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The Journal of Immunology, 2003, 171: 5389-5395.
Copyright © 2003 by The American Association of Immunologists

Fibrinogen Depletion Attenuates Staphyloccocus aureus Infection by Preventing Density-Dependent Virulence Gene Up-Regulation 1

Jacob M. Rothfork*,{dagger}, Sophie Dessus-Babus*,{dagger}, Willem J. B. Van Wamel{ddagger}, Ambrose L. Cheung{ddagger} and Hattie D. Gresham2,*,{dagger}

* Research Service, Albuquerque Veterans Affairs Medical Center, Albuquerque, NM 87108; {dagger} Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM 87131; and {ddagger} Department of Microbiology, Dartmouth Medical School, Hanover, NH 03755


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Staphylococcus aureus undergoes a density-dependent conversion in phenotype from tissue-adhering to tissue-damaging and phagocyte-evading that is mediated in part by the quorum-sensing operon, agr, and its effector, RNAIII. Contributions of host factors to this mechanism for regulating virulence have not been studied. We hypothesized that fibrinogen, as a component of the inflammatory response, could create spatially constrained microenvironments around bacteria that increase density independently of bacterial numbers and thus potentiate quorum-sensing-dependent virulence gene expression. Here we show that transient fibrinogen depletion significantly reduces the bacterial burden and the consequential morbidity and mortality during experimental infection with wild-type S. aureus, but not with bacteria that lack expression of the quorum-sensing operon, agr. In addition, it inhibits in vivo activation of the promoter for the agr effector, RNAIII, and downstream targets of RNAIII, including {alpha} hemolysin and capsule production. Moreover, both in vitro and in vivo, the mechanism for promoting this phenotypic switch in virulence involves clumping of the bacteria, demonstrating that S. aureus responds to fibrinogen-mediated bacterial clumping by enhancing density-dependent virulence gene expression. These data demonstrate that down-modulation of specific inflammatory components of the host that augment bacterial quorum sensing can be a strategy for enhancing host defense against infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Staphylococcus aureus is a major human pathogen causing significant morbidity and mortality in both community- and hospital-acquired infections (1). Because antibiotic resistance is increasing in both the community and the hospital (2), and antibiotic-resistant strains have emerged that are clinically very aggressive (2, 3), there is renewed interest in developing strategies for enhancing host defense against this pathogen and in understanding the regulation of bacterial virulence factors that contribute to disease.

S. aureus is a member of a growing list of medically important bacterial pathogens that regulate virulence gene expression by a method of bacterial communication called quorum sensing (4, 5, 6, 7, 8, 9, 10). In this type of cell-to-cell communication, bacteria sense each other and their numbers via genetically regulated mechanisms that trigger behaviors that are primarily effective at high population densities (4). The triggers in this method of communication are small, diffusible compounds (autoinducers) secreted by the bacteria that accumulate extracellularly. At the appropriate concentration threshold that reflects a sufficient number of bacteria, these autoinducers signal gene expression programs that direct the coordinated action of the population. While the genetic systems that regulate quorum sensing are undergoing intensive investigation (5, 6, 7, 8, 9, 10), the contribution of the host environment to this mechanism for activating virulence gene expression has not been examined. Because the host niche in which the bacteria survive could provide an additional source of environmental triggers for the potentiation of quorum sensing, we have focused on identifying host factors that promote density-dependent virulence.

The acute inflammatory response as a component of innate immunity is an early and essential barrier to infection by extracellular bacterial pathogens such as S. aureus. As part of the acute phase response, the plasma protein fibrinogen plays a critical role in promoting neutrophil migration and adhesion, inducing cytokine and chemokine synthesis, coating foreign bodies, walling off the site of infectious injury, and initiating wound healing (11, 12, 13, 14, 15). In addition, fibrin/fibrinogen contributes to the generation of type 1 immunity that can be critical for effective clearance of some pathogens (16). In contrast, the interaction of some bacteria with fibrinogen contributes to the pathogenesis of the infection (17). In this regard, S. aureus expresses at least seven cell-bound and secreted proteins with direct fibrinogen/fibrin-binding activity (18, 19, 20), and several of these contribute to pathogenesis, indicating that the interaction of S. aureus with fibrinogen is important for optimal virulence (20, 21, 22, 23, 24). However, a role for fibrinogen in regulating density-dependent virulence gene expression has not been described. Clumping is a well-recognized response of S. aureus to soluble fibrinogen/fibrin (25, 26) and could represent a mechanism for increasing bacterial density independently of the absolute number of bacteria. Therefore, we hypothesized that fibrinogen-mediated bacterial clumping could create a microenvironment within the host that promotes density-dependent virulence.

