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Commensal bacteria augment Staphylococcus aureus infection by inactivation of phagocyte-derived reactive oxygen species

['Josie F. Gibson', 'Department Of Molecular Biology', 'Biotechnology', 'University Of Sheffield', 'Sheffield', 'United Kingdom', 'Florey Institute', 'The Bateson Centre', 'Grace R. Pidwill', 'Oliver T. Carnell']

Date: 2021-10

Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection.

S. aureus is a commensal inhabitant of the human skin and nares. However, it can cause serious diseases if it is able to breach our protective barriers such as the skin, often via wounds or surgery. If infection occurs via a wound, this initial inoculum contains both the pathogen, other members of the microflora and also wider environmental microbes. We have previously described “augmentation”, whereby this other non-pathogenic material can enhance the ability of S. aureus to lead to a serious disease outcome. Here we have determined the breadth of augmenting material and elucidated the cellular and molecular basis for its activity. Augmentation occurs via shielding of S. aureus from the direct bactericidal effects of reactive oxygen species produced by macrophages. This initial protection enables the effective establishment of S. aureus infection. Understanding augmentation not only explains an important facet of the interaction of S. aureus with our innate immune system, but also provides a platform for the development of novel prophylaxis approaches.

Augmentation is a recently described phenomenon whereby human skin commensals enhance S. aureus pathogenesis [ 12 ]. S. aureus bloodstream infection in mice can be augmented by either Gram-positive commensals, their purified peptidoglycan (PGN) or a natural mix of skin flora [ 12 ]. In this example of microbial crowdsourcing, only S. aureus benefits, not the non-pathogenic commensals, which succumb. During murine sepsis, augmenting material is co-phagocytosed with S. aureus in Kupffer cells, resulting in increased bacterial survival and the subsequent formation of clonal liver microabscesses [ 12 ], with the potential to seed other organs in the body [ 13 ]. In Kupffer cells, augmentation is associated with reduced oxidation and, importantly, augmentation is not observed in transgenic mice lacking functional NOX2, defining a pivotal role for ROS in this phenomenon. However, major signalling receptor-mediated mechanisms (including NOD1, NOD2, TLRs and the inflammasome) did not account for augmentation [ 12 ]. To elucidate the molecular mechanism(s) underpinning augmentation, we sought to define the breadth of materials able to enhance S. aureus infection and investigate whether augmentation occurs for other human pathogens. Using in vitro and in vivo studies, we demonstrate that the molecular basis for augmentation is absorption of ROS by augmenting material, shielding S. aureus from macrophage-mediated killing.

Staphylococcus aureus exists in a polymicrobial environment, primarily as a human commensal organism [ 1 – 3 ], but can also cause disease after a breach in host defences, often via localised tissue injury [ 4 ]. S. aureus causes a spectrum of disease, from minor skin infections to life-threatening bacteraemia: infections that are increasingly difficult to treat due to antibiotic resistance [ 5 ]. Human innate immune defences, primarily phagocytes, play a crucial role in preventing serious S. aureus disease. However, during infection S. aureus can reside within, and escape from, an intraphagocyte niche [ 6 – 8 ]. Similar to other intracellular pathogens [ 9 , 10 ], this can lead to a population bottleneck, where most bacteria are effectively killed by phagocytes, but a small proportion survive, enabling continued infection [ 11 ]. This results in the emergence of clonal bacterial populations, which expand from the small numbers surviving the population bottleneck. In the murine sepsis model, liver-resident macrophages known as Kupffer cells are the basis of this population bottleneck and subsequent bacterial clonality [ 12 , 13 ]. Macrophages are crucial for defence against S. aureus, exposing bacteria to an array of bactericidal mechanisms, including ROS, deleterious enzymes and antimicrobial peptides [ 14 ]. After phagocytosis, NADPH oxidase (NOX2) produces superoxide (O 2 - ) [ 15 ], which is converted to hydrogen peroxide (H 2 O 2 ) and hydroxyl radical ( · OH). Hypochlorous acid (HOCl) is generated from H 2 O 2 via the enzyme myeloperoxidase (MPO) [ 16 ]. Reactive nitrogen species (RNS) are produced by inducible nitric oxide synthase (iNOS), creating nitric oxide (NO · ) which can then react with O 2 - to form peroxynitrite (ONOO - ) [ 17 ]. All reactive species cause bacterial damage, but HOCl and H 2 O 2 may be key in vivo, as both are efficacious against biofilms [ 18 , 19 ]. S. aureus uses several approaches to resist ROS/RNS: two superoxide dismutases detoxify O 2 - [ 20 ], catalase removes H 2 O 2 [ 21 ] , alkyl hydroperoxidase acts to reduce H 2 O 2, ONOO - and organic peroxides [ 22 ] , and staphylococcal peroxidase inhibitor (SPIN) inhibits MPO therefore blocking HOCl formation [ 23 ]. Many S. aureus-ROS studies focus on neutrophils, since chronic granulomatosis disease (CGD) highlights ROS as vital in neutrophil bacterial clearance [ 24 ]. Nevertheless, ROS are also important in tissue macrophages [ 25 ] for defence against S. aureus [ 26 ].

