The Electron Transport Chain Sensitizes Staphylococcus aureus and Enterococcus faecalis to the Oxidative Burst

Abstract

Small-colony variants (SCVs) of Staphylococcus aureus typically lack a functional electron transport chain and cannot produce virulence factors such as leukocidins, hemolysins, or the antioxidant staphyloxanthin. Despite this, SCVs are associated with persistent infections of the bloodstream, bones, and prosthetic devices. The survival of SCVs in the host has been ascribed to intracellular residency, biofilm formation, and resistance to antibiotics. However, the ability of SCVs to resist host defenses is largely uncharacterized. To address this, we measured the survival of wild-type and SCV S. aureus in whole human blood, which contains high numbers of neutrophils, the key defense against staphylococcal infection. Despite the loss of leukocidin production and staphyloxanthin biosynthesis, SCVs defective for heme or menaquinone biosynthesis were significantly more resistant to the oxidative burst than wild-type bacteria in human blood or the presence of purified neutrophils. Supplementation of the culture medium of the heme-auxotrophic SCV with heme, but not iron, restored growth, hemolysin and staphyloxanthin production, and sensitivity to the oxidative burst. Since Enterococcus faecalis is a natural heme auxotroph and cause of bloodstream infection, we explored whether restoration of the electron transport chain in this organism also affected survival in blood. Incubation of E. faecalis with heme increased growth and restored catalase activity but resulted in decreased survival in human blood via increased sensitivity to the oxidative burst. Therefore, the lack of functional electron transport chains in SCV S. aureus and wild-type E. faecalis results in reduced growth rate but provides resistance to a key immune defense mechanism.

Keywords: Enterococcus faecalis; Staphylococcus aureus; bacteremia; enterococcus; neutrophil; oxidative burst; small-colony variant.

FIG 1

Survival of SCV S. aureus in blood is greater than that of wild-type (WT) bacteria. (A) Survival of wild-type S. aureus USA300 in blood from individual donors. Data are the mean survival from three independent experiments from each donor. (B and C) Survival of wild-type S. aureus USA300 and ΔhemB (B) and ΔmenD (C) mutants, and complemented strains, in human blood. Data are the mean of results of four independent experiments using blood from at least three different donors. (D) Images of pelleted stationary-phase S. aureus strains highlighting differences in pigmentation. Images are representative of three independent assays. (E and F) Growth profiles of S. aureus wild-type and ΔhemB (E) and ΔmenD (F) mutants and complemented strains. Data are the mean of results of three independent experiments. Where shown, error bars represent the standard deviations of the mean. Data in panels B and C were analyzed by a two-way repeated-measures analysis of variance (ANOVA) and Sidak's post hoc test. *, P < 0.01, compared with the wild type. Since data points overlap in panels E and F, error bars were omitted for clarity, but standard deviations were within 5% of the mean.

FIG 2

SCVs survive the oxidative burst better than wild-type S. aureus. (A) Survival of wild-type S. aureus USA300 (WT) and ΔhemB and ΔmenD mutants in the presence of PMNs purified from human blood. (B) Percentages of S. aureus USA300 wild-type, ΔhemB, and ΔmenD bacteria internalized into phagocytic cells 2 h after inoculation into whole human blood. (C) Percentages of phagocytic cells that contained S. aureus strains, and had impaired membrane integrity, as determined using the Zombie Violet reagent after 6 h in whole human blood. (D) Survival of S. aureus USA300 wild-type, ΔhemB, and ΔmenD bacteria after 6 h in blood pretreated with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) or an identical volume of DMSO solvent alone (DMSO). In all cases, data are the mean of results of four independent experiments using blood from at least three different donors. Data in panel A were analyzed by a two-way repeated-measures ANOVA and Sidak's post hoc test. *, P < 0.01, compared with the wild type. For panels B to D, data were analyzed via a one-way ANOVA with Tukey's post hoc test. This revealed no significant differences between values in panels B and C. In panel D, an asterisk indicates a P of <0.01 and NS (nonsignificant) indicates a P of >0.05 when the indicated comparisons were made.

FIG 3

Survival of wild-type but not SCV S. aureus is enhanced by Agr. (A) Survival of S. aureus USA300 wild-type (WT), ΔagrA, ΔagrC, and ΔRNAIII strains in whole human blood over 6 h. (B) Survival of S. aureus USA300 ΔagrC transformed with pCL55 (CTL), pCL55 containing the wild-type agrC gene (WT), and three mutated variants of agrC that result in Q305H, M234L, or R238H substitutions conferring a constitutively active phenotype. (C) Survival of S. aureus USA300 wild-type (WT), ΔhemB, ΔhemB ΔagrA, ΔhemB ΔagrC, and ΔhemB ΔRNAIII strains in whole human blood over 6 h. For all panels, data are the mean of results of four independent experiments using blood from at least three different donors. Data were analyzed by a two-way repeated-measures ANOVA with Dunnett's post hoc test to compare strains to the WT (A), CTL (B), or the ΔhemB mutant (C). *, P < 0.01. In panel A, all mutants were significantly more susceptible to immune defenses than the wild type at 4 and 6 h. In panel B, all strains expressing agrC (wild type or mutated) survived better than the ΔagrC mutant at the 4- and 6-h time points. In panel C, all ΔhemB mutants (with or without agr) survived equally well and significantly better than the wild type.

FIG 4

Heme promotes growth and virulence factor production of the ΔhemB mutant but decreases survival in blood. (A) Growth profiles (as determined by OD₆₀₀ readings) of the WT and ΔhemB and ΔmenD mutants in metal-adjusted TSB containing iron in the form of 1 or 10 μM FeCl₃ or 10 μM heme. Note that the open triangles are largely obscured by the filled triangles. (B) Graph showing hemolytic activities of the WT and ΔhemB and ΔmenD mutants grown in the presence of 1 or 10 μM FeCl₃ or 10 μM heme. The panel above the graph illustrates the pigmentation of the ΔhemB mutant grown in the absence or presence of 10 μM heme. The WT is shown for comparison. There was no effect of heme on the pigmentation of the WT or ΔmenD strain. (C) Survival of the WT and ΔhemB and ΔmenD mutants, grown in the presence of 1 or 10 μM FeCl₃ or 10 μM heme, after 6 h of incubation in whole human blood. Data in panels B and C were analyzed via a one-way ANOVA with Tukey's post hoc test. For each strain, comparisons were made between 1 μM FeCl₃ and 10 μM FeCl₃ or 10 μM heme. *, P < 0.01.

FIG 5

Heme promotes susceptibility of E. faecalis to host defenses. (A and B) Growth profiles (as determined by OD₆₀₀ readings) of E. faecalis JH2-2 (A) and OG1X (B) grown in the absence or presence of 10 μM heme. (C and D) Catalase activity (expressed as mM H₂O₂ degraded in 1 h by 10⁷ CFU) of E. faecalis JH2-2 (C) and OG1X (D) grown in the absence or presence of 10 μM heme. (E and F) Survival of E. faecalis JH2-2 (E) and OG1X (F) after 6 h in blood pretreated with DPI or an identical volume of DMSO solvent alone. In each case, data are the mean of results of four experiments in duplicate. For panels E and F, three different blood donors were used. Error bars represent the standard deviations of the mean. Data were analyzed by a one-way ANOVA with Tukey's post hoc test, which revealed significant differences (P < 0.01) in panels C and D between bacteria grown in the absence or presence of heme. In panels E and F, an asterisk indicates a P of <0.01 and NS (nonsignificant) indicates a P of >0.05 when the indicated comparisons were made.