Quorum sensing in S. aureus is regulated at least in part by the agr operon (9, 10, 24, 27, 28). The agr locus combines secretion of an octapeptide thiolactone ligand (autoinducer) and a two-component regulatory pathway to generate a regulatory RNA transcript, RNAIII, that is the effector of the operon. Under conditions of high autoinducer concentration, i.e., high bacterial density, RNAIII down-regulates gene expression encoding for surface adhesins while up-regulating those encoding for capsule production, secreted toxins, and proteases (24, 27, 29, 30, 31). This conversion from a tissue-adhering to a tissue-damaging and phagocyte-evading phenotype is thought to be important for in vivo pathogenesis and the development of invasive infection (10, 24, 27, 32). In this work we show for the first time that fibrinogen-mediated bacterial clumping significantly potentiates density-dependent virulence gene expression and the resultant bacterial infection. Moreover, our data demonstrate that removal of a host niche (i.e., fibrin/fibrinogen) that potentiates quorum sensing can ameliorate infection-dependent morbidity and mortality.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

C57BL/6 and BALB/c female mice (8–16 wk) were purchased from Charles River Breeding Laboratories (Wilmington, MA). The appropriate institutional committees approved all experiments. To transiently deplete fibrinogen, mice were given 5–6 U of ancrod (Sigma-Aldrich, St. Louis, MO) vs sterile saline i.p. 18 h before and at the time of infection (12, 33). At this dose and route of administration, plasma fibrinogen levels recovered within ~24 h (33). Plasma fibrinogen levels were measured by thrombin-mediated clotting (Diagnostica Stago, Parsippany, NJ) and were 0.934 ± 0.06 mg/ml in control mice (n = 5) and undetectable in ancrod-treated mice (<150 µg/ml; n = 4) at the time of infection.

Bacteria

Wild-type (WT) 3 RN6390 and agr- RN6911 bacteria, containing plasmids encoding the promoters for RNAIII, RNAII, and cap5 driving the expression of green fluorescent protein (GFP) were prepared as previously described (30, 34, 35). For the hemolysin (hla) promoter driving expression of GFP, a 440-bp fragment upstream of the Shine Dalgarno sequence was amplified by PCR and cloned into the EcoRI/Xba site in pSK236 as previously described (34, 35). Strain Newman, and a low passage (<10), recent clinical isolate were additional WT bacteria used for study. To generate early exponential phase, nonfluorescent bacteria for infection, they were grown from frozen stocks generated with the appropriate selection antibiotics at 107 CFU/25 ml in Columbia broth for 12 h with shaking, washed, diluted 1/75 and grown for 4 h, diluted 1/75 for 2 h, and finally diluted for 2 h at 1/75. The mean channel of fluorescence of the bacteria cultured by this method was 15–20 as measured by flow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ). The overnight culture supernatants from WT and agr- bacteria were filtered (Millipore, Bedford, MA) and used immediately as sources of autoinducer or spent supernatant devoid of autoinducer.

In vivo infection models and in vivo promoter activation

We infected air pouches, created as previously described (36), with early exponential phase, nonfluorescent bacteria (7 x 107 CFU for WT and 1 x 108 CFU for agr- bacteria) to initiate acute abscess formation. After 4–28 h, the abscess was lavaged (5 ml) and sonicated, the tissue surrounding it was excised (surface area, 5.01 ± 0.1 cm2; n = 8) and sonicated, and the kidneys were homogenized and sonicated in PBS/0.1% Triton X-100 before CFU evaluation. The percentage and number of neutrophils in the lavage were determined as previously described (37). macrophage inflammatory protein 2 (MIP-2) levels in the lavage were determined by ELISA (R&D Systems, Minneapolis, MN). Clumping was determined by culturing the lavage pre- and postsonication (26). In vivo promoter activation was measured by flow cytometry. Sonicates from the various sites were centrifuged (12,500 x g), and the bacteria were washed, sonicated, and fixed in 2% paraformaldehyde. Profiles were based on 10,000–50,000 gated events, which were confirmed to be S. aureus by Ab staining (IgM anti-peptidoglycan; Sanbio, Uden, The Netherlands) (34). Morbidity was measured as weight loss over 7 days. For mortality studies, mice were injected i.p, with 1–2.5 x 108 CFU of WT or 2.5–5.0 x 108 agr- bacteria and observed for 7 days. Mice were sacrificed when moribund (37).