The test vaccine consisted of 1 μg ClfA, 50 μg CpG and 1% w/v Alhydrogel an aluminium based adjuvant, components which have been used previously [ 37 – 39 ]. Mice were vaccinated on days 0, 14 and 21 before S. aureus infection on day 28, with blood drawn before and after vaccinations ( Fig 6A ). The vaccine was tested for efficacy, alongside PBS control injections, in three S. aureus infection scenarios; low dose (1x10 6 S. aureus), high dose (1x10 7 S. aureus) and augmented low dose (1x10 6 S. aureus +/- M. luteus PGN), in 2 independent experiments (Figs 6 and S7 ). Low dose infection caused low numbers (an average of 38 CFU) of bacteria in the liver, with more observed in the high dose infection (an average of 1.43x10 6 CFU), and as expected, the augmentation groups had very high liver bacterial numbers (an average of 4.45x10 7 CFU) ( Fig 6B ). Interestingly, vaccination reduced S. aureus liver and kidney bacterial numbers only for the augmentation groups (Figs 6B and S7A , S7B ). The second independent experiment also showed that the vaccine was only effective in reducing S. aureus pathogenesis in the augmented scenario ( S7C–S7F Fig ). These data suggest that using augmentation to examine vaccine efficacy may be a useful strategy, as it mimics natural infection.

To examine the importance of ROS production, the mass formation assay was evaluated following treatment with NOX2 inhibitors DPI or apocynin, using concentrations which did not inhibit S. aureus growth ( S6B and S6C Fig ). Addition of DPI or apocynin significantly reduced the level of augmentation compared to the untreated controls ( Fig 5G and 5H ), but treatment with a specific scavenger of mitochondrial superoxide (mitoTEMPO) did not ( Fig 5I ). This confirms that ROS production, specifically in phagosomes, is important for augmentation of S. aureus infection within macrophages. Our in vitro assays showed that augmenting material protects S. aureus from ROS. Therefore, we used a specific fluorescent probe, Hydrop, to examine levels of H 2 O 2 within infected RAW264.7 macrophages. The Hydrop assay showed significantly reduced H 2 O 2 levels in RAW264.7 cells infected with S. aureus and HK M. luteus, in comparison to S. aureus alone (Figs 5J and S6D ). To further examine how augmentation affects oxidation of the bacteria, RAW264.7 macrophages were infected with GFP S. aureus stained with CellROX, a dye which becomes fluorescent when oxidised by ROS. There were significantly more oxidised S. aureus events in macrophages infected with S. aureus alone than those infected with S. aureus alongside HK M. luteus (Figs 5K and S6E ). Together these data demonstrate ROS levels are reduced in the presence of augmenting material, suggesting this material acts to inactivate ROS.

