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Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Liu Z, Reba S, Chen WD, Porwal SK, Boom WH, Petersen RB, Rojas R, Viswanathan R, Devireddy L - J. Exp. Med. (2014)

Bottom Line: Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron.In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro.Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

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Affiliation: Case Comprehensive Cancer Center; Department of Pathology; Department of Medicine, Tuberculosis Research Institute and Division of Infectious Diseases; and Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106Case Comprehensive Cancer Center; Department of Pathology; Department of Medicine, Tuberculosis Research Institute and Division of Infectious Diseases; and Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106.

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2,5-DHBA-mediated growth augmentation of E. coli in vitro. (A–D) E. coli W3110 (A), E. coli W3110 mut aroB (B), E. coli W3110ΔFepA (C) or E. coli W3110ΔFepA/ΔaroB (D; 104 CFU/ml each) was cultured in the presence of the indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01, ***, P < 0.001. (E and F) E. coli W3110 mut aroB (104 CFU/ml) was cultured in the presence of 0.1 µM FeCl3 and increasing concentrations of 2,5-DHBA (E) or 2,3-DHBA (F) overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G, right) Schematic showing chromium substitution in 2,5-DHBA. (left) E. coli W3110 mut aroB (104 CFU/ml) was cultured in the presence of 0.1 µM CrCl3 and increasing concentrations of 2,5-DHBA overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01. (H) E. coli H9049 (104 CFU/ml) was cultured as stated in A. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (I and J) E. coli H9049 (104 CFU/ml) was cultured in the presence of 0.1 µM FeCl3 and increasing concentrations of 2,5-DHBA (H) or 2,3-DHBA (I) and CFU were determined 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (K) DHBA supplementation has no effect on S. aureus growth. S. aureus strain 25923 at 105 CFU/ml was cultured in the presence of indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and CFU were determined at 12 h after culture by plating onto tryptic soy agar plates. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons. P < 0.05 was considered significant. (L) C. albicans is impervious to DHBA supplementation. C. albicans strain 562 at 106 CFU/ml was cultured in the presence of indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and their number was enumerated in a hemocytometer 24 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance, followed by the Tukey HSD test for multiple comparisons. P < 0.05 was considered significant.
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fig1: 2,5-DHBA-mediated growth augmentation of E. coli in vitro. (A–D) E. coli W3110 (A), E. coli W3110 mut aroB (B), E. coli W3110ΔFepA (C) or E. coli W3110ΔFepA/ΔaroB (D; 104 CFU/ml each) was cultured in the presence of the indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01, ***, P < 0.001. (E and F) E. coli W3110 mut aroB (104 CFU/ml) was cultured in the presence of 0.1 µM FeCl3 and increasing concentrations of 2,5-DHBA (E) or 2,3-DHBA (F) overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G, right) Schematic showing chromium substitution in 2,5-DHBA. (left) E. coli W3110 mut aroB (104 CFU/ml) was cultured in the presence of 0.1 µM CrCl3 and increasing concentrations of 2,5-DHBA overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01. (H) E. coli H9049 (104 CFU/ml) was cultured as stated in A. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (I and J) E. coli H9049 (104 CFU/ml) was cultured in the presence of 0.1 µM FeCl3 and increasing concentrations of 2,5-DHBA (H) or 2,3-DHBA (I) and CFU were determined 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (K) DHBA supplementation has no effect on S. aureus growth. S. aureus strain 25923 at 105 CFU/ml was cultured in the presence of indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and CFU were determined at 12 h after culture by plating onto tryptic soy agar plates. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons. P < 0.05 was considered significant. (L) C. albicans is impervious to DHBA supplementation. C. albicans strain 562 at 106 CFU/ml was cultured in the presence of indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and their number was enumerated in a hemocytometer 24 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance, followed by the Tukey HSD test for multiple comparisons. P < 0.05 was considered significant.

Mentions: Previously, both 2,3-DHBA and enterobactin were shown to confer a growth advantage to E. coli by promoting iron uptake (Hancock et al., 1977). The mammalian siderophore, 2,5-DHBA, resembles 2,3-DHBA, so we reasoned that 2,5-DHBA may also promote bacterial growth. We tested this hypothesis using an E. coli strain harboring a mutation in the aroB gene (W3110 mut aroB) whose gene product catalyzes one of the early steps in the shikimate pathway preventing enterobactin synthesis (Braun et al., 1983; Chaudhuri et al., 1986; Bentley, 1990). In iron-restrictive medium (RPMI), the aroB mutant grows poorly compared with the parental E. coli W3110 strain (Braun et al., 1983). As expected, addition of 2,3-DHBA augmented growth of the aroB mutant (Fig. 1 B). The growth promoting activity of 2,3-DHBA on the aroB mutant is less effective than that observed in parental E. coli W3110 (Fig. 1 A). However, 2,5-DHBA also augmented the growth of the aroB mutant in a dose-dependent manner (Fig. 1 B). Supplementation with 2,5-DHBA augmented the growth of parental W3110 strain, although less efficiently than 2,3-DHBA (Fig. 1 A). In contrast, addition of benzoic acid, a chemical paralogue of 2,5-DHBA, had a minimal effect on the growth of both parental or aroB mutant E. coli strains (Fig. 1 B). Thus, supplementation with 2,5-DHBA rescues the growth deficit caused by the absence of enterobactin.


Regulation of mammalian siderophore 2,5-DHBA in the innate immune response to infection.