In vitro promoter activation

Early exponential phase bacteria (107 CFU/ml) were cultured with shaking in polystyrene tubes in broth for 5 h unless indicated otherwise in the presence or the absence of the following: 1% human serum or plasma; 1% mouse serum, plasma, or fibrinogen-depleted plasma; purified human fibronectin (BD Biosciences); purified human fibrinogen (American Diagnostica, Greenwich, CT); or 2.5–50% culture supernatant containing autoinducer from WT bacteria made up to 50% supernatant with agr- supernatant to control for nutrient depletion. At the specified times, the bacteria were washed in PBS/0.1% Triton X-100, sonicated, and fixed in 2% paraformaldehyde, and their fluorescence was measured by flow cytometry.

Statistical evaluation

Data are depicted as the mean ± SEM. Survival was evaluated by Fisher’s exact test, and other parameters were evaluated by Mann-Whitney U test for nonparametrics or Student’s t test using StatView for Macintosh.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Fibrinogen depletion prevents morbidity and mortality after infection with WT, but not quorum-sensing-deficient, S. aureus

To test our hypothesis, we determined the effect of transient fibrinogen depletion on the morbidity and mortality induced during experimental S. aureus infection. Fibrinogen can be depleted by the toxin, ancrod, which cleaves fibrinogen at the thrombin cleavage site, resulting in soluble fibrin monomers that are removed by the reticuloendothelial system (33). Ancrod has been used to specifically deplete fibrinogen in experimental models of inflammation and for treatment of acute ischemic stroke in clinical medicine (12, 38). We evaluated morbidity in an air pouch abscess model of skin infection by measuring daily weight loss after infection. In this model, fibrinogen depletion significantly reduced mobidity (weight loss) in mice infected with WT bacteria (Fig. 1A). In addition, this same protection was observed after infection with a recent clinical S. aureus isolate and in BALB/c mice, indicating that this protection was not limited to a single strain of bacteria or mice (data not shown). To address whether fibrinogen would protect against a lethal infection, we evaluated survival in a peritonitis abscess model that results in sepsis (38). After infection with WT bacteria, transient fibrinogen depletion significantly improved survival (Fig. 1B).



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  		FIGURE 1. Measurement of morbidity (weight loss) (A and C) and mortality (B and D) of saline-treated and fibrinogen-depleted mice infected with either wild-type (A and B) or agr-deficient S. aureus (C and D). A, Weight loss after infection of air pouches with WT bacteria in saline-treated ({blacksquare}) and fibrinogen-depleted mice ({circ}; n = 8 for each group). *, p < 0.05; p = 0.0087 (at 2 days). B, Survival of saline-treated (dashed line) and fibrinogen-depleted mice (solid line) infected i.p. with WT bacteria (n = 10–11 for each group). *, p = 0.032 (on day 7). C, Weight loss of saline-treated ({blacksquare}) and fibrinogen-depleted mice ({circ}) infected with agr- bacteria in the air pouch model (n = 8 in each group). D, Survival of saline-treated (dashed line) and fibrinogen-depleted mice (solid line) after i.p. injection of agr- bacteria (n = 9–10 for each group). *, p = 0.0113 (on day 1); p = NS (on day 7).

 
If this protection from morbidity and mortality relates to quorum sensing, fibrinogen depletion should not affect these parameters in mice infected with agr-deficient bacteria. While agr-deficient bacteria are less virulent than WT (1, 24), they can cause serious infection and death when given at increased inocula (39, 32, 40). Therefore, we measured both morbidity and mortality in mice infected with an appropriate challenge dose of agr-deficient bacteria and found that fibrinogen depletion affected neither morbidity (Fig. 1C) nor mortality (Fig. 1D). In fact, transient fibrinogen depletion increased mortality 1 day after infection with agr-deficient bacteria, in contrast to its protective effect on infection with WT bacteria (Fig. 1, B vs D). These data indicate that transient fibrinogen depletion attenuates host morbidity and mortality and that its protective effect applies only to infection with S. aureus containing an intact quorum-sensing apparatus.