A Images of GFP-S. aureus mass formation within RAW264.7 cells, scale 20 μm B RAW264.7 cells infected with GFP S. aureus (MOI 5) with or without heat-killed M. luteus (MOI 50), (n = 4), ****p<0.0001 C RAW264.7 cells infected with GFP S. aureus (MOI 5) with or without heat-killed M. luteus (ratio to S. aureus, 10, 5, 2.5, 0.5 or media control), (n = 4), **p<0.008; ****p<0.0001 D RAW264.7 cells infected with GFP S. aureus (MOI 5, 2.5, 1.25, 0.25, 0.025 or media control) with or without of heat-killed M. luteus (MOI 50), (n = 4), **p<0.003; ***p<0.0008; ****p<0.0001 E-F MDMs infected with GFP S. aureus (MOI 5) with or without heat-killed M. luteus (MOI 50) E images of GFP S. aureus mass formation within human MDMs, scale 20 μm F number of S. aureus masses observed (n = 3), **p<0.003 G RAW264.7 cells infected with GFP S. aureus (MOI 5) in the presence or absence of heat-killed M. luteus (MOI 50), either with or without DPI (2 μM), (n = 4), *p<0.05;***p<0.0004; ****p<0.0001 H RAW264.7 cells infected with GFP S. aureus (MOI 5) in the presence or absence of heat-killed M. luteus (MOI 50), either with or without apocynin (500 μM), (n = 4), ****p<0.0001 I RAW264.7 cells infected with GFP S. aureus (MOI 5) in the presence or absence of heat-killed M. luteus (MOI 50), either with or without mitoTEMPO (1 μM), (n = 4, non-significant) J RAW264.7 cells infected with GFP S. aureus (MOI 5) with or without heat-killed M. luteus (MOI 50) with Hydrop used to visualise hydrogen peroxide (n = 4, violin plot with median values shown), **p<0.007; ***p<0.0004 K RAW264.7 cells infected with CellROX-stained GFP S. aureus (MOI 50) to visualise intracellular oxidation in the presence or absence of heat-killed M. luteus (MOI 50), (n = 4, violin plot with median values shown), ****p<0.0001. In panels B, F, H, I and K, a two-tailed Mann Whitney test was used, in panels C, D, G, and J, a Kruskal-Wallis test with Dunn’s post hoc test was used. Where used, error bars show mean +/- SD.

Augmentation occurs at the initiation of infection by circumventing the deleterious effects of ROS in vivo. To determine how these manifest at the cellular level, we used a murine macrophage cell line. Time-lapse imaging of RAW264.7 cells infected with fluorescent S. aureus were used to examine whether bacteria surviving within macrophages may represent the source of the microabcesses that occur as a product of augmentation [ 12 ]. In the presence of augmenting material, intracellular S. aureus survival and growth were observed within individual macrophages, which eventually led to host cell death and formation of large extracellular accumulations of bacteria, referred to here as bacterial masses ( Fig 5A ). Using a high-throughput assay to examine S. aureus mass formation, RAW264.7 cells were infected with S. aureus with or without HK M. luteus at a ratio of 1:10, a lower ratio than was used in the preceding in vitro and in vivo work, to limit cell toxicity. Despite this, the number of masses was significantly increased in the augmented group in comparison to S. aureus infection alone ( Fig 5B ). We next examined the ratio of augmenting material to S. aureus, using an augmenting material ratio of 10, 5, 2.5, 0.5 and 0.05 to S. aureus, with increased numbers of masses forming in the presence of higher concentrations of augmenting material ( Fig 5C ). Higher S. aureus levels also led to increased mass formation ( Fig 5D ). These data demonstrate dose-dependent augmentation by HK bacteria of S. aureus survival and proliferation within macrophages. Finally, we used human monocyte-derived macrophages (MDMs) in the time-lapse S. aureus mass formation assay. S. aureus was able to survive, proliferate and escape from MDMs ( Fig 5E ). Similarly, S. aureus mass formation from MDMs was significantly increased in the presence of augmenting material (Figs 5F and S6A ). Augmenting material therefore increases the capacity of S. aureus to overwhelm human macrophages.

To test whether ROS-susceptible S. aureus could be augmented in vivo, a low dose (1x10 6 CFU) of katA ahpC was injected with or without HK M. luteus (1x10 8 CFU). In the presence of augmenting material, the katA ahpC strain had an exceptionally large and significant increase in liver bacterial numbers from 0 CFU to ~3x10 7 CFU, levels seen in augmented wild-type S. aureus infections, but no significant change in weight loss or kidney bacterial numbers (Figs 4C and S5J , S5K ). Thus S. aureus katA ahpC can not only be augmented but also this leads to loss of attenuation in the liver. This further supports the assertion that augmentation occurs in the liver and is associated with the ability of the bacteria to survive ROS, as well as that augmentation also occurs during the initiation of infection. ROS resistance is additionally required for later infection stages, as S. aureus katA ahpC in the presence of augmenting material does not recover to parental bacterial numbers in the kidney.

