New Results

The electron transport chain sensitises Staphylococcus aureus and Enterococcus faecalis to the oxidative burst

Kimberley L. Painter, Alex Hall, Kam Pou Ha, Andrew M. Edwards
doi: https://doi.org/10.1101/165589

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 anti-oxidant 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 defences is largely uncharacterised. To address this, we measured 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 leukcocidin production and staphyloxanthin biosynthesis, SCVs defective for heme or menquinone biosynthesis were significantly more resistant to the oxidative burst than wild-type bacteria in a whole human blood model. 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 defence mechanism.

Introduction

Staphylococcus aureus is responsible for a raft of different infections of humans and animals [1-3]. The key host defence against infection is the neutrophil, which phagocytoses S. aureus and exposes it to a cocktail of reactive oxygen species (ROS) during a process known as the oxidative (or respiratory) burst [4-6]. Whilst this is often sufficient to clear infection, invasive staphylococcal diseases frequently lead to persistent or recurrent infections of the bones, joints, heart or implanted devices [1, 7–9]. The development of these hard to treat infections is often associated with the presence of small colony variants (SCVs) [10–17]. As the name suggests, SCVs form small colonies on agar plates, typically due to metabolic defects caused by mutations that abrogate the electron-transport chain or biosynthetic pathways [16–21]. For example, several clinical studies have isolated SCVs with mutations in genes required for heme or menaquinone biosynthesis, including from the bloodstream [17–20]. The slow growth of SCVs provides a strong selection pressure for reversion to the wild-type, either by repair of the causative mutation or the acquisition of a suppressor mutation [18,19,22]. This presents challenges to their study and so targeted deletion of genes within the hem or men operons, which confer a phenotype that is identical to that of clinical SCVs, has been used to enable their study without the problem of reversion to the wild-type [23–26], SCVs can also arise in the absence of mutation, resulting in a very unstable phenotype, although the molecular basis for this is unknown [27], The emergence of SCVs is a rare but consistent consequence of S. aureus replication, which generates a small sub-population of the variants [22]. However, SCV emergence is significantly increased in response to diverse environmental stresses including antibiotics, reactive oxygen species, low pH within host cell vacuoles and exoproducts from Pseudomonas, which frequently causes co-infections with S. aureus [26–33].

Despite their diverse molecular basis, most SCVs have similar phenotypic characteristics. For example, activity of the Agr quorum-sensing system is weak or absent, and therefore cytolytic toxin production is negligible whilst surface proteins are strongly expressed [25,34–36]. These properties enable SCVs to persist in non-immune host cells and form robust biofilms, which has been hypothesised to contribute to their ability to persist in host tissues [27,37–39]. Furthermore, SCVs are typically resistant to antibiotics including the aminoglycosides, sulphonamides or fusidic acid and are often less susceptible to other antibiotics compared to wild-type bacteria [40–44].

Whilst these phenotypic properties very likely contribute to staphylococcal persistence in the host, the ability of SCVs to resist phagocytic cells, the key host defence against S. aureus, is poorly understood. Although SCVs are resistant to the ROS H2O2, they lack several defences used by wild-type bacteria to protect against immune cells [26]. For example, staphyloxanthin pigment, which promotes wild-type survival of both the oxidative burst and antimicrobial peptides, is absent in SCVs [15,18,45–47]. Furthermore, wild-type bacteria secrete numerous cytolytic toxins that kill neutrophils and enable bacterial survival, but this is absent in SCVs [15,18,25,34]. SCVs also exhibit reduced coagulase activity and some isolates lack catalase, both of which have been linked to survival of wild-type bacteria in the host [15,18,26,48–50]. Therefore, the effect of a defective electron transport chain on the susceptibility of SCV S. aureus to the oxidative burst of neutrophils is unclear.

Enterococcus faecalis, another major cause of bloodstream infections, shares some of the phenotypic properties of S. aureus SCVs since it is naturally defective for heme production and therefore lacks a functional electron-transport chain [51–53]. However, E. faecalis encodes type a and b cytochromes, and the presence of exogenous heme promotes E. faecalis growth in air, confirming the presence of an otherwise intact respiratory chain [51–53]. Exogenous heme also restores catalase activity, which has been shown to promote H2O2 resistance [54–55]. As such, it is unclear what whether E. faecalis gains an advantage from being defective for heme biosynthesis, particularly with respect to host defences that generate reactive oxygen species such as neutrophils.

Therefore, the aim of this work was to determine how the absence of the electron transport chain affects the survival of S. aureus and E. faecalis exposed to the oxidative burst of neutrophils.

Methods

Bacterial strains and culture conditions

The bacterial strains used in this study are detailed in table 1. Staphylococci were grown in tryptic soy broth (TSB) at 37 °C with shaking (180 RPM) for 18 h to late stationary phase. Enterococci were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) at 37 °C with shaking (180 RPM) for 18 h to late stationary phase. For assays involving human blood, bacteria were plated onto Columbia blood agar (CBA) or THY supplemented with 5% sterile defibrinated sheep’s blood to neutralise any remaining oxidants from the assay. For some experiments iron (and other cations) was removed from TSB (100 ml) by incubation with Chelex resin (6 g) for 16 h at 4 °C with stirring. The following individual metals were then replaced: ZnCI2 (25 µM), CaCI2 (1 mM), MgCI2 (1 mM), MnCI2 (25 µM). Iron was added in the form of FeCI3 (1 or 10 µM) or heme (10 µM).

View this table:
Table 1. Bacterial strains and plasmids used in this study.

Genetic manipulation of S. aureus

The construction of ΔmenD, ΔhemB ΔagrA, ΔagrC and ΔRNAIII mutants was achieved using pIMAY as described previously [25,26,56]. To construct the double ΔhemBΔagr mutants, the three agr mutants (ΔagrA, ΔagrC and ΔRNAIII) were made electrocompetent and the hemB gene deleted using pIMAY as described previously [25].

Mutants lacking ΔhemB or ΔmenD were complemented with pCL55 containing the relevant gene under the control of the hem or men operon promoters respectively [26]. To control for pleiotropic effects of plasmid insertion into geh, pCL55 alone was transformed into hemB and menD mutant strains. The ΔagrC mutant was complemented with pCN34 containing a copy of the agrC gene under the control of the agr P3 promoter, and pCN34 alone (pEmpty) was used to control for pleitropic effects of the plasmid. In addition to wild-type agrC, plasmids containing mutated forms of agrC which confer a constitutively active phenotype were also transformed into the ΔagrC mutant strain [57].