Fibrinogen enhances agr-dependent activation of quorum sensing in vitro

The above data suggested that fibrinogen may play a role in mediating the density-dependent phenotypic switch in virulence during S. aureus infection. To address this, we determined the effect of fibrinogen on the activation of the two promoters of the quorum-sensing agr locus. Agr contains two promoters; P2 which initiates the RNAII transcript that encodes the autoinducing peptide and the two-component sensor/regulator, and P3, which initiates the RNAIII transcript, the effector of the operon (9, 10, 24, 27). Activation of the P3 promoter can be used as a proxy for density-dependent conversion in virulence gene expression (27, 34). To test our hypothesis, we used WT and agr-deficient S. aureus that contained constructs of the promoters for RNAIII, RNAII, and downstream targets of RNAIII, {alpha} hemolysin (hla) and capsule production (cap5), driving expression of GFP (34, 35). By incubating early exponential phase, nonfluorescent bacteria with various agents in vitro, promoter activation was quantified by the magnitude of fluorescence. At a time (5 h) and an initial bacterial density (107/ml) that did not result in significant RNAIII promoter activation with broth, RNAIII activation was significantly increased by the inclusion of 1% serum or 1% plasma from both humans and mice (Fig. 2A). Plasma from mice that had been fibrinogen depleted by ancrod was equivalent to serum for RNAIII activation, demonstrating that fibrinogen was primarily responsible for plasma-enhanced RNAIII activation, although serum components also contributed (Fig. 2A). Purified human fibrinogen, compared with human albumin, significantly and dose-dependently enhanced RNAIII promoter activation, reaching optimal activation at 10–100 µg/ml (Fig. 2B); concentrations equivalent to those in 1% plasma and in inflammatory exudates. Kinetically, fibrinogen-enhanced RNAIII promoter activation was not observed until a time that also resulted in promoter activation in broth (3 h), suggesting that fibrinogen was enhancing density-dependent activation (Fig. 1C). Fibrinogen did not result in significant early activation (<2.5 h) of the upstream RNAII promoter of the agr locus (Fig. 1D) indicating that its effect was on facilitating peptide delivery to the two-component signaling system required to generate RNAIII, rather than increasing the mechanism for producing and recognizing the autoinducer. RNAII activation was enhanced at a time after RNAIII activation, confirming the positive feedback loop of RNAIII on RNAII (31).



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  		FIGURE 2. In vitro measurement of the effects of fibrinogen, serum, and plasma on activation of the RNAIII, RNAII, and hla promoters of S. aureus. A, RNAIII promoter activation (fluorescence units) in broth ({square}), serum (horizontal lines), plasma ({blacksquare}), and fibrinogen-depleted plasma () from humans and mice (n = 4). *, p < 0.01; **, p < 0.001 (vs broth). B, Dose-dependent increase in RNAIII promoter activation with purified human fibrinogen (open symbols, dashed line) but not human serum albumin (closed symbols, solid line; n = 3). *, p < 0.001. C, Kinetics of RNAIII promoter activation with 10 µg/ml fibrinogen (open symbols, dashed line) or broth (closed symbols, solid line; n = 3). D, Kinetics of RNAII promoter activation with 10 µg/ml of fibrinogen (open symbols, dashed line) or broth (closed symbols, solid line; n = 4–12). E, RNAIII promoter activation at 5 h in wild-type and agr- bacteria with 10 µg/ml fibrinogen ({blacksquare}) or broth ({square}; n = 4). F, Kinetics and dose dependence of hla promoter activation in broth (solid line), 10 µg/ml (small dashed line), or 100 µg/ml of fibrinogen (large dashed line; n = 4). *, p < 0.02.

