The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain - PubMed (original) (raw)

The Agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain

Vera Pader et al. Infect Immun. 2014 Oct.

Abstract

Staphylococcus aureus is responsible for numerous chronic and recurrent infections, which are frequently associated with the emergence of small-colony variants (SCVs) that lack a functional electron transport chain. SCVs exhibit enhanced expression of fibronectin-binding protein (FnBP) and greatly reduced hemolysin production, although the basis for this is unclear. One hypothesis is that these phenotypes are a consequence of the reduced Agr activity of SCVs, while an alternative is that the lack of a functional electron transport chain and the resulting reduction in ATP production are responsible. Disruption of the electron transport chain of S. aureus genetically (hemB and menD) or chemically, using 2-n-heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibited both growth and Agr activity and conferred an SCV phenotype. Supplementation of the culture medium with synthetic autoinducing peptide (sAIP) significantly increased Agr expression in both hemB mutant strains and S. aureus grown with HQNO and significantly reduced staphylococcal adhesion to fibronectin. However, sAIP did not promote hemolysin expression in hemB mutant strains or S. aureus grown with HQNO. Therefore, while Agr regulates fibronectin binding in SCVs, it cannot promote hemolysin production in the absence of a functional electron transport chain.

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Figures

FIG 1

FIG 1

Loss of the electron transport chain reduces growth and Agr expression. Shown are growth (A and D), P3 expression (B and E), and P3 expression corrected for growth (C and F) of S. aureus SH1000 (A to C) and USA300 (D to F) and derived strains. (A to C) SH1000 grown in the absence or presence of HQNO and a hemB::Tn mutant in TSB as determined by the OD600 (growth) and A_520 (GFP fluorescence) measurements. (D to F) USA300 wild type, Δ_hemB, and Δ_menD_ grown in TSB. The measurements are as described for panels A to C. The data represent the means of at least 4 independent experiments, each in triplicate. The error bars represent the standard deviations of the mean. RFU, relative fluorescence units.

FIG 2

FIG 2

Loss of the electron transport chain results in enhanced fibronectin binding. (A) Attachment of wild-type (WT) S. aureus SH1000 grown in the absence or presence (+HQ) of HQNO and a hemB::Tn mutant (hemB) to immobilized human fibronectin. Also shown are values relating to the attachment of agr_-defective SH1001 (Δ_agr) grown in the absence or presence of HQNO and of a hemB Δ_agr_ strain. (B) Attachment of WT USA300 and hemB and menD mutants. The values represent the mean averages of at least 4 experiments done in triplicate, and the error bars represent the standard deviations of the mean. Values that are significantly greater (P = 0.05 by Student's t test) than that of the wild type are indicated by asterisks.

FIG 3

FIG 3

Loss of the electron transport chain results in significantly reduced hemolytic activity. Shown are hemolytic activities of culture supernatants of wild-type S. aureus SH1000 and derived strains (A) and USA300 and derived mutants (B). Culture supernatants were used undiluted (U) or after serial 2-fold dilutions, as indicated. The data represent the means of at least 4 independent experiments, each in triplicate. The error bars represent the standard deviations of the mean. Values that are significantly different (P < 0.05 by Student's t test) from those of the wild type at equivalent dilutions are indicated by asterisks.

FIG 4

FIG 4

Synthetic AIP enhances agr expression in the absence of a functional electron transport chain. Shown are growth (A and D), P3 expression (B and E), and P3 expression corrected for growth (C and F) of SH1000 and derived strains (A to C) and USA300 and derived strains (D to F). (A to C) Wild-type S. aureus grown in the absence or presence of HQNO and that of a hemB::Tn mutant in TSB only or TSB supplemented with 1 μM or 10 μM sAIP. (D to F) Wild-type S. aureus and a hemB mutant grown in the absence or presence of 10 μM sAIP. The data represent the means of at least 4 independent experiments, each in triplicate. The error bars were omitted to enhance clarity but were all within 5% of the mean.

FIG 5

FIG 5

Agr regulates fibronectin binding in both the presence and absence of the electron transport chain. (A) Fibronectin binding of wild-type S. aureus SH1000 grown in the absence (WT) or presence (WT + HQ) of HQNO and a hemB::Tn mutant (hemB). Also shown are the fibronectin-binding levels of agr_-deficient mutants grown in the absence (Δ_agr) or presence (Δ_agr_ + HQ) of HQNO or in the hemB::Tn mutant background (Δ_agr hemB_). The culture medium consisted of TSB only or TSB supplemented with 1 μM sAIP or 10 μM sAIP. (B) Fibronectin binding of wild-type S. aureus USA300 and a hemB mutant grown in the absence or presence of 10 μM sAIP. Experiments were repeated 4 times in triplicate. Values that are significantly different (P < 0.05 by Student's t test) from those seen in the absence of sAIP are indicated by asterisks.

FIG 6

FIG 6

Agr activation by sAIP does not promote hemolytic activity in the absence of a functional electron transport chain. (A) Hemolytic activities of culture supernatants of wild-type SH1000 grown in the absence or presence of HQNO and of a hemB::Tn mutant in the absence (open bars) or presence (filled bars) of 10 μM sAIP. (B) Hemolytic activities of culture supernatants of wild-type USA300 and a hemB mutant in the absence (open bars) or presence (filled bars) of 10 μM sAIP. The data represent the mean averages of 4 experiments performed in triplicate, and the error bars represent the standard deviations of the mean. None of the values obtained from cultures containing sAIP were significantly different from those without sAIP at identical dilutions.

FIG 7

FIG 7

Agr activation by sAIP does not promote hemolytic activity in concentrated cultures in the absence of a functional electron transport chain. Wild-type SH1000 and a hemB::Tn mutant were concentrated to equivalent bacterial densities and cultured in TSB in the absence or presence of HQNO and the absence (open bars) or presence (filled bars) of 10 μM sAIP. The data represent the mean averages of 4 experiments performed in triplicate, and the error bars represent the standard deviations of the mean. None of the values obtained from cultures containing sAIP were significantly different from those without sAIP at identical dilutions.

FIG 8

FIG 8

Hemin and menadione restore growth and Agr activity in hemB and menD mutants, respectively. (A, B, D, and E) SH1000 hemB::Tn (A and D) and USA300 hemB (B and E) were grown in the absence or presence of 1 μg ml−1 hemin, and growth (A and B) was measured via OD600 readings, while P3 activity (corrected for OD600 values) was measured to determine Agr activity (D and E). (C and F) USA300 menD was grown in the absence or presence of 1 μg ml−1 menadione, and growth (C) and P3 activity (F) were measured as described for the hemB mutants. The data represent the means of at least 4 independent experiments, each in triplicate. The error bars were omitted to enhance clarity but were all within 5% of the mean.

FIG 9

FIG 9

Hemin and menadione restore hemolytic activity in hemB and menD mutants, respectively. (A) SH1000 hemB::Tn was grown in the absence or presence of 1 μg ml−1 hemin, and the hemolytic activity of the culture supernatant was determined. Culture supernatant from wild-type SH1000 is included for comparison. (B) USA300 hemB and menD were grown in the absence or presence of 1 μg ml−1 hemin or menadione, and the hemolytic activity of the culture supernatant was determined. Culture supernatant from wild-type USA300 is included for comparison. The data represent the means of 4 experiments done in triplicate, and the error bars represent the standard deviations of the mean. Values that are significantly different (P < 0.05 by Student's t test) from those of the wild type at the same dilution are indicated by asterisks.

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