Whole human blood survival assay

The survival of bacteria in whole human blood was done as described previously [58]. Ethical approval for drawing and using human blood was obtained from the Regional Ethics committee and Imperial NHS trust tissue bank (REC Wales approval: 12/WA/0196, ICHTB HTA licence: 12275). Blood was drawn from healthy human donors into tubes containing EDTA and used immediately in assays based on a previously described protocol [4]. Suspensions of bacteria (105 CFU in 10 µl PBS) were mixed with blood (90 µl) and incubated for up to 6 h at 37 °C with mixing. At indicated time points aliquots were taken, diluted serially in PBS and plated onto CBA plates to enumerate CFU. In some assays blood was pre-treated (10 min) with Diphenyleneiodonium (DPI) or an identical volume of DMSO alone to control for solvent effects [4].

Measurement of bacterial growth

Stationary-phase bacteria were diluted 1:50 into a final volume of 200 µl TSB in microtitre plates (Corning) before incubation at 37 °C with shaking (500 RPM) in a POLARstar Omega multiwell plate reader. Bacterial growth was measured using OD600 measurements every 30 min for a total of 17 h [57].

Hemolysin production

The hemolytic activity of bacterial culture supernatants was determined as described previously [25]. Briefly, culture supernatants were recovered by centrifugation (13,000 X g, 10 min) of stationary-phase cultures. The supernatant was then diluted in 2-fold steps using fresh TSB. Aliquots from each dilution (100 µl) were mixed with an equal volume of 2 % sheep blood suspension in PBS and incubated at 37 °C for 1 h in a static incubator. Subsequently, unlysed blood cells were removed by centrifugation and the supernatant containing lysed erythrocytes transferred to a new microtitre plate. The degree of erythrocyte lysis was quantified by measuring the absorbance of the supernatant at A450 and reference to controls. Erythrocytes incubated with TSB alone or TSB containing 1 % TX-100 served as negative and positive controls respectively.

Measurement of phagocytosis and immune cell viability

Phagocytosis of bacteria in whole human blood was determined using a protocol based on that described previously [59]. Stationary-phase bacteria (1 ml) were pelleted (17,000 X g, 3 min) and washed twice with PBS. The pellet was then resuspended in 200 µl of 1.5 mM Fluorescein isothiocyanate (FITC) dissolved in freshly prepared carbonate buffer (0.05 M NaCO3 and 0.1 M NaCI). Bacteria were then incubated for 60 min (room-temperature with tumbling) in the dark. FITC-labelled bacteria were then washed three times in carbonate buffer and adjusted to 1 × 106 CFU ml-1 in PBS. FITC-labelled bacteria (10 µl, 1 × 104 CFU) were added to 96-well plates prior to the addition of 90 µl of freshly isolated blood, as described for the whole blood killing assay. At each time point (0, 2, 4 and 6 h), the blood/bacteria mixture (100 µl) was added to 900 µl red blood cell lysis solution (eBioscience) and incubated at room temperature in the dark for 10 min. Samples were then centrifuged (500 × g, 10 min) and the resulting pellet washed once in PBS (1 ml) before a final centrifugation step (500 × g, 10 min) and then the pellet containing immune cells and bacteria was resuspended in 100 µl PBS or 1% paraformaldehyde (PFA; Affymetrix) if no further staining was required. Where samples were to be analysed for host cell death, samples were incubated in PBS containing the Zombie Violet live-dead dye (Biolegend) at a 1:500 dilution in the dark. Free primary amine groups were quenched using 1.4 ml 1% bovine serum albumin (BSA) and samples were centrifuged (500 × g, 10 min) before resuspension in 100 µl 1% PFA. Positive controls were generated by heat-killing host cells (100 °C, 10 min) prior to Zombie staining. Samples were then fixed overnight (12-16 h) in 1% paraformaldehyde at 4 °C. Immune cell/bacteria samples were analysed on a Fortessa flow cytometer (BD) and at least 10,000 events were captured. Green (FITC-bacteria) and violet (Zombie-labelled host cells) fluorescence were detected at 488/530 (30) nm and 404/450 nm, respectively. Based on preliminary analyses and using the methodology of Surewaard et al. (2013) [60], free bacteria (i.e. bacteria not phagocytosed) were identified as events with a side scatter of < 50K. By contrast, host cells were identified as events with a side scatter of > 50K. Samples were analysed alongside controls, which consisted of bacteria without FITC labelling, host cells with or without Zombie stain, uninfected host cells and heat-killed host cells as appropriate. Data were analysed using FlowJo software (Version 10). Compensation was not necessary as the spectra of the fluorescent signals did not overlap.

Catalase assay

Catalase activity of bacterial cells was determined as described previously [26]. Overnight bacterial cultures (1 ml) were washed three times in PBS and 107 CFU added to 100 µM H2O2 in PBS (1 ml). Bacteria were incubated in the H2O2 in the dark at 37 °C. At the start of the assay and every 15 min, 200 µl of sample was pelleted (17,000 × g) and 20 µl added to a 96 well microtitre plate. The concentration of remaining H2O2 was determined using a Pierce Quantitative Peroxide Assay (Aqueous Compatible) kit.

Results

The loss of the electron transport chain promotes survival of S. aureus in human blood

To study the susceptibility of electron transport chain-deficient SCVs to the oxidative burst, we employed the well-established ex vivo whole human blood model of infection. This is model is appropriate because S. aureus is a major cause of bacteraemia and blood contains a high density of neutrophils, as well as the required opsonins and other relevant immune factors such as platelets [4,61,62]. In this model system, S. aureus is rapidly phagocytosed by neutrophils and exposed to the oxidative burst [4,61,62].

Freshly-drawn human blood containing anti-coagulant (EDTA) was incubated with wild-type S. aureus USA300, or mutants with deletions of hemB or menD, and survival determined over time by CFU counts. Preliminary experiments determined that individual donors had slightly different anti-staphylococcal activity and so at least 3 different donors were used for each experiment (Fig. 1A). However, for each of the 5 donors we observed a consistent decrease in CFU counts of wild-type bacteria over time with just 1-5% of the inoculum surviving after 6 h (Fig. 1A). By contrast, SCVs defective for heme-or menaquinone-biosynthesis survived at much higher levels than the wild type over the entire duration of the assay with 70% of the ΔhemB mutant inoculum and 69% of ΔmenD viable after 6 h incubation in blood (Fig. 1B,C).

Figure 1.
Figure 1. Survival of SCV S. aureus in blood is greater than that of wild-type bacteria.