 
Activation of the RNAIII promoter occurs independently of the intact agr locus in bacterial vegetations in a rabbit endocarditis model (34). Therefore, fibrinogen-enhanced activation of RNAIII could be independent of agr. However, we did not detect RNAIII promoter activation in either the presence or the absence of fibrinogen in bacteria deficient in the agr locus (Fig. 1E). Moreover, if our hypothesis is correct, then fibrinogen should enhance promoter activation for virulence factors that are downstream targets of RNAIII, such as hla and cap5. Fibrinogen significantly stimulated activation of the promoter for hla at a dose equivalent for RNAIII activation, but at a later time than that observed for RNAIII (Fig. 1F). In addition, it increased the concentration of {alpha} hemolysin measured in the culture supernatant at 12 h from 3- to 8-fold from WT bacteria (RN6390 and Newman), but not from bacteria lacking the agr operon, even though these bacteria clump readily in the presence of fibrinogen (data not shown). Equivalent data were obtained for activation of the cap5 promoter (data not shown). These data demonstrate that the agr locus is important for fibrinogen-stimulated {alpha} hemolysin activity and capsule production.

Fibrinogen-enhanced quorum sensing requires the autoinducer and clumping

The above data demonstrated that fibrinogen-potentiated quorum sensing required the agr locus, but occurred after activation of the RNAII transcript, suggesting that fibrinogen-induced clumping facilitates delivery of the secreted autoinducer. If true, then addition of exogenous autoinducer to fibrinogen-clumped bacteria should increase RNAIII promoter activation. Moreover, at saturating concentrations of autoinducer, there should be minimal benefit of fibrinogen-induced clumping on promoter activation. At 2.5 h, when minimal promoter activation occurred in the presence or the absence of fibrinogen (Fig. 2C), addition of 2.5% autoinducer-containing supernatant to fibrinogen-clumped bacteria significantly enhanced promoter activation (Fig. 3A). As the concentration of autoinducer increased to 25%, the fold increase in promoter activation diminished (Fig. 3A), indicating that in the presence of excess autoinducer, there is no benefit of fibrinogen on quorum sensing.



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  		FIGURE 3. Role of the autoinducer and fibrinogen- and fibronectin-mediated clumping in potentiation of RNAIII promoter activation. A, Fold increase in RNAIII promoter activation in fibrinogen-clumped bacteria over broth at 2.5 h in the presence of increasing concentrations of autoinducer supernatant (n = 4). B, Clumping measured by a reduction in CFU (open symbols) in the presence of fibronectin (triangles, solid line) or fibrinogen ({square}, dashed line) compared with total CFU after sonication (closed symbols; n = 4). C, RNAIII promoter activation in the presence of broth or increasing concentrations of fibrinogen () or fibronectin ({blacksquare}; n = 8). D–J, Model of clumping-induced potentiation of quorum sensing. Wild-type bacteria in broth by light microscopy (D), fluorescence microscopy (E), and depicted graphically (F) with the autoinducer drawn as a pinwheel. Fibrinogen-clumped bacteria by light microscopy (G), fluorescence microscopy (H), and graphically depicted (I).

 
These data suggest that clumping is sufficient to concentrate the autoinducer and thus promote quorum sensing. Fibronectin is another adhesive protein that clumps S. aureus (41). We compared its ability with fibrinogen for clumping and potentiating RNAIII promoter activation. We measured clumping as a reduction in CFU assessed before and after sonication of the culture (26). Fibronectin dose-dependently induced clumping of the bacteria (Fig. 3B) and RNAIII promoter activation (Fig. 3C) equivalent to fibrinogen at a time (5 h) when there was minimal promoter activation with broth alone. Importantly, the ability of both fibronectin and fibrinogen to enhance promoter activation resulted from a relative increase in bacterial density (due to clumping) and not from an increase in absolute bacterial number (Fig. 3B). These data demonstrate that clumping by either fibrinogen or fibronectin in the presence of the autoinducer is sufficient to potentiate quorum sensing in this pathogen. Analysis by fluorescence microscopy revealed that while WT bacteria in broth formed loose aggregates (Fig. 3D), this was not sufficient for RNAIII promoter activation at this time (Fig. 3E and graphically depicted in Fig. 3F). In contrast, in the presence of 10 µg/ml of fibrinogen, the bacteria were densely aggregated (Fig. 3G), and this was sufficient for optimal RNAIII promoter activation (Fig. 3, H and I).

