Abstract

Staphylococcus aureus is a major cause of nosocomial and community-acquired infection. It expresses several factors that promote avoidance of phagocytosis by polymorphonuclear leucocytes. Clumping factor A (ClfA) is a fibrinogen-binding surface protein of S. aureus that is an important virulence factor in several infection models. This study investigated whether ClfA is an antiphagocytic factor, and whether its antiphagocytic properties were based on its ability to bind fibrinogen. In S. aureus, ClfA was shown to be of equal importance to protein A, the antiphagocytic properties of which are well established. ClfA expressed in a surrogate Gram-positive host was also found to be antiphagocytic. A ClfA mutant that was unable to bind fibrinogen had a similar antiphagocytic effect to native ClfA in the absence of fibrinogen. ClfA inhibited phagocytosis in the absence of fibrinogen, and showed enhanced inhibition in the presence of fibrinogen.

Introduction

Staphylococcus aureus causes a wide variety of diseases, including abscesses, septicaemia, septic arthritis and infective endocarditis. Immunity to staphylococcal disease involves the binding of complement and opsonizing antibodies to the bacterial surface, which leads to activation of phagocytosis by human polymorphonuclear leucocytes (PMNL) (Cunnion et al., 2003; Rooijakkers et al., 2005). Once S. aureus penetrates the barrier of the skin PMNL are the critical first line of defence (Verdrengh & Tarkowski, 1997).

Staphylococcus aureus expresses several factors that promote avoidance of phagocytosis by PMNL (Foster, 2005; Rooijakkers et al., 2005). Protein A is a virulence factor in experimental infection models of subcutaneous infection, murine sepsis and septic arthritis and staphylococcal pneumonia (Patel et al., 1987; Gomez et al., 2004; Palmqvist et al., 2002). It is believed to be antiphagocytic due to its ability to bind the Fc region of immunoglobulin G (IgG) (Gemmell et al., 1991). Capsular polysaccharide is a virulence factor in a murine bacteraemia model (Thakker et al., 1998). The antiphagocytic effect of capsule is due to its ability to impede binding by opsonizing antibodies that recognize antigens on the cell surface, as well as to inhibit complement fixation (Thakker et al., 1998; Cunnion et al., 2003). This contributes to the virulence of S. aureus by preventing bacterial clearance from the bloodstream, liver and spleen (Luong & Lee, 2002). Poly-N-acetylglucosamine also contributes to virulence in animal models of bacteraemia and renal abscess formation by inhibiting complement-mediated phagocytic uptake by PMNL (Kropec et al., 2005).

Clumping factor A (ClfA) is a fibrinogen-binding surface protein of S. aureus (McDevitt et al., 1997). It contains an N-terminal secretory signal peptide, followed by a 520-residue region A that contains the ligand-binding domain, region R consisting primarily of SD dipeptide repeats and a C-terminal domain that allows anchoring to the cell wall peptidoglycan (McDevitt et al., 1995).

Clumping factor A is an important virulence factor in several infection models, including rat experimental endocarditis (Moreillon et al., 1995), murine sepsis and septic arthritis (Josefsson et al., 2001) and rabbit infective endocarditis (Vernachio et al., 2003). By comparing wild-type S. aureus with ClfA-deficient mutants and by transfer of clfA to Streptococcus gordonii (Stutzmann Meier et al., 2001) and to Lactococcus lactis, ClfA was identified as an important factor in mediating endovascular infection (Que et al., 2001). ClfA-deficient strains are also attenuated in a murine model of sepsis and septic arthritis (Josefsson et al., 2001; Vernachio et al., 2003). In this model immunization with recombinant region A of ClfA was protective, as was passive immunization with human IgG containing a high titre against ClfA (Josefsson et al., 2001).

It is possible that ClfA acts as a virulence factor in certain infections by inhibiting phagocytosis, as well as promoting adhesion to fibrin and fibrinogen. This study investigated whether ClfA is antiphagocytic and if this effect is due to its ability to bind fibrinogen.