(A) Survival of wild-type S. aureus USA300 in blood from individual donors. Data represent the mean survival from 3 independent experiments from each donor. (B, C) Survival of wild-type S. aureus USA300 and ΔhemB (B) or ΔmenD (C), and complemented strains, in human blood. Data represent the mean of 4 independent experiments using blood from at least 3 different donors. (D) Images of pelleted stationary-phase S. aureus strains highlighting differences in pigmentation. Images are representative of 3 independent assays. (E, F) Growth profiles of S. aureus wild-type and ΔhemB (E) or ΔmenD (F), and complemented strains. Data represent the mean of 3 independent experiments. Where shown, error bars represent the standard deviation of the mean. Data in B and C were analysed by 2-way repeated measures ANOVA and Dunnett’s post-hoc test. *represents p = <0.01 compared with wild-type.

Complementation of the hemB or menD mutations conferring the SCV phenotype restored the wild-type phenotype for growth and staphyloxanthin production, and resulted in significantly decreased survival in blood (Fig. 1B,C,D,E,F). This confirmed that enhanced SCV survival in blood was due to the loss of heme or menaquinone biosynthesis, rather than the acquisition of adventitious mutations during genetic manipulation. Therefore, despite the lack of staphyloxanthin pigment and cytolysin production, loss of the electron transport chain confers a survival advantage to S. aureus in blood.

Wild-type S. aureus is more sensitive to the oxidative burst than SCVs

Having demonstrated that survival of SCVs in blood is greater than that of the wild-type, we sought to understand why. Several previous studies have shown that incubation of S. aureus in whole human blood results in rapid phagocytic uptake of the bacterium by polymorphonuclear leukocytes (PMNs) [4,61,62]. We confirmed those findings and found no differences in the phagocytosis of wild-type, ΔhemB or ΔmenD mutants (Fig. 2A). We also demonstrated that the viability of neutrophils that phagocytosed S. aureus did not vary between wild-type and SCVs (Fig. 2B). Therefore, both immune evasion and killing of immune cells by SCVs were ruled out as an explanation for their ability to survive in human blood.

Figure 2.
Figure 2. SCVs survive the oxidative burst better than wild-type S. aureus.

(A) The percentage of S. aureus wild-type, ΔherriB, or ΔmenD 5. aureus USA300 internalised into phagocytic cells 2 h after inoculation into whole human blood. (B) The percentage 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. (C) Survival of S. aureus wild-type, ΔhemB, or ΔmenD S. aureus USA300 after 6 h in blood pre-treated with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) or an identical volume of DMSO solvent alone (DMSO). Data were analysed via a one-way ANOVA with Tukey’s post hoc test, which revealed no significant differences in (A) or (B). In (C), * indicates p = < 0.01, NS indicates p = > 0.05 when the indicated comparisons were made.

The principle mechanism by which neutrophils kill S. aureus is the oxidative burst [4–6]. To confirm that this was the case in our model system we measured bacterial viability in human blood treated with diphenyleneiodonium (DPI), which blocks the oxidative burst, or the DMSO solvent alone. Suppression of NADPH with DPI, but not DMSO alone, resulted in significantly elevated survival of wild-type S. aureus, confirming that the oxidative burst is the key defence against S. aureus in human blood (Fig. 2C) [4–6]. The addition of DPI to blood did not significantly alter SCV CFU counts, since survival was already very high (Fig. 2C). Therefore, SCV S. aureus appears to be significantly less susceptible to the oxidative burst than wild-type bacteria. This is in agreement with our previously reported finding that both the ΔhemB and ΔmenD SCVs were more resistant to H2O2 than wild-type bacteria, and provides an explanation for the increased survival of SCVs in blood [26].

Agr activity promotes the survival of wild-type but not SCV S. aureus in blood

Although Agr-regulated toxins have been shown to kill neutrophils, several clinical studies have shown an association of Agr dysfunction with persistent bacteremia [63]. Therefore, we considered the possibility that the weak Agr activity of SCVs contributed to their survival in blood.

To test this, we compared the survival of wild-type and Agr-defective strains in whole human blood. This revealed a significantly greater loss of viability of agr mutants compared with the wild-type (Fig. 3A). In particular, mutants lacking quorum-sensing components of Agr (ΔagrA or ΔagrC) were approximately 4-fold more susceptible to immune cells in blood than the wild-type, whilst the RNAIII mutant was 2-fold more susceptible than the wild-type (Fig. 3A). This finding is in keeping with previous work that showed that AgrA-regulated PSMs contribute to survival of S. aureus within the phagocytic vacuole of neutrophils, in addition to RNAIII-regulated toxins [60]. Therefore, a functional Agr system promotes the survival of wild-type bacteria in human blood.

Figure 3.
Figure 3. Survival of wild-type but not SCV S. aureus is enhanced by Agr.

(A) Survival of wild-type (WT), ΔagrA, ΔagrC or ΔRNAIII S. aureus USA300 in whole human blood over 6 h. (B) Survival of S. aureus USA300 ΔagrC mutant transformed with pCL55 (CTL), pCL55 containing the wild-type agrC gene (WT), or 3 mutated variants of agrC that result in Q305H, M234L or R238H substitutions conferring a constitutively-active phenotype. (C) Survival of wild-type (WT), ΔhemB, ΔhemBΔagrA, ΔhemBΔagrC or ΔhemBΔRNAIII S. aureus USA300 in whole human blood over 6 h. For all panels, data represent the mean of 4 independent experiments using blood from at least 3 different donors. Data were analysed by 2-way repeated measures ANOVA with Dunnett’s post-hoc test to compare strains to WT (A), CTL (B) or to ΔhemB (C). * indicates p = < 0.01. In panel A, all mutants were significantly more susceptible to immune defences 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 (+/-agr) survived equally well and significantly better than the wild-type.

Complementation of the ΔagrC mutant with a wild-type copy of the gene increased survival in blood (Fig. 3B). However, complementation of ΔagrC with mutant copies of agrC which confer constitutive Agr activity, even in the presence of serum [57], did not promote bacterial survival above that of the wild-type gene (Fig. 3B).

Although Agr activity is extremely weak in SCVs, we explored whether this contributed to their survival by generating ΔhemB mutants defective for agrA, agrC or RNAIII, and measuring their survival in blood (Fig. 3C). This revealed that survival of each of the ΔhemBΔagr-mutants was as high as for the ΔhemB mutant with an intact agr operon. Therefore, whilst loss of Agr activity in the wild-type reduces survival in human blood, the lack of Agr activity in SCVs is not detrimental for their survival. This indicates that toxin production is an important mechanism by which wild-type S. aureus survives phagocytosis. By contrast, since SCVs can survive the oxidative burst they do not need toxins to survive phagocytosis.