Fibrinogen depletion reduces quorum sensing-dependent virulence gene expression in vivo

To confirm in vivo our in vitro observation that fibrinogen facilitates activation of the promoter for RNAIII and a downstream target of RNAIII, {alpha} hla, we examined the timing and magnitude of RNAIII promoter activation after infection of control and fibrinogen-depleted mice in the skin abscess infection model. Following infection with early exponential, nonfluorescent bacteria for 4–28 h, we isolated the bacteria from a lavage of the abscess and determined the percentage of fluorescent bacteria and the magnitude of fluorescence as a measure of RNAIII promoter activation. In control mice the percentage of fluorescent bacteria was modest at 4 h (~12%), but it increased rapidly over the next 24 h to ~70%. The magnitude of fluorescence also increased (Fig. 4, A–C) and was significantly different at 28 h (Fig. 4D), indicating substantial activation of quorum sensing by this time in the infection. In contrast, bacteria isolated from fibrinogen-depleted mice were minimally fluorescent (~3–5%) at all time points, indicating significantly reduced RNAIII promoter activation in vivo (Fig. 4, A–C). Moreover, repletion with 10 mg of purified human fibrinogen given i.p. at the time of infection restored RNAIII promoter activation in fibrinogen-depleted mice (Fig. 4B).



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  		FIGURE 4. In vivo measurement of RNAIII and hla promoter activation in bacteria isolated from infected air pouch lavages from saline-treated and fibrinogen-depleted mice. A, Flow cytometric profile of RNAIII promoter activation in WT bacteria isolated at 4 h from saline-treated (medium line) and fibrinogen-depleted mice (heavy line). B, RNAIII promoter activation at 24 h in saline-treated (medium line), fibrinogen-depleted (heavy line), and fibrinogen-repleted (light dashed line) mice. C, RNAIII promoter activation at 28 h in saline-treated (medium line) and fibrinogen-depleted (heavy line) mice. D, Quantitative assessment of RNAIII promoter activation in fluorescence units from control ({square}), fibrinogen-depleted ({blacksquare}), and fibrinogen-repleted () mice at 4, 24, and 28 h. (n = 8–12). *, p < 0.05 (at 24 h) and p < 0.001 (at 28 h). E, Flow cytometric profile of RNAIII promoter activation in agr- bacteria at 28 h in saline (medium line) and fibrinogen-depleted mice (heavy line). F, Flow cytometric profile of hla promoter activation in WT bacteria at 28 h in saline-treated (medium line) and fibrinogen-depleted (heavy line) mice.

 
Bacteria recovered from mice infected with agr-deficient S. aureus did not demonstrate RNAIII promoter activation in either the presence or the absence of fibrinogen even at 28 h (Fig. 4E). Fibrinogen depletion even prevented activation of the hla promoter observed at 28 h (Fig. 4F) as well as {alpha} hemolysin activity detected in the abscess lavages (data not shown). These data demonstrate that transient fibrinogen depletion significantly attenuates activation of quorum sensing and the resulting switch in virulence phenotype in an experimental abscess infection model.

Fibrinogen depletion attenuates bacterial burden after infection with WT, but not quorum sensing-deficient, S. aureus

We measured bacterial burden in the abscess lavage, the tissue matrix surrounding the abscess, and the kidney as a distant metastasis site. At 4 h after infection, bacterial burden was equivalent in control and fibrinogen-depleted mice at all three sites (Fig. 5, A–C), indicating that fibrinogen depletion did not affect initial seeding or adherence of the bacteria at the site of infection. However, over the next 24 h, bacterial numbers in control mice increased significantly in abscess lavage and kidney, but not in the tissue matrix surrounding the abscess. The most significant increase in bacterial growth occurred after we detected initiation of RNAIII activation. In contrast, bacterial burden in the fibrinogen-depleted mice was relatively constant over this time at all sites, indicating that fibrinogen depletion was not enhancing host defense by promoting bacterial clearance but, rather, by preventing bacterial growth (Fig. 5, A–C). To measure the effect of fibrinogen on in vivo clumping, the abscess lavage was cultured pre- and postsonication. Only bacteria isolated from control mice, not those from fibrinogen-depleted mice, exhibited statistically significant clumping (postsonication CFU, 8.18 ± 0.18 vs 7.19 ± 0.26 for presonication; n = 6; p < 0.005, by paired t test).