S. aureus mutants lacking oxidative stress resistance mechanisms are susceptible to ROS and attenuated in pathogenesis [ 20 , 21 ]. We used a S. aureus katA ahpC to map which ROS resistance mechanisms are important for S. aureus survival and to test the ability of augmenting material to rescue this strain. The place of the ROS resistance enzymes investigated here in detoxifying the oxidative burst is shown in Fig 4A . S. aureus katA ahpC would be expected to have a reduced ability to detoxify H 2 O 2 , organic peroxides and peroxynitrite, and is more sensitive to peroxides in vitro [ 21 ]. S. aureus katA ahpC was protected from H 2 O 2 by live M. luteus, but not by HK or H 2 O 2 -treated cells ( Fig 4B ), as were individual katA or ahpC mutants ( S5A and S5B Fig ). When katA ahpC was exposed to HOCl, katA ahpC survival was significantly increased from ~0.2% to ~100% with the addition of M. luteus and to ~68% with HK M. luteus, but not ROS-treated M. luteus which remained at ~4.4% survival ( S5C Fig ). The role of ROS resistance was then tested in vivo using the murine sepsis model. The katA, ahpC and katA ahpC strains were attenuated, with significantly fewer liver bacteria recovered for ahpC and katA ahpC, and kidney bacteria for katA ahpC and katA, with all strains causing significantly reduced weight loss in comparison to wild-type ( S5D–S5I Fig ).

Thus, augmenting material may act as a buffer to react with, and therefore detoxify, ROS. If this is so, pre-treatment of augmenting material with ROS would diminish its effect. To test this, live M. luteus were pre-treated with ROS prior to inclusion in the in vitro liquid culture assay. HOCl pre-treated M. luteus showed a clear loss of protective ability, with no surviving bacteria, in comparison to live or HK M. luteus ( Fig 3E ). For both H 2 O 2 and peroxynitrite, the level of S. aureus survival with addition of ROS-treated M. luteus was ~10–20%, whereas with HK M. luteus this was ~100% ( Fig 3F and 3G ). This indicates that augmenting material has a finite capacity to react with ROS and, in so doing, loses its ability to protect S. aureus.

We have previously shown that live bacteria augment S. aureus infection [ 12 ], as such, we hypothesised that M. luteus would promote S. aureus survival in the presence of ROS. M. luteus was used at 100 times the concentration of S. aureus. Addition of live M. luteus led to significantly increased survival of S. aureus after H 2 O 2 , HOCl and peroxynitrite treatments, but not methyl viologen ( S4B–S4E Fig ). It was possible that live M. luteus was mediating augmentation via production of ROS defence enzymes, such as catalase. Addition of HK M. luteus increased S. aureus survival when exposed to HOCl but not H 2 O 2 and peroxynitrite (Figs 3E and S4B–S4D ). To determine if the lack of effectiveness of HK M. luteus was due to the availability of ROS active moieties the ratio of augmenting material was raised (ratio of 1:2500), which significantly increased S. aureus survival following exposure to H 2 O 2 or peroxynitrite ( Fig 3F and 3G ). Thus, both live and HK M. luteus can protect S. aureus from ROS. It appears that H 2 O 2 or peroxynitrite are effectively deactivated by enzymes present in live M. luteus; although these enzymes promote augmentation they are not required.

Since augmentation does not occur in the absence of NOX2, and Kupffer cells have reduced ROS levels in augmented S. aureus infection [ 12 ], we hypothesised that inactivation of ROS by augmenting material could be the mechanism by which S. aureus survival is enhanced with an ensuing increase in pathogenesis. We therefore tested whether augmenting material protects S. aureus from specific ROS and RNS in vitro, using H 2 O 2 , sodium hypochlorite (a source of HOCl), peroxynitrite, and methyl viologen (a source of superoxide) ( Fig 3A ). S. aureus survival in liquid culture in vitro was measured following ROS exposure, with or without M. luteus PGN. Exposure to each ROS led to a significant reduction in S. aureus numbers, while addition of PGN significantly increased S. aureus survival in the presence of H 2 O 2 , HOCl and peroxynitrite, but not methyl viologen (Figs 3B–3D and S4A ).