Restoration of the electron transport chain with heme results in decreased survival of SCVs in blood

During infection, S. aureus acquires iron from the host, predominantly via the acquisition of heme liberated from erythrocytes via hemolytic toxins [64]. In addition to acting as an iron source, heme can also be utilised by heme-auxotrophic SCVs to restore the electron transport chain [18,26]. To determine how heme influenced the phenotype of heme- and menquinone-defective SCVs, and their susceptibility to the oxidative burst, we grew wild-type or SCV S. aureus in media deficient for heme and containing minimal free iron (1 µM FeCI3), abundant iron (10 µM FeCI3), or in the presence of heme (10 µM).

The growth rate of wild-type S. aureus was not significantly affected by the presence of the higher concentration of FeCI3 or heme, although the latter led to a slight increase in the length of the lag phase (Fig. 4A). Similarly, abundant iron did not affect growth of the ΔmenD SCV, but heme caused slight growth retardation (Fig. 4A). By contrast, abundant iron slightly promoted the growth rate of the ΔhemB SCV, whilst heme enhanced the growth almost to wild-type levels (Fig. 4A). In addition to the growth rate, heme supplementation restored hemolytic activity and staphyloxanthin biosynthesis to the ΔhemB mutant (Fig. 4B). However, heme supplementation of the ΔhemB mutant also resulted in significantly increased susceptibility to the oxidative burst of neutrophils in blood (Fig. 4C), which is in keeping with our previous finding that heme supplementation renders heme-auxotrophic SCVs sensitive to H2O2 [26]. By contrast, supplementation of the medium with iron had no effect on susceptibility of the ΔhemB mutant to the oxidative burst or H2O2 (Fig. 4C). This is in agreement with previous work showing that iron-loading of S. aureus does not alter susceptibility to the oxidative burst of neutrophils [65,66]

Figure 4.
Figure 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 WT, ΔherriB and ΔmenD in metal-adjusted TSB containing iron in the form of 1 or 10 µM FeCI3 or 10 µM heme. Please note that the open triangles are largely obscured by the filled triangles. (B) the graph shows hemolytic activity of WT, ΔhemB and ΔmenD grown in the presence of 1 or 10 µM FeCI3 or 10 µM heme. The panel illustrates the pigmentation of the ΔhemB mutant grown in the absence or presence of 10 µM heme. WT is shown for comparison. There was no effect of heme on the pigmentation of the WT or ΔmenD strain. (C) Survival of WT, &hemB and hmenD, grown in the presence of 1 or 10 µM FeCI3 or 10 µM heme, after 6 h incubation in whole human blood. Data (B) and (C) were analysed via a one-way ANOVA with Tukey’s post hoc test. For each strain, comparisons were made between 1 µM FeCI3 and 10 µM FeCI3 or 10 µM heme. * indicates p = <0.01.

By contrast to the ΔhemB mutant, the susceptibility of both the wild-type and ΔmenD mutant to the oxidative burst was unchanged by growth in the presence of heme. Therefore, at the concentration used (10 µM), heme does not directly sensitise S. aureus to the oxidative burst. Rather, it is the restoration of the electron-transport chain in the ΔhemB mutant that confers sensitivity to the oxidative burst.

The absence of an electron-transport chain enables survival of Enterococcus faecalis in human blood

The elevated survival of the S. aureus ΔhemB mutant, relative to wild-type, led us to consider whether a similar phenomenon occurred with Enterococcus faecalis, which despite producing cytochromes lacks a functional electron transport chain due to an inability to synthesise heme [51–53]. However, E. faecalis employs heme uptake systems to scavenge heme from the environment and therefore supplementation of the culture medium with heme results in increased growth under aerobic conditions. We confirmed this in two different E. faecalis strains (Fig. 5A,B), which grew to a higher optical density in the presence of heme. In addition, E. faecalis grown in the presence of heme produce a functional catalase, which we observed in both of the strains examined (Fig. 5C,D). However, as observed for the ΔhemB SCV, growth of E. faecalis in the presence of heme led to significantly diminished survival in human blood by increasing sensitivity to the oxidative burst (Fig. 5E,F). Therefore, as for SCV S. aureus, the absence of the electron-transport chain in E. faecalis promotes survival in the bloodstream by reducing sensitivity to oxidative stress generated by host immune cells.

Figure 5.
Figure 5. Heme promotes susceptibility of E. faecalis to host defences.

(A,B) growth profiles (as determined by OD600 readings) of E. faecalis JH2-2 (A) or OG1X (B) grown in the absence or presence of 10 µM heme. (C,D) Catalase activity (expressed as mM H2O2 degraded in 1 hr by 107 CFU) of E. faecalis JH2-2 (C) or OG1X (D) grown in the absence or presence of 10 µM heme. (E,F) Survival of E. faecalis JH2-2 (E) or OG1X (F) after 6 h in blood pre-treated with DPI or an identical volume of DMSO solvent alone. Data were analysed by one-way ANOVA with Tukey’s post hoc test, which revealed no significant differences (p = < 0.01) in (A) and (B) between bacteria grown in the absence or presence of heme. In (E) and (F), * indicates p = < 0.01, NS indicates p = > 0.05 when the indicated comparisons were made.

Discussion

During infection S. aureus faces two major threats: host defences and antibiotic therapy. Previous work has shown that SCVs of S. aureus are less susceptible to antibiotics than wild-type bacteria. Our data demonstrate that the SCV S. aureus is also less susceptible to host immune defences. These data fit with a previous study that revealed that SCVs are less sensitive than wild-type to host-derived antimicrobial peptides [67]. However, the resistance of SCVs to both the oxidative burst and AMPs is surprising given the lack of staphyloxanthin pigment, which contributes to resistance of wild-type S. aureus to both ROS and AMPs [4,68].

We do not currently understand the molecular basis of ROS resistance in SCVs. However, the damaging effects of ROS are proposed to occur via the Fenton reaction, which involves the reaction of H2O2 with free iron leading to the generation of highly-reactive hydroxyl radicals [69,70]. The lack of an electron-transport chain, together with the associated decreased TCA activity (which utilises iron-containing enzymes such as aconitase) in SCVs is hypothesised to result in decreased iron content relative to wild-type bacteria. Furthermore, there is evidence that the electron transport chain generates superoxide radicals that liberate iron from iron-sulphur clusters, making it available for the Fenton reaction [71].