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  		FIGURE 5. Measurement of bacterial burden over time in saline-treated and fibrinogen-depleted mice infected with WT (A–C) and agr- (D–F) S. aureus. Log CFU from saline-treated ({square}) and fibrinogen-depleted ({blacksquare}) mice at 4, 24, and 28 h after infection from the skin air pouch lavage (A), tissue surrounding the air pouch abscess (B), and the kidney (C; n = 8–12 mice/group). *, p < 0.04. Comparison of bacterial burden at 28 h in the air pouch abscess lavage (D), air pouch abscess tissue (E), and the kidney (F) between saline-treated ({square}) and fibrinogen-depleted ({blacksquare}) mice infected with either WT or agr- bacteria (n = 11–12 for each group). *, p < 0.03.

 
If transient depletion of fibrinogen was enhancing host defense by inhibiting activation of quorum sensing, it should have no effect on infection with bacteria that lack the quorum-sensing agr operon. At 28 h, when fibrinogen depletion significantly reduced the number of WT bacteria at all three sites, it had no effect on bacterial burden at these sites in mice infected with an equivalent dose of agr-deficient bacteria (Fig. 5, D–F). These data demonstrate that the agr locus is required to observe the protective effect of transient fibrinogen depletion on bacterial burden.

Fibrinogen depletion did not enhance host defense either by increasing the number of neutrophils present at the site of infection (at 4 h, 8.1 ± 2.2 x 105 neutrophils/ml of lavage for saline-treated mice vs 8.8 ± 3.8 x 105 for fibrinogen-depleted mice; n = 8–12) or by increasing the concentration of the neutrophil-recruiting chemokine, MIP-2 (at 4 h, 2215 ± 634 pg/ml of lavage for saline-treated mice vs 994 ± 547 pg/ml for fibrinogen-depleted mice; n = 4). In fact, MIP-2 levels in the fibrinogen-depleted mice were one-third to one-half those in the control mice even at 24 and 28 h after infection and were equivalent to those present 28 h after infection with the quorum-sensing deficient agr- strain (1080 ± 247; n = 7). The positive effect of fibrinogen on chemokine secretion is consistent with other in vitro observations (14, 15). These data indicate that fibrinogen depletion promotes host survival and limits morbidity and bacterial burden after infection with quorum-sensing-sufficient S. aureus by preventing activation of the promoter for the quorum-sensing effector, RNAIII, and not by promoting neutrophil recruitment and destruction of the pathogen.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These data demonstrate that fibrinogen-induced clumping of S. aureus creates a microenvironment within the host that facilitates quorum sensing-dependent virulence, resulting in increased bacterial burden and host morbidity and mortality. Host microenvironments could potentiate quorum sensing either by increasing the concentration of the autoinducer, as we showed with clumping, or by limiting diffusion of the autoinducer, as may occur within an intracellular vacuole (39). Thus, virulence as a quorum sensing-regulated behavior may not represent exclusively the absolute bacterial number but, rather, the microenvironment in the host surrounding the bacteria. In this way, even small numbers of bacteria could survive transit through the blood and into other tissues sites or even within a phagosome. Host potentiation of quorum sensing could apply to other medically relevant pathogens such as P. aeruginosa, which exists in intraluminal macrocolonies (clumps) in the lungs of patients with cystic fibrosis (40), and Enterococcus faecalis, which survives within macrophages (42) and responds to cues in urine and serum for modulation of virulence (43).

While the contribution of fibrinogen to the pathogenesis of S. aureus infections has been long recognized (1, 19, 20, 21, 22, 23, 24), our data are the first to show a role for it in the density-dependent activation of the quorum-sensing effector RNAIII and two downstream targets of RNAIII, {alpha} hemolysin and capsule production. Because these factors have been implicated in tissue damage and phagocyte evasion, fibrinogen depletion prevents conversion of the bacteria from an adherent to an invasive phenotype. Both in vitro and in vivo data indicate that clumping is the primary mechanism for this potentiation of quorum sensing. The magnitude of clumping involved is quite dramatic (Fig. 3, G and H), and low levels of agglutination are not sufficient (Fig. 3, D and E). While fibronectin was able to achieve equivalent clumping in vitro, its concentration in plasma is significantly lower than that of fibrinogen, perhaps explaining why fibrinogen depletion was sufficient in vivo for protection. Whether other serum-derived factors also work in vivo as well as in vitro (Fig. 2A) or whether tissue fluids not present in plasma could also facilitate quorum sensing is unknown. Clumping is known to protect S. aureus from neutrophil-dependent (25) and extracellular phospholipase A2-dependent killing (26), suggesting that it acts as a barrier to penetration of these cidal agents. Because we found that fibrinogen depletion only protected against infection with WT and not agr-deficient bacteria, fibrinogen-clumped WT bacteria may be protected from killing by up-regulating virulence factors that impede the function of these cidal agents. Many genes, some as yet undefined, are up-regulated by RNAIII and could contribute to survival of S. aureus in this environment (31). In addition, fibrinogen depletion significantly reduced MIP-2 levels, which could affect the ability of the pathogen to survive inside neutrophils at the site of infection, as we have shown previously (37). This possibility is currently being investigated.