Liu Z, Reba S, Chen WD, Porwal SK, Boom WH, Petersen RB, Rojas R, Viswanathan R, Devireddy L - J. Exp. Med. (2014)

2,5-DHBA-mediated growth augmentation of E. coli in vitro. (A–D) E. coli W3110 (A), E. coli W3110 mut aroB (B), E. coli W3110ΔFepA (C) or E. coli W3110ΔFepA/ΔaroB (D; 104 CFU/ml each) was cultured in the presence of the indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01, ***, P < 0.001. (E and F) E. coli W3110 mut aroB (104 CFU/ml) was cultured in the presence of 0.1 µM FeCl3 and increasing concentrations of 2,5-DHBA (E) or 2,3-DHBA (F) overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G, right) Schematic showing chromium substitution in 2,5-DHBA. (left) E. coli W3110 mut aroB (104 CFU/ml) was cultured in the presence of 0.1 µM CrCl3 and increasing concentrations of 2,5-DHBA overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01. (H) E. coli H9049 (104 CFU/ml) was cultured as stated in A. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (I and J) E. coli H9049 (104 CFU/ml) was cultured in the presence of 0.1 µM FeCl3 and increasing concentrations of 2,5-DHBA (H) or 2,3-DHBA (I) and CFU were determined 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (K) DHBA supplementation has no effect on S. aureus growth. S. aureus strain 25923 at 105 CFU/ml was cultured in the presence of indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and CFU were determined at 12 h after culture by plating onto tryptic soy agar plates. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons. P < 0.05 was considered significant. (L) C. albicans is impervious to DHBA supplementation. C. albicans strain 562 at 106 CFU/ml was cultured in the presence of indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and their number was enumerated in a hemocytometer 24 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance, followed by the Tukey HSD test for multiple comparisons. P < 0.05 was considered significant.
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fig1: 2,5-DHBA-mediated growth augmentation of E. coli in vitro. (A–D) E. coli W3110 (A), E. coli W3110 mut aroB (B), E. coli W3110ΔFepA (C) or E. coli W3110ΔFepA/ΔaroB (D; 104 CFU/ml each) was cultured in the presence of the indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01, ***, P < 0.001. (E and F) E. coli W3110 mut aroB (104 CFU/ml) was cultured in the presence of 0.1 µM FeCl3 and increasing concentrations of 2,5-DHBA (E) or 2,3-DHBA (F) overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G, right) Schematic showing chromium substitution in 2,5-DHBA. (left) E. coli W3110 mut aroB (104 CFU/ml) was cultured in the presence of 0.1 µM CrCl3 and increasing concentrations of 2,5-DHBA overnight and CFU were determined at 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01. (H) E. coli H9049 (104 CFU/ml) was cultured as stated in A. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (I and J) E. coli H9049 (104 CFU/ml) was cultured in the presence of 0.1 µM FeCl3 and increasing concentrations of 2,5-DHBA (H) or 2,3-DHBA (I) and CFU were determined 12 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (K) DHBA supplementation has no effect on S. aureus growth. S. aureus strain 25923 at 105 CFU/ml was cultured in the presence of indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and CFU were determined at 12 h after culture by plating onto tryptic soy agar plates. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance followed by the Tukey HSD test for multiple comparisons. P < 0.05 was considered significant. (L) C. albicans is impervious to DHBA supplementation. C. albicans strain 562 at 106 CFU/ml was cultured in the presence of indicated amounts of 2,5-DHBA, 2,3-DHBA, or benzoic acid overnight and their number was enumerated in a hemocytometer 24 h after culture. Data are the means of three independent experiments ± SD. Statistical analysis by one-way analysis of variance, followed by the Tukey HSD test for multiple comparisons. P < 0.05 was considered significant.
Mentions: Previously, both 2,3-DHBA and enterobactin were shown to confer a growth advantage to E. coli by promoting iron uptake (Hancock et al., 1977). The mammalian siderophore, 2,5-DHBA, resembles 2,3-DHBA, so we reasoned that 2,5-DHBA may also promote bacterial growth. We tested this hypothesis using an E. coli strain harboring a mutation in the aroB gene (W3110 mut aroB) whose gene product catalyzes one of the early steps in the shikimate pathway preventing enterobactin synthesis (Braun et al., 1983; Chaudhuri et al., 1986; Bentley, 1990). In iron-restrictive medium (RPMI), the aroB mutant grows poorly compared with the parental E. coli W3110 strain (Braun et al., 1983). As expected, addition of 2,3-DHBA augmented growth of the aroB mutant (Fig. 1 B). The growth promoting activity of 2,3-DHBA on the aroB mutant is less effective than that observed in parental E. coli W3110 (Fig. 1 A). However, 2,5-DHBA also augmented the growth of the aroB mutant in a dose-dependent manner (Fig. 1 B). Supplementation with 2,5-DHBA augmented the growth of parental W3110 strain, although less efficiently than 2,3-DHBA (Fig. 1 A). In contrast, addition of benzoic acid, a chemical paralogue of 2,5-DHBA, had a minimal effect on the growth of both parental or aroB mutant E. coli strains (Fig. 1 B). Thus, supplementation with 2,5-DHBA rescues the growth deficit caused by the absence of enterobactin.

Bottom Line: Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron.In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro.Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

View Article: PubMed Central - HTML - PubMed

Affiliation: Case Comprehensive Cancer Center; Department of Pathology; Department of Medicine, Tuberculosis Research Institute and Division of Infectious Diseases; and Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106Case Comprehensive Cancer Center; Department of Pathology; Department of Medicine, Tuberculosis Research Institute and Division of Infectious Diseases; and Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106.

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Related in: MedlinePlus