The ability of S. aureus SCVs to survive the oxidative burst comes at a cost. The electron-transport chain enables aerobic respiration, rapid bacterial growth and toxin production. These toxins include hemolysins that enable S. aureus to access heme, the bacterium’s primary source of iron during infection [72]. Therefore, the absence of hemolysin production by the ΔhemB mutant enables maintenance of the SCV phenotype in the presence of red blood cells. The menaquinone-defective SCV cannot restore the wild-type phenotype using host-derived materials and therefore maintains its phenotype regardless of hemolysin production.

E. faecalis lacks the necessary biosynthetic machinery to synthesise heme making it a heme auxotroph [51–53]. However, some strains secrete a cytolysin with hemolytic activity that provides a mechanism of heme acquisition[73]. The liberation of heme from erythrocytes would be expected to promote growth and restore catalase activity, but would also increase susceptibility to host defences. The maintenance of cytochromes and catalase that are restored by exogenous heme suggests that heme acquisition is a consistent and beneficial event during colonisation and/or infection. What is not clear however, is when and where heme acquisition occurs. For example, isolates recovered from patients with infective endocarditis, an infection of the heart valves that persists despite a robust immune response, are typically defective for hemolysin production [73,74]. This may indicate that hemolysin production, and thus heme acquisition, is undesirable at this site. By contrast, 30-40% of E. faecalis isolates carried in the gut or isolated from urinary-tract infections are hemolytic [74]. However, further work is needed to understand the basis for this observation and whether heme-mediated susceptibility to the oxidative burst plays a role.

Previous work reported that heme supplementation enabled E. faecalis to survive H2O2 challenge by restoring catalase activity [54–55]. However, whilst we also observed restoration of catalase activity in E. faecalis supplied with heme, this did not correlate with increased resistance to the oxidative burst. We have shown previously that the ΔhemB mutant is catalase-deficient but was much less susceptible to the oxidative burst than wild-type bacteria [26]. This indicates that catalase is not required for survival of the oxidative burst, a finding that is supported by previous work with S. aureus that showed a catalase mutant was as virulent as the wild-type [75].

In summary, SCV S. aureus sacrifices fast growth and toxin production for enhanced resistance to host defences and antibiotics. This dramatic change in phenotype may enable the transition from highly-damaging, acute infection to a less pathogenic but persistent infection type. Our data indicate that the lack of heme production in E. faecalis also promotes survival in human blood, suggesting a common survival mechanism between these two pathogens.

Acknowledgements

The following are gratefully acknowledged for providing bacterial strains, phage or reagents: Ruth Massey (University of Bath), Malcolm Horsburgh (University of Liverpool), Tim Foster (Trinity College Dublin), Angela Nobbs (University of Bristol), and the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) Program: under NIAID/NIH Contract No. HHSN272200700055C. A.M.E. acknowledges funding from the Royal Society, Department of Medicine (Imperial College), and from the Imperial NIHR Biomedical Research Centre, Imperial College London. K.L.P. was supported by a PhD studentship from the Faculty of Medicine, Imperial College London. K.P.H. is supported by an MRC-funded PhD studentship awarded to the Centre for Molecular Bacteriology and Infection, Imperial College London.

