. It has been reported that Escherichia coli and Salmonella use the two-component system proteins QseC and QseE to recognise catecholamines and so act as bacterial adrenergic receptors. In this study, we mutated the E. coli O157:H7 and Salmonella enterica serovar Typhimurium genes encoding QseC and QseE and found that this did not block stress hormone responsiveness in either species. Motility, biofilm formation, and analysis of virulence of the mutants using two infection models were similar to the wild-type strains. The main differences in phenotypes of the qseC and qseE mutants were responses to changes in temperature and growth in different media particularly with respect to salt, carbon, and nitrogen salt sources. In this physiological respect, it was also found that the phenotypes of the qseC and qseE mutants differed between E. coli and Salmonella. These findings collectively suggest that QseC and QseE are not essential for E. coli and Salmonella to respond to stress hormones and that the proteins may be playing a role in regulating metabolism.

Keywords: E. coli; Salmonella; QseC; QseE; catecholamines; stress.

Figure 1.

Growth induction of wild-type E. coli and Salmonella and their ΔqseC and ΔqseE mutants in serum-SAPI medium +/− catecholamines. Overnight cultures of wild-type E. coli(A) or Salmonella(B) and their ΔqseC and ΔqseE mutants were diluted to around 100 CFU/ml into serum-SAPI with no additions (negative control) or with the catecholamines shown (all at 50 µM), and grown for 18 h statically at 37°C. Iron in the form of ferric nitrate (50 µM) was used as a positive growth control. After incubation, bacteria were enumerated by serial dilution and growth on LA plates. Values shown represent the means and standard deviations of triplicate viable count determinations from triplicate cultures (n = 3). To measure incorporation of ⁵⁵Fe from ⁵⁵Fe-labelled transferrin into wild-type strains and ΔqseC and ΔqseE mutants (left hand in (C) and (D)), bacteria were incubated for 18 h at 37°C in serum-SAPI supplemented with 2.1 × 10⁵ cpm of ⁵⁵Fe-labelled transferrin in the presence or absence of 50 µM of NE as described in Materials and methods. Bacterial uptake of radioactively labelled iron [⁵⁵Fe] by wild-type and mutant strains were measured using scintillation counting. To measure ³H-NE internalisation of wild-type strains and ΔqseC and ΔqseE mutant cultures were incubated statically at 37°C in duplicate in 3 ml of 50 mM SAPI-Tris pH7.5 supplemented with ³H-NE (1 × 10⁵ cpm/ml) and measured for ³H-NE uptake after 90 min incubation as described in Materials and methods (right hand in (C) and (D)). One way ANOVA was used to calculate the level of significance. The assay was performed in triplicate on three independent occasions. Key: WT, wild-type; ∆C, ∆qseC mutant; ∆E, and ∆qseE mutant; asterisks indicate statistical significance of * P < .05, **P < .01, and ***P < .001.

Figure 2.

Adrenergic antagonist inhibition of NE growth induction of wild-type E. coli and Salmonella and their ΔqseC and ΔqseE mutants Overnight cultures of wild-type E. coli(A) or Salmonella(B) and their ΔqseC and ΔqseE mutants were diluted to around 100 CFU/ml into serum-SAPI without (control) or with 50 µM NE plus concentrations of the phentolamine shown (10–300 μM) and grown for 18 h statically at 37°C. Note phentolamine by itself was not toxic at 300 μM (data not shown). Cultures were then enumerated for viable cells as described for Fig. 1. Values represent the means and standard deviations of triplicate viable count determinations from triplicate cultures (n = 3). Key: lines show data comparisons. Asterisks indicate statistical significance of * P < .05, **P < .01, and ***P < .001.

Figure 3.

Effect of catecholamines on the motility and biofilm formation of wild-type E. coli and Salmonella and their ΔqseC and ΔqseE mutants The histograms in (A) and (B) show the motility in soft agar of wild-type E. coli (A) and Salmonella (B) and their ΔqseC and ΔqseE mutants in the absence and presence of catecholamines (50 µM, NE, Dop, or Epi) as described in Materials and methods; (n = 3). (C) and (D) show biofilm formation of E. coli and Salmonella ΔqseC or ΔqseE mutants in DMEM and serum-SAPI. Overnight cultures of wild-type E. coli (C) or Salmonella (D) and their ΔqseC or ΔqseE mutants were diluted 1:1000 into DMEM or serum-SAPI without and with 50 µM, NE, Dop, or Epi and incubated for 18 h statically at 37°C; attachment was measured using crystal violet staining. Culture values were corrected for staining due to media only. Data represent means and SEM of four biological replicates. Key: lines show comparisons. Asterisks and the symbols * indicate statistical significance of * P < .05, **P < .01; and ***P < .001.

Figure 4.

Virulence-associated phenotypes of wild-type E. coli and Salmonella ΔqseC and their ΔqseE mutants (A) and (B) show the attachment to, and invasion of, Caco-2 cells by wild-type (WT) E. coli (A) and Salmonella (B) and their ΔqseC and ΔqseE mutants (ΔC and ΔE) after 3 h incubation at 37°C; n = 3. Bacterial numbers were quantified as described in Materials and methods. (C) (E. coli) and (D) (Salmonella) show the virulence (LD50) of the wild-type and ΔqseC and ΔqseE mutant strains in a Galleria model of infection (n = 3). Data are expressed as % survival, and are representative results of at least three independent experiments.

Figure 5.

Growth of wild-type E. coli and Salmonella and their ΔqseC and ΔqseE mutants in C or N modified M9 medium. Overnight cultures of wild-type E. coli or Salmonella and their ΔqseC and ΔqseE mutants were diluted 1:1000 in M9 medium supplemented with 0.4% glucose (A), 0.4% glycerol (B) or 8.55 mM of ammonium sulphate instead of ammonium chloride (C). To measure the growth kinetics of E. coli and Salmonella ΔqseC and ΔqseE mutants in high and low osmotic strength media, cultures were diluted 1:100 in M9 medium with 0.4% glucose as carbon source with either 0.6 M NaCl (D) or no (0) M NaCl (E). Growth kinetics were measured at 37°C over 24 h in a Varioscan spectrophotometer. Values shown represent the means and standard deviations of values from triplicate cultures (n = 3).

Figure 6.

Temperature sensitivity of wild-type E. coli and Salmonella and their ΔqseC and ΔqseE mutants Overnight cultures of wild-type E. coli or Salmonella and their ΔqseC or ΔqseE mutants were diluted 1:1000 in M9 medium supplemented with 0.4% glucose, and growth kinetics over the 20–40°C range measured for 24 h in a Varioscan spectrophotometer. Values represent the means and standard deviations of values from triplicate cultures (n = 3).