The requirement for concomitant administration of S. aureus and augmenting material suggested that they are likely co-phagocytosed. To examine this, the amount of augmenting material within individual phagocytes in vivo was examined. A zebrafish transgenic line with fluorescent macrophages was used: Tg(mpeg:mCherry.CAAX)sh378 [ 35 ]. Augmentation has previously been shown to occur during systemic infection of zebrafish larvae [ 12 ]. Larvae were infected with GFP fluorescent S. aureus and/or fluorescently stained PGN. Macrophages phagocytosed injected material in each individually injected group ( Fig 2G and 2H ) and S. aureus and PGN were co-localised when present within the same macrophage (Figs 2I and S3E ). Macrophages were imaged and the area of phagocytosed fluorescent materials was quantified using Fiji. The area taken up by S. aureus within individual macrophages was not altered when PGN was present ( Fig 2J ). However, the area of PGN was significantly increased in the presence of S. aureus, in comparison to PGN injected alone ( Fig 2K ). Thus, augmentation does not alter the level of S. aureus phagocytosis in vivo, however, it appears that macrophages which engulf S. aureus also phagocytose more augmenting material.

A-C Mice were intravenously injected with 500 μg M. luteus PGN 24 hours, 6 hours or 1 hour before infection with 1x10 6 S. aureus, or at the same time as S. aureus, or with S. aureus alone A Diagram of experimental protocol B liver CFUs, enumerated at 72 hpi (n = 7 per group, median value shown, Kruskal-Wallis test with Dunn’s post-test), *p<0.05 C Summary heat-map for augmenting ability of PGN added before S. aureus infection, showing significant changes in liver CFUs and weight change D-F Mice were intravenously injected with 500 μg M. luteus PGN 48 hours, 24 hours or 6 hours after infection with 1x10 6 S. aureus, or at the same time as S. aureus, or with S. aureus alone D Diagram of experimental protocol E liver CFUs, enumerated at 72 hpi (n = 5 per group, median value shown, Kruskal-Wallis test with Dunn’s post-test) *p<0.05 F Summary heat-map for augmenting ability of PGN added after S. aureus infection, showing significant changes in liver CFUs and weight change G-K Zebrafish larvae injected with 400 CFU S. aureus, 5 ng of M. luteus PGN, or both. The larvae have fluorescent macrophages (red) and were injected with fluorescent S. aureus (green) and/or fluorescently labelled M. luteus PGN (blue) G-I Images of infected larvae at 2 hpi showing macrophages containing S. aureus, scale 6.9 μm, greyscale insets depict location of fluorescence signal within the hatched box of the main image, for ease of visualisation ( G ), M. luteus peptidoglycan scale 6.9 μm ( H ), or both scale 10 μm ( I ), J Area of macrophage taken up by S. aureus at 2 hpi (n = 3, 14–21 larvae per group, unpaired t-test) K Area of macrophage taken up by M. luteus PGN at 2hpi (n = 3, 11–21 larvae per group, two-tailed unpaired t-test, ***p<0.0004).

Next, we tested whether augmenting material was able to increase the virulence of a range of human pathogens: Enterococcus faecalis, an opportunist pathogen capable of residing within macrophages [ 31 ]; Streptococcus pneumoniae, which is able to survive within phagocytes and experiences a population bottleneck which seeds further infection [ 32 ]; Pseudomonas aeruginosa, which survives within macrophages [ 33 ]; and Streptococcus pyogenes, which can survive and escape from within host cells [ 34 ]. During murine sepsis, live M. luteus augmented E. faecalis infection (of 5x10 7 CFU) with a significant increase in liver and lung bacteria compared to E. faecalis alone (Figs 1F, 1G and S1H–S1L ). Furthermore, M. luteus PGN augmented a larger E. faecalis inoculum (1x10 8 CFU), although it did not at 5x10 7 CFU ( S1M–S1R Fig ). In both cases of E. faecalis augmentation, the liver bacterial number never increased higher than the inoculum. Pathogenesis of S. pneumoniae and P. aeruginosa was not increased by the presence of M. luteus PGN in sepsis models (Figs 1G and S2A–S2F ). Also M. luteus PGN did not alter mouse weight or S. pyogenes numbers in an intra-muscular leg infection model ( S2G and S2H Fig). These data suggest that augmented infections which result in increased pathogen numbers from the inoculum may be specific to S. aureus and a facet of its particular interaction mechanism with the host.