References

  1. 1.↵
    Lowy FD. 1998. Staphylococcus aureus infections. N Engl J Med 339:520–532.
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.
    Gordon RJ, Lowy FD. 2008. Pathogenesis of methicillin-resistant Staphylococcus aureus infection. Clin Infect Dis. 5:S350–359.
    OpenUrlCrossRef
  3. 3.↵
    Aires-de-Sousa M. 2017. Methicillin-resistant Staphylococcus aureus among animals: current overview. Clin Microbiol Infect 23:373–380.
    OpenUrlCrossRef
  4. 4.↵
    Liu GY, Essex A, Buchanan JT, Datta V, Hoffman HM, Bastian JF, Fierer J, Nizet V. 2005. Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity. J Exp Med 202:209–215.
    OpenUrlAbstract/FREE Full Text
  5. 5.
    Buvelot H, Posfay-Barbe KM, Linder P, Schrenzel J, Krause KH. 2017. Staphylococcus aureus, phagocyte NADPH oxidase and chronic granulomatous disease. FEMS Microbiol Rev 41:139–157.
    OpenUrlCrossRef
  6. 6.↵
    Ellson CD, Davidson K, Ferguson GJ, O’Connor R, Stephens LR, Hawkins PT. 2006. Neutrophils from p40phox-/-mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing. J Exp Med. 203:1927–1937.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Chen CJ, Su LH, Lin TY, Huang YC. 2010. Molecular analysis of repeated methicillin-resistant Staphylococcus aureus infections in children. PLoS ONE 5: e14431.
    OpenUrlCrossRefPubMed
  8. 8.
    Sreeramoju P, Porbandarwalla NS, Arango J, Latham K, Dent DL, Stewart RM, Patterson JE. 2011. Recurrent skin and soft tissue infections due to methicillin-resistant Staphylococcus aureus requiring operative debridement. Am J Surg 201:216–220.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Peyrani P, Allen M, Seligson D, Roberts C, Chen A, Haque N, Zervos M, Wiemken T, Harting J, Christensen D, Ramirez R. 2012. Clinical outcomes of osteomyelitis patients infected with methicillin-resistant Staphylococcus aureus USA-300 strains. Am J Orthop 41:117–122.
    OpenUrlPubMed
  10. 10.↵
    Abele-Horn M, Schupfner B, Emmerling P, Waldner H, Göring H. 2000. Persistent wound infection after herniotomy associated with small-colony variants of Staphylococcus aureus. Infection 2: 53–54.
    OpenUrlCrossRef
  11. 11.
    Acar JF, Goldstein FW, Lagrange P. 1978. Human infections caused by thiamine-or menadione-requiring Staphylococcus aureus. J Clin Microbiol 8:142–147.
    OpenUrlAbstract/FREE Full Text
  12. 12.
    Agarwal H, Verrall R, Singh SP, Tang YW, Wilson G. 2007. Small colony variant Staphylococcus aureus multiorgan infection. Pediatr Infect Dis J 26:269–271.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.
    Besier S, Ludwig A, Ohlsen K, Brade V, Wichelhaus TA. 2007. Molecular analysis of the thymidine-auxotrophic small colony variant phenotype of Staphylococcus aureus. Int J Med Microbiol 297:217–225.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.
    Kahl B, Herrmann M, Everding AS, Koch HG, Becker K, Harms E, Proctor RA, Peters G. 1998. Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J Infect Dis 177:1023–1029.
    OpenUrlCrossRefPubMedWeb of Science
  15. 15.↵
    Kahl BC. 2014. Small colony variants (SCVs) of Staphylococcus aureus - A bacterial survival strategy. Infect Genet Evol 21:515–522.
    OpenUrlCrossRefPubMed
  16. 16.↵
    Proctor RA, van Langevelde P, Kristjansson M, Maslow JN, Arbeit RD. 1995. Persistent and relapsing infections associated with small-colony variants of Staphylococcus aureus. Clin Infect Dis 20:95–102.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    Kim NH, Kang YM, Han WD, Park KU, Park KH, Yoo JI, Lee DG, Park C, Song KH, Kim ES, Park SW, Kim NJ, Oh MD, Kim HB. 2016. Small-Colony Variants in Persistent and Recurrent Staphylococcus aureus Bacteremia. Microb Drug Resist 22:538–544.
    OpenUrlCrossRef
  18. 18.↵
    Proctor RA, von Eiff C, Kahl BC, Becker K, McNamara P, Herrmann M, Peters G. 2006. Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4:295–305.
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    Dean MA, Olsen RJ, Long SW, Rosato AE, Musser JM. 2014. Identification of Point Mutations in Clinical Staphylococcus aureus Strains that Produce Small Colony Variants Auxotrophic for Menadione. Infect Immun 82:1600–1605.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    Lannergård J, von Eiff C, Sander G, Cordes T, Seggewiss J, Peters G, Proctor RA, Becker K, Hughes D. 2008. Identification of the genetic basis for clinical menadione-auxotrophic small-colony variant isolates of Staphylococcus aureus. Antimicrob Agents Chemother 52:4017–4022.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    Kriegeskorte A, Block D, Drescher M, Windmüller N, Mellmann A, Baum C, Neumann C, Lorè NI, Bragonzi A, Liebau E, Hertel P, Seggewiss J, Becker K, Proctor RA, Peters G, Kahl BC. 2014. Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression. MBio 5:e01447–14.
    OpenUrlCrossRefPubMed
  22. 22.↵
    Edwards AM. 2012. Phenotype switching is a natural consequence of Staphylococcus aureus replication. J Bacteriol 194:5404–5412.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    von Eiff C, Heilmann C, Proctor RA, Woltz C, Peters G, Götz F. 1997. A site-directed Staphylococcus aureus hemB mutant is a small-colony variant which persists intracellularly. J Bacteriol 179:4706–4012.
    OpenUrlAbstract/FREE Full Text
  24. 24.
    Bates DM, von Eiff C, McNamara PJ, Peters G, Yeaman MR, Bayer AS, Proctor RA. 2003. Staphylococcus aureus menD and hemB mutants are as infective as the parent strains, but the menadione biosynthetic mutant persists within the kidney. J Infect Dis 187:1654–1661.
    OpenUrlCrossRefPubMed
  25. 25.↵
    Pader V, James EH, Painter KL, Wigneshweraraj S, Edwards AM. 2014. The agr quorum-sensing system regulates fibronectin binding but not hemolysis in the absence of a functional electron transport chain. Infect Immun 82:4337–4347.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    Painter KL, Strange E, Parkhill J, Bamford KB, Armstrong-James D, Edwards AM. 2015. Staphylococcus aureus adapts to oxidative stress by producing H2O2-resistant small-colony variants via the SOS response. Infect Immun. 83:1830–1844.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    Tuchscherr L, Medina E, Hussain M, Völker W, Heitmann V, Niemann S, Holzinger D, Roth J, Proctor RA, Becker K, Peters G, Löffler B. 2011. Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection. EMBO Mol Med 3:129–141.
    OpenUrlAbstract/FREE Full Text
  28. 28.
