Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli

Abstract

The molecular mechanisms that define asymptomatic bacteriuria (ABU) Escherichia coli colonization of the human urinary tract remain to be properly elucidated. Here, we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness, and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin, and yersiniabactin and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient medium. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient medium but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with strain 83972, there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a "cheater" and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization.

Fig 1

Total siderophore activity of ABU E. coli isolates (n = 47). Solid CAS assays were performed with glucose or glycerol as the carbon source and at pH 5.5 or pH 7.0. The radius of the halo formed around each colony corresponds with the amount of siderophore activity. The data are presented as box plots with crosses representing the medians. ABU E. coli siderophore activity was significantly lower when cultures were grown with glucose at pH 5.5 than under the other growth conditions (P < 0.001). The siderophore activity of ABU E. coli 83972 is indicated.

Fig 2

Siderophores synthesized by ABU E. coli strain 83972. (A) HPLC analysis showing siderophores synthesized by ABU E. coli strain 83972 wild-type (WT) and 83972 siderophore biosynthetic deletion mutants in MM9 medium at pH 7.0. The identity of each peak was confirmed by LC-MS. (B) Chemical structures of the siderophores enterobactin, salmochelin, yersiniabactin, and aerobactin. (DHBS)₂ is the 2,3-dihydroxybenzoylserine dimer. Abs, absorbance.

Fig 3

Liquid CAS assays for ABU E. coli strain 83972 wild-type and siderophore biosynthetic deletion mutants in MM9 medium at pH 5.5 and pH 7.0. Results are the means of biological triplicates plus standard deviations. Bar graph shows percent siderophore units, calculated as [(Ar − As)/Ar] × 100, where Ar is the absorbance of MM9/CAS solution and As is the sample absorbance. The quadruple mutant E. coli 83972 EASY had negligible siderophore activity at both pHs.

Fig 4

Growth of ABU E. coli strain 83972 wild type and siderophore biosynthetic deletion mutants in MM9 medium with 50 μM DIP at pH 7.0 (A) and pH 5.5 (B). E. coli 83972 siderophore mutants with the same growth profile as the wild-type strain are not shown. Growth rates were calculated from the exponential phase (2 to 5 h), and cell densities were determined from the 10-h time point. Results are the means of biological triplicates ± standard deviations. Strains 83972 Y, 83972 SY, 83972 ES, 83972 EAS, and 83972 EASY had significantly different growth rates and cell densities after 10 h of growth than the wild-type E. coli 83972 at both pHs (P < 0.05). Strain 83972 ESY had a significantly lower growth rate and final cell density than the wild-type E. coli 83972 at pH 7.0 (P < 0.05).

Fig 5

Relative fitness of E. coli 83972 siderophore biosynthetic deletion mutants compared to E. coli 83972^AMP in a mixed 1:1 growth experiment in MM9 medium at pH 7.0 containing 50 μM DIP. Fitness indexes were calculated after 7 h of growth by dividing the CFU/ml of each mutant by the CFU/ml of 83972^AMP. The dashed line represents a fitness index of 1.0, which indicates no difference in fitness between the two strains. The relative fitness of each E. coli 83972 biosynthetic mutant was not significantly different from 1.

Fig 6

Growth and relative fitness of E. coli 83972 siderophore receptor deletion mutants. (A) Growth in MM9 medium, pH 7.0, containing 50 μM DIP. E. coli 83972 siderophore mutants with the same growth profile as the wild-type strain are not shown. Strains 83972 ES^R, 83972 EAS^R, 83972 ESY^R, and 83972 EASY^R had significantly lower growth rates and lower cell densities after 10 h of growth than wild-type E. coli 83972 (P < 0.001). (B) Relative fitness of 83972 siderophore receptor mutants compared to 83972^AMP after a mixed 1:1 growth experiment in MM9 medium at pH 7.0 containing 50 μM DIP. Results are the means of biological triplicates ± standard deviations. The dashed line represents a fitness index of 1.0, which indicates no difference in fitness between the two strains. Strains 83972 ES^R, 83972 EAS^R, 83972 ESY^R, and 83972 EASY^R were severely outcompeted by E. coli 83972^AMP (P < 0.05). Strains 83972 S^R, 83972 E^R, 83972 AS^R, 83972 SY^R, 83972 AY^R, 83972 EY^R, 83972 EA^R, 83972 ASY^R, and 83972 EAY^R were also significantly outcompeted by E. coli 83972^AMP (P < 0.05).

Fig 7

Growth and relative fitness of E. coli 83972 iron acquisition deletion mutants in human urine. (A) Growth of 83972 wild-type, 83972 F, 83972 EASY, and 83972 EASYF in human urine with 50 μM DIP. Strain 83972 EASYF had a significantly lower growth rate (P < 0.001) and final cell density (P = 0.001) than wild-type E. coli 83972. (B) Growth of E. coli 83972 siderophore receptor mutants in human urine (no DIP). E. coli 83972 siderophore receptor mutants with the same growth profile as the wild-type strain are not shown. Strains 83972 ES^R, 83972 EAS^R, 83972 ESY^R, and 83972 EASY^R displayed reduced growth rates and had lower cell densities after 10 h of growth than the wild-type E. coli 83972 (P < 0.05). (C) Relative fitness of 83972 siderophore receptor mutants compared to 83972^AMP after a mixed 1:1 growth experiment in human urine (no DIP). Results are the means of biological triplicates ± standard deviations. The dashed line represents a fitness index of 1.0, which indicates no difference in fitness between the two strains. Strains 83972 ES^R, 83972 EAS^R, 83972 ESY^R, and 83972 EASY^R were significantly outcompeted by strain 83972^AMP (P < 0.05).

Fig 8

Fitness of E. coli 83972 siderophore receptor mutants during in vivo competition with E. coli 83972^AMP. C3H/HeJ mice were challenged with a 1:1 mixture of 83972^AMP and 83972 ES^R, 83972 EAS^R, 83972 ESY^R, or 83972 EASY^R. (A) Each dot represents total number of CFU recovered from each mouse per ml of urine (i), per 0.1 g of bladder tissue (ii), or per 0.1 g of kidney tissue (iii). Lines connect data points for the same mouse, and horizontal bars represent median values. WT, E. coli 83972^AMP. (B) Each symbol represents the log₁₀ fitness index calculated for each individual mouse. Fitness indices were calculated by dividing the mutant CFU/ml of urine (i) or 0.1 g of bladder tissue (ii) or 0.1g of kidney tissue (iii) by that of 83972^AMP. Dashed lines represent a log₁₀ fitness index of 0, which indicates no difference in fitness between the two strains. Horizontal bars represent group medians, and each competition group had 7 to 8 mice. All mutants were significantly outcompeted by 83972^AMP in all three niches examined (P < 0.05).