Our data define the contribution of soluble fibrinogen/fibrin at abscess sites in the potentiation of quorum sensing and do not address its role in the adhesion of bacteria to foreign bodies and injured tissues. In these situations the interaction of fibrinogen with the bacteria may not lead to activation of RNAIII even though virulence factor expression (hla) can be altered (44, 45). In agreement with this, we found that adhesion of bacteria to tissue in the kidney did not lead to significant RNAIII activation even at CFUs equivalent to those in the pouch where activation could be detected (J. Rothfork and H. Gresham, unpublished observation). In contrast, adhesion within vegetations in an endocarditis model was sufficient for activation of RNAIII (34). These data indicate that S. aureus within different host niches may express different genetic programs. We believe our data with the air pouch model of skin infection may be most relevant for the initiation of acute abscess formation. Because capsule production is a target of RNAIII (29, 30), and capsular polysaccharides contribute to abscess formation (46), fibrinogen-dependent clumping may precede the contribution of the capsule to this pathologic process, suggesting that abscess formation is a dynamic process involving contributions from both the host and the pathogen.

Normal humans have a high degree of resistance to S. aureus infection, giving it a reputation as an opportunistic pathogen (1). Importantly, many of the patients that are highly susceptible to S. aureus infection are those with chronic illnesses such as cystic fibrosis, heart disease, diabetes, and kidney failure (1, 47), in which plasma fibrinogen levels are elevated consequential to the acute phase response (47, 48, 49). It is intriguing to speculate that elevated fibrinogen levels could increase susceptibility to infection by enhancing density-dependent virulence gene expression. Moreover, the coagulation system is becoming increasingly recognized as a contributor to the most severe consequences of infection (50). In this regard, the efficacy of activated protein C was tested in an experimental model of S. aureus arthritis and did not enhance survival or limit sepsis (51). These data, in addition to ours, suggest that stabilizing fibrinogen levels is sufficient to permit some of the most severe consequences of this infection and is not beneficial to the host. Further experimentation will be required to confirm whether limiting plasma fibrinogen levels increased as a consequence of the acute phase response will reduce the susceptibility to S. aureus infection.

Pharmacologic intervention in quorum-sensing-regulated virulence is being investigated as a potential therapy for treatment of specific infectious diseases (52, 53, 54, 55). Whereas these strategies are based on developing competitive antagonists of the autoinducer or on production of agents that destroy the autoinducer, our observation is the first to demonstrate that altering a contribution of the host to quorum sensing can have an equally beneficial effect (Fig. 1, A and B). Our data suggest that eliminating the environment in the host that contributes to density-dependent virulence gene expression may be an additional mechanism for enhancing host defense against infection.


   Acknowledgments
 
We acknowledge the expert technical assistance of Susan Alexander, Beth McKown, and Cheryl Pulaski.


   Footnotes
 
1 This work was supported by grants from the National Institutes of Health (to H.D.G. and A.L.C.) and the Merit Review Program of the Department of Veterans Affairs (to H.D.G.). Back

2 Address correspondence and reprint requests to Dr. Hattie D. Gresham, Department of Molecular Genetics and Microbiology, MSC08 4660, 1 University of New Mexico, Albuquerque, NM 87131-0001. E-mail address: hgresham{at}salud.unm.edu Back

3 Abbreviations used in this paper: WT, wild type; cap5, capsule production gene; GFP, green fluorescent protein; hla, hemolysin gene; MIP, macrophage inflammatory protein. Back

Received for publication June 25, 2003. Accepted for publication September 12, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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