    Vesga O, Groeschel MC, Otten MF, Brar DW, Vann JM, Proctor RA. 1996. Staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu. J Infect Dis 173:739–742.
    OpenUrlCrossRefPubMedWeb of Science
  29. 29.
    Hoffman LR, Dèziel E, D’Argenio DA, Lépine F, Emerson J, McNamara S, Gibson RL, Ramsey BW, Miller SI. 2006. Selection for Staphylococcus aureus small-colony variants due to growth in the presence of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 103:19890–19895.
    OpenUrlAbstract/FREE Full Text
  30. 30.
    Massey RC, Buckling A, Peacock SJ. 2001. Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus. Curr Biol 11:1810–1814.
    OpenUrlCrossRefPubMedWeb of Science
  31. 31.
    Schaaff F, Bierbaum G, Baumert N, Bartmann P, Sahl HG. 2003. Mutations are involved in emergence of aminoglycoside-induced small colony variants of Staphylococcus aureus. Int J Med Microbiol 293:427–435.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.
    Vestergaard M, Paulander W, Ingmer H. 2015. Activation of the SOS response increases the frequency of small colony variants. BMC Res Notes. 8:749.
    OpenUrlCrossRef
  33. 33.↵
    Leimer N, Rachmühl C, Palheiros Marques M, Bahlmann AS, Furrer A, Eichenseher F, Seidl K, Matt U, Loessner MJ, Schuepbach RA, Zinkernagel AS. 2016. Nonstable Staphylococcus aureus Small-Colony Variants Are Induced by Low pH and Sensitized to Antimicrobial Therapy by Phagolysosomal Alkalinization. J Infect Dis 213:305–313.
    OpenUrlCrossRefPubMed
  34. 34.↵
    Moisan H, Brouillette E, Jacob CL, Langlois-Bégin P, Michaud S, Malouin F. 2006. Transcription of virulence factors in Staphylococcus aureus small-colony variants isolated from cystic fibrosis patients is influenced by SigB. J Bacteriol 188:64–76.
    OpenUrlAbstract/FREE Full Text
  35. 35.
    Mitchell G, Fugère A, Pépin Gaudreau K, Brouillette E, Frost EH, Cantin AM, Malouin F. 2013. SigB is a dominant regulator of virulence in Staphylococcus aureus small colony variants. PLoS One 8: e65018.
    OpenUrlCrossRefPubMed
  36. 36.↵
    Vaudaux P, Francois P, Bisognano C, Kelley WL, Lew DP, Schrenzel J, Proctor RA, McNamara PJ, Peters G, Von Eiff C. 2002. Increased expression of clumping factor and fibronectin-binding proteins by hemB mutants of Staphylococcus aureus expressing small colony variant phenotypes. Infect Immun 70:5428–5437.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    von Eiff C, Becker K, Metze D, Lubritz G, Hockmann J, Schwarz T, Peters G. 2001. Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with Darier’s disease. Clin Infect Dis 32:1643–1647.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.
    Singh R, Ray P, Das A, Sharma M. 2010. Enhanced production of exopolysaccharide matrix and biofilm by a menadione-auxotrophic Staphylococcus aureus small-colony variant. J Med Microbiol 59: 521–527.
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    Tuchscherr L, Heitmann V, Hussain M, Viemann D, Roth J, von Eiff C, Peters G, Becker K, Löffler B. 2010. Staphylococcus aureus small-colony variants are adapted phenotypes for intracellular persistence. J Infect Dis 202:1031–1040.
    OpenUrlCrossRefPubMed
  40. 40.↵
    Baumert N, von Eiff C, Schaaff F, Peters G, Proctor RA, Sahl HG. 2002. Physiology and antibiotic susceptibility of Staphylococcus aureus small colony variants. Microb Drug Resist 8:253–260.
    OpenUrlCrossRefPubMedWeb of Science
  41. 41.
    Brouillette E, Grondin G, Lefebvre C, Talbot BG, Malouin F. 2004. Mouse mastitis model of infection for antimicrobial compound efficacy studies against intracellular and extracellular forms of Staphylococcus aureus. Vet Microbiol 101:253–262.
    OpenUrlCrossRefPubMed
  42. 42.
    Norstrom T, Lannergard J, Hughes D. 2007. Genetic and phenotypic identification of fusidic acid-resistant mutants with the small-colony-variant phenotype in Staphylococcus aureus. Antimicrob Agents Chemother 51:4438–4446.
    OpenUrlAbstract/FREE Full Text
  43. 43.
    Tsuji BT, von Eiff C, Kelchlin PA, Forrest A, Smith PF. 2008. Attenuated vancomycin bactericidal activity against Staphylococcus aureus hemB mutants expressing the small-colony-variant phenotype. Antimicrob Agents Chemother 52:1533–1537.
    OpenUrlAbstract/FREE Full Text
  44. 44.↵
    Garcia LG, Lemaire S, Kahl BC, Becker K, Proctor RA, Denis O, Tulkens PM, Van Bambeke F. 2013. Antibiotic activity against small-colony variants of Staphylococcus aureus: review of in vitro, animal and clinical data. J Antimicrob Chemother 68:1455–1464.
    OpenUrlCrossRefPubMedWeb of Science
  45. 45.↵
    Liu CI, Liu GY, Song Y, Yin F, Hensler ME, Jeng WY, Nizet V, Wang AH, Oldfield E. 2008. A cholesterol biosynthesis inhibitor blocks Staphylococcus aureus virulence. Science 319:1391–1394.
    OpenUrlAbstract/FREE Full Text
  46. 46.
    Clauditz A, Resch A, Wieland KP, Peschel A, Götz F. 2006. Staphyloxanthin plays a role in the fitness of Staphylococcus aureus and its ability to cope with oxidative stress. Infect Immun 74:4950–4953.
    OpenUrlAbstract/FREE Full Text
  47. 47.↵
    Mishra NN, Liu GY, Yeaman MR, Nast CC, Proctor RA, McKinnell J, Bayer AS. 2011. Carotenoid-related alteration of cell membrane fluidity impacts Staphylococcus aureus susceptibility to host defense peptides. Antimicrob Agents Chemother 55:526–531.
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    Cosgrove K, Coutts G, Jonsson IM, Tarkowski A, Kokai-Kun JF, Mond JJ, Foster SJ. 2007. Catalase (KatA) and alkyl hydroperoxide reductase (AhpC) have compensatory roles in peroxide stress resistance and are required for survival, persistence, and nasal colonization in Staphylococcus aureus. J Bacteriol 189:1025–1035.
    OpenUrlAbstract/FREE Full Text
  49. 49.
    Gaupp R, Ledala N, Somerville GA. 2012. Staphylococcal response to oxidative stress. Front Cell Infect Microbiol 2:33.
    OpenUrl
  50. 50.↵
    McAdow M, Kim HK, Dedent AC, Hendrickx AP, Schneewind O, Missiakas DM. 2011. Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood. PLoS Pathog 7:e1002307.
    OpenUrlCrossRefPubMed
  51. 51.↵
    Bryan-Jones DG, Whittenbury R. 1969. Hematin-dependent oxidative phosphorylation in Streptococcus faecalis. J Gen Microbiol 58:247–60.
    OpenUrlCrossRefPubMedWeb of Science
  52. 52.
    Ritchey TW, Seeley HW. 1974. Cytochromes in Streptococcus faecalis var. zymogenes grown in a hematin-containing medium. J Gen Microbiol 85:220–228.
    OpenUrlCrossRefPubMedWeb of Science
  53. 53.↵
    Winstedt L, Frankenberg L, Hederstedt L, von Wachenfeldt C. 2000. Enterococcus faecalis V583 contains a cytochrome bd-type respiratory oxidase. J Bacteriol 182:3863–3866.