Previously, we have shown that S. aureus pathogenesis can be augmented by live Gram-positive skin commensals, purified PGN, or natural skin flora [ 12 ]. To determine the breadth of material able to augment S. aureus pathogenesis, we used the murine sepsis model and co-injection of a low S. aureus infectious dose with potential augmenting materials. Increased bacterial numbers in the liver is a key marker of augmented infection, with accompanying weight loss and/or increased kidney bacterial load in severe cases [ 12 ]. We first tested Gram-negative bacteria Escherichia coli and Roseomonas mucosa, as part of the human microflora [ 27 , 28 ], with heat-killed (HK) Micrococcus luteus as a positive control. Addition of HK M. luteus, E. coli or R. mucosa significantly increased S. aureus counts in the liver in comparison to S. aureus-only infected mice (Figs 1A–1C, 1E and S1A–S1C ). On average S. aureus liver counts are greatly increased from the inocula (of 1x10 6 CFU) to 1.25x10 8 CFU in augmented infections, in comparison to 1x10 6 in control infections. Interestingly, E. coli benefits from the presence of S. aureus, with an increase in E. coli counts in the liver, although these CFU counts are reduced in comparison to the injected E. coli inoculum ( Fig 1B ). In order to assess whether cross-kingdom materials could augment staphylococcal infection, we tested HK Cryptococcus neoformans and live fungi in the murine sepsis model. Addition of HK C. neoformans significantly increased S. aureus liver numbers (Figs 1D, 1E and S1D ). In contrast, Saccharomyces cerevisiae, an occasional human commensal which rarely becomes pathogenic [ 29 , 30 ], did not increase S. aureus liver or kidney numbers, but did enhance mouse weight loss (Figs 1E and S1E–S1G ). Together these data demonstrate that S. aureus pathogenicity can be enhanced by a wider range of microorganisms than has previously been shown.

Discussion

S. aureus is an insidious pathogen made more concerning due to the spread of antimicrobial resistance and the lack of an available vaccine. Understanding infection dynamics provides a route to the identification of disease breakpoints where interventions might be most effective. An effective vaccine should be able to prevent disease establishment, and so understanding the status of the pathogen at this infection initiation stage is crucial. All pathogens exist within a polymicrobial environment from which they emerge to cause disease. S. aureus lives as a human commensal, primarily in the nares where even in this niche it forms only a small proportion of the microbiome [1–3]. Thus, all S. aureus infections are initiated from an inoculum that is mostly not the pathogen. With this backdrop, we have identified the augmentation phenomenon, where human-skin commensals or derivatives enhance S. aureus pathogenesis, acting at the level of initial macrophage interaction [12]. The amount of material required to augment S. aureus infection is comparable to the number of bacteria located on human skin or vascular catheters [40,41]. Here we find that augmentation is not specific to S. aureus as it occurs with other opportunist pathogens. Both E. coli and E. faecalis, which survive intracellularly within macrophages [42,43], benefitted from augmenting material. However, in both cases augmentation was evidenced by reduced clearance rather than an increase in pathogen load, suggesting the increase in pathogenesis resulting in increased bacterial burden may be peculiar to S. aureus. We also show S. aureus disease can be augmented by a range of particulate materials from whole bacteria to fungal cell walls, suggesting that augmentation is not mediated by a response to specific components. This is supported by our previous work that demonstrated augmentation not to require any of the major host response pathways such as NOD1 and NOD2 [12]. The hypothesis that augmentation occurs at the initiation of infection was further supported by the requirement for co-inoculation of S. aureus and augmenting material.