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    Frankenberg L, Brugna M, Hederstedt L. 2002. Enterococcus faecalis heme-dependent catalase. J Bacteriol 184:6351–6356.
    OpenUrlAbstract/FREE Full Text
  55. 55.↵
    Baureder M, Reimann R, Hederstedt L. 2012. Contribution of catalase to hydrogen peroxide resistance in Enterococcus faecalis. FEMS Microbiol Lett 331:160–164.
    OpenUrlCrossRefPubMed
  56. 56.↵
    Monk IR, Shah IM, Xu M, Tan MW, Foster TJ. 2012. Transforming the untransformable: application of direct transformation to manipulate genetically Staphylococcus aureus and Staphylococcus epidermidis. MBio 3:e00277–11.
    OpenUrlCrossRefPubMed
  57. 57.↵
    James EH, Edwards AM, Wigneshweraraj S. 2013. Transcriptional downregulation of agr expression in Staphylococcus aureus during growth in human serum can be overcome by constitutively active mutant forms of the sensor kinase AgrC. FEMS Microbiol Lett 349:153–162.
    OpenUrlCrossRefPubMedWeb of Science
  58. 58.↵
    Edwards AM, Potts JR, Josefsson E, Massey RC. 2010. Staphylococcus aureus host cell invasion and virulence in sepsis is facilitated by the multiple repeats within FnBPA. PLoS Pathogens 6: e1000964.
    OpenUrlCrossRefPubMed
  59. 59.↵
    Ko YP, Kuipers A, Freitag CM, Jongerius I, Medina E, van Rooijen WJ, Spaan AN, van Kessel KP, Höök M, Rooijakkers SH. 2013. Phagocytosis escape by a Staphylococcus aureus protein that connects complement and coagulation proteins at the bacterial surface. PLoS Pathog 9: e1003816.
    OpenUrlCrossRefPubMed
  60. 60.↵
    Surewaard BG, de Haas CJ, Vervoort F, Rigby KM, DeLeo FR, Otto M, van Strijp JA, Nijland R. 2013. Staphylococcal alpha-phenol soluble modulins contribute to neutrophil lysis after phagocytosis. Cell Microbiol 15:1427–37.
    OpenUrlCrossRefPubMed
  61. 61.↵
    Smith EJ, Visai L, Kerrigan SW, Speziale P, Foster TJ. 2011. The Sbi protein is a multifunctional immune evasion factor of Staphylococcus aureus. Infect Immun. 79:3801–3809.
    OpenUrlAbstract/FREE Full Text
  62. 62.↵
    Malachowa N, Whitney AR, Kobayashi SD, Sturdevant DE, Kennedy AD, Braughton KR, Shabb DW, Diep BA, Chambers HF, Otto M, DeLeo FR. 2011. Global changes in Staphylococcus aureus gene expression in human blood. PLoS One. 6:e18617
    OpenUrlCrossRefPubMed
  63. 63.↵
    Painter KL, Krishna A, Wigneshweraraj S, Edwards AM. 2014. What role does the quorum-sensing accessory gene regulator system play during Staphylococcus aureus bacteremia? Trends Microbiol. 22:676–685.
    OpenUrlCrossRefPubMed
  64. 64.↵
    Skaar EP, Humayun M, Bae T, DeBord KL, Schneewind O. 2004. Iron-source preference of Staphylococcus aureus infections. Science. 2004 305:1626–8.
    OpenUrlAbstract/FREE Full Text
  65. 65.↵
    Repine JE, Fox RB, Berger EM, Harada RN. 1981. Effect of staphylococcal iron content on the killing of Staphylococcus aureus by polymorphonuclear leukocytes. Infect Immun 32:407–410.
    OpenUrlAbstract/FREE Full Text
  66. 66.↵
    Hoepelman IM, Bezemer WA, Vandenbroucke-Grauls CM, Marx JJ, Verhoef J. 1990. Bacterial iron enhances oxygen radical-mediated killing of Staphylococcus aureus by phagocytes. Infect Immun 58:26–31.
    OpenUrlAbstract/FREE Full Text
  67. 67.↵
    Gläser R, Becker K, von Eiff C, Meyer-Hoffert U, Harder J. 2014. Decreased susceptibility of Staphylococcus aureus small-colony variants toward human antimicrobial peptides. J Invest Dermatol 134: 2347–2350.
    OpenUrlCrossRefPubMed
  68. 68.↵
    Mishra NN, Liu GY, Yeaman MR, Nast CC, Proctor RA, McKinnell J, Bayer AS. 2011. Carotenoid-related alteration of cell membrane fluidity impacts Staphylococcus aureus susceptibility to host defense peptides. Antimicrob Agents Chemother 55: 526–531.
    OpenUrlAbstract/FREE Full Text
  69. 69.↵
    Fang FC. 2011. Antimicrobial actions of reactive oxygen species. MBio 2: e00141–11.
    OpenUrlCrossRefPubMed
  70. 70.↵
    Imlay JA. 2013. The molecular mechanisms and physiological consequences of oxidative stress: lessons from a model bacterium. Nat Rev Microbiol 11:443–454.
    OpenUrlCrossRefPubMed
  71. 71.↵
    Keyer K, Imlay JA. 1996. Superoxide accelerates DNA damage by elevating free-iron levels. Proc Natl Acad Sci U S A 93:13635–13640.
    OpenUrlAbstract/FREE Full Text
  72. 72.↵
    Skaar EP, Humayun M, Bae T, DeBord KL, Schneewind O. 2004. Iron-source preference of Staphylococcus aureus infections. Science 305: 1626–1628.
    OpenUrlAbstract/FREE Full Text
  73. 73.↵
    Coque TM, Patterson JE, Steckelberg JM, Murray BE. 1995. Incidence of hemolysin, gelatinase, and aggregation substance among enterococci isolated from patients with endocarditis and other infections and from feces of hospitalized and community-based persons. J Infect Dis 171: 1223–1229.
    OpenUrlCrossRefPubMedWeb of Science
  74. 74.↵
    Creti R, Imperi M, Bertuccini L, Fabretti F, Orefici G, Di Rosa R, Baldassarri L. 2004. Survey for virulence determinants among Enterococcus faecalis isolated from different sources. J Med Microbiol 53: 13–20.
    OpenUrlCrossRefPubMedWeb of Science
  75. 75.↵
    Horsburgh MJ, Clements MO, Crossley H, Ingham E, Foster SJ. 2001. PerR controls oxidative stress resistance and iron storage proteins and is required for virulence in Staphylococcus aureus. Infect Immun 69: 3744–3754.
    OpenUrlAbstract/FREE Full Text
  76. 76.
    Pader V, Hakim S, Painter KL, Wigneshweraraj S, Clarke TB, Edwards AM. 2016. Staphylococcus aureus inactivates daptomycin by releasing membrane phospholipids. Nat Microbiol 2: 16194.
    OpenUrl
  77. 77.
    Jacob AE, Hobbs SJ. 1974. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J Bacteriol 117: 360–372.
    OpenUrl
  78. 78.
    Ike Y, Craig RA, White BA, Yagi Y, Clewell DB. 1983. Modification of Streptococcus faecalis sex pheromones after acquisition of plasmid DNA. Proc Natl Acad Sci U S A 80: 5369–5373.
    OpenUrlAbstract/FREE Full Text
  79. 79.
    Lee CY, Buranen SL, Ye ZH. 1991. Construction of single-copy integration vectors for Staphylococcus aureus. Gene 103: 101–105.
    OpenUrlCrossRefPubMedWeb of Science
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Posted July 19, 2017.
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The electron transport chain sensitises Staphylococcus aureus and Enterococcus faecalis to the oxidative burst
Kimberley L. Painter, Alex Hall, Kam Pou Ha, Andrew M. Edwards
bioRxiv 165589; doi: https://doi.org/10.1101/165589
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