Augmentation has a profound effect on S. aureus disease, resulting in the ability to reduce the required inoculum by 1000-fold to cause systemic disease in the murine sepsis model [12]. To determine what the molecular mechanism might be, we homed in on those events which occur within the macrophage after phagocytosis, where ROS production is known to be required for augmentation [12]. In vitro, augmenting material protected S. aureus from H 2 O 2 , HOCl and peroxynitrite, suggesting that augmenting material reacts with ROS acting as a buffer, allowing continued S. aureus survival. Augmenting material showed a variable, protective capacity against different ROS. As an example, low dose HK M. luteus (equivalent CFU 5x106) protected WT S. aureus from killing by HOCl but not the other ROS tested (Figs 3E and S4C, S4D). Conversely, PGN (25 mg/ml) was able to protect S. aureus from H 2 O 2 , peroxynitrite and HOCl (Fig 3B–3D). Use of a higher dose of HK M. luteus (equivalent CFU 1.25x108), which was comparable to the number of live cells from which the PGN was derived gave protection to all 3 ROS demonstrating parity (Fig 3F and 3G). Live M. luteus, at a concentration of 106 CFU, was able to protect S. aureus from ROS killing by HOCl, H 2 O 2 and peroxynitrite, which is likely due in part to M. luteus ROS resistance enzymes, such as catalase. A variety of biological entities present on augmenting material hold the potential to react with ROS resulting in, for example, oxidation or chlorination [44,45]. Furthermore, pre-treatment of augmenting material with ROS inhibited its protective ability, defining a finite capacity for ROS detoxification. The in vitro data was obtained in an environment very different from that experienced by the bacteria inside phagocytes, let alone in vivo, therefore it was important to make analyses in these more complex milieu. There are a range of ROS, all ultimately originating from superoxide as a product of NADPH oxidase, but which are directly involved in S. aureus killing in macrophages is unknown [26]. S. aureus katA ahpC is susceptible to H 2 O 2 in vitro and is attenuated in vivo. Interestingly, augmentation had a dramatic effect on S. aureus katA ahpC pathogenesis resulting in extremely boosted virulence, to a level compatible to its parent. This embeds the role of ROS resistance at the very earliest stages of disease in order to pass the initial threshold of infection establishment. H 2 O 2 is produced early during oxidative burst [16,17,46] and may therefore constitute a key ROS in controlling S. aureus. We also found that augmenting material protects S. aureus from HOCl and that pre-treatment of the augmentor with this ROS abrogated its protective effect. HOCl is derived from H 2 O 2 by MPO within macrophages but at a proposed lower level than in neutrophils [47], leading to a higher H 2 O 2 concentration in macrophages [48]. Nevertheless, here we demonstrated that MPO is an important host defence enzyme in vivo, where its loss resulted in increased bacterial load in the liver highlighting HOCl as an important ROS in the control of infection. Lack of MPO did not prevent augmentation, in contrast to the loss of NOX2 activity in mice which did [12]. It is therefore likely that augmenting material acts as a sink for ROS in general thereby protecting S. aureus and allowing it to survive this crucial phase in host innate defences.

The effect of augmenting material is to allow S. aureus to survive the ROS assault in the macrophage. Inhibition of ROS in the absence of augmenting material did not greatly enhance S. aureus mass formation in isolated macrophages, possibly indicating that other killing mechanisms, of which there are a variety [26], may compensate in vitro. When S. aureus infection is augmented, absorption of ROS by augmenting material may prevent further maturation of the phagosome and thus activation of downstream bactericidal mechanisms. However, the importance of host ROS in controlling S. aureus infection real-life infections is clearly demonstrated with increased S. aureus pathogenicity in MPO (or NOX2 [12]) deficient mice, as well as the attenuation of S. aureus katA ahpC infection in vivo. A model for the molecular mechanism of augmentation is shown in S8 Fig, where phagocytosis of a threshold number of S. aureus leads to activation of ROS production and bacterial killing. Augmentation results in a bolus of phagocytosed material in addition to the S. aureus that acts to detoxify ROS and so increase the chance of bacterial survival, subsequent proliferation and lysis of the phagocyte, releasing a cluster of bacteria able to further multiply to form a microabscess. It is these microabscesses that can then go onto seed other sites in the host leading to a systemic and potentially fatal infection. Augmentation may act, therefore, to increase chances of infection spread by expanding the number of macrophages that are ineffective at controlling the initial infective dose. As the initiation of human infection will come from a polymicrobial environment, augmentation provides a framework to test prophylactic regimen. Indeed, under an augmentation scenario, an experimental S. aureus vaccine reduced bacterial burden. Understanding infection dynamics and the interplay between pathogen, host and other organisms is beginning to give insight into disease progression, and how novel interventions to sway the outcome in the favour for the host may be derived.

[END]

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