Materials and methods

Bacterial strains and growth conditions

Bacterial strains are listed in Table 1. Staphylococcus aureus strains were cultured on trypticase soy agar or broth (Oxoid, Basingstoke, UK) at 37°C with shaking at 200 r.p.m. for liquid cultures. Lactococcus lactis strains were cultured in M17 medium (Difco, Detroit, MI) containing 0.5% glucose without shaking at 30°C. Antibiotics were incorporated where appropriate at the following concentrations: 10 μg mL−1 chloramphenicol, 5 μg mL−1 erythromycin and 10 μg mL−1 kanamycin.

1

Strains and plasmids used in this study

 Properties References 
Strain 
Staphylococcus aureus 
Newman NCTC 8178 Duthie & Lorenz (1952) 
Newman clfA clfA::ErmR McDevitt (1994) 
Newman spa spa::KanR This study 
Newman spa clfA spa::KanRclfA::ErmR This study 
Lactococcus lactis 
MG1363 Plasmid-free derivative of NCD 0712 Gasson (1983) 
NZ9800 MG1363 bearing nisin resistance plasmid [CamRKuipers (1993) 
Plasmid 
pKS80 L. lactis expression vector [ErmRWells (1993) 
pKS80 clfA pKS80 containing clfA coding sequence [ErmRO'Brien (2002) 
pKS80 spa pKS80 containing spa coding sequence [ErmRO'Brien (2002) 
pNZ8037 Nisin-inducible L. lactis expression vector [CamRde Ruyter (1996) 
pNZ8037 clfA pNZ8037 containing clfA coding sequence [CamRLoughman (2005) 
pNZ8037 clfAPY pNZ8037 containing clfA coding sequence, incorporating mutations [CamRLoughman (2005) 
 Properties References 
Strain 
Staphylococcus aureus 
Newman NCTC 8178 Duthie & Lorenz (1952) 
Newman clfA clfA::ErmR McDevitt (1994) 
Newman spa spa::KanR This study 
Newman spa clfA spa::KanRclfA::ErmR This study 
Lactococcus lactis 
MG1363 Plasmid-free derivative of NCD 0712 Gasson (1983) 
NZ9800 MG1363 bearing nisin resistance plasmid [CamRKuipers (1993) 
Plasmid 
pKS80 L. lactis expression vector [ErmRWells (1993) 
pKS80 clfA pKS80 containing clfA coding sequence [ErmRO'Brien (2002) 
pKS80 spa pKS80 containing spa coding sequence [ErmRO'Brien (2002) 
pNZ8037 Nisin-inducible L. lactis expression vector [CamRde Ruyter (1996) 
pNZ8037 clfA pNZ8037 containing clfA coding sequence [CamRLoughman (2005) 
pNZ8037 clfAPY pNZ8037 containing clfA coding sequence, incorporating mutations [CamRLoughman (2005) 
*

ErmR, KanR and CamR; resistance to erythromycin, kanamycin and chloramphenicol, respectively.

Transduction

A null mutation in protein A was transduced from S. aureus 8325-4 spa::KanR (Patel et al., 1987) by generalized phage transduction using phage 85 (Foster, 1998) to strains Newman (Duthie & Lorenz, 1952) and Newman clfA (McDevitt et al., 1994) with selection for kanamycin resistance. The fidelity of transductants was verified by Southern blotting and by Western immunoblotting of solublized cell wall proteins with anti-ClfA polyclonal antisera and with horseradish peroxidase-conjugated goat IgG.

PMNL isolation

Fresh whole blood was obtained from healthy volunteers, heparinized and mixed with an equal volume of phosphate-buffered saline (PBS). This was centrifuged through step gradients of Histopaque (ρ=1.077, Sigma, St Louis, MO) and Ficoll-paque (ρ=1.119, Amersham, Chalfont St Giles, UK) and PMNL were aspirated from the buffy coat between the Ficoll and Histopaque layers. Cells were washed in RPMI 1640 medium [containing 10 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 25 mM glutamine and 0.05% (v/v) human serum albumin] and resuspended in water. After a 30 s osmotic shock to lyse contaminating red blood cells, 10 × PBS was added and the cells were again washed in RPMI. PMNL were counted in a haemocytometer (bright line, Neubauer) and adjusted to 5 × 106 cells per mL in RPMI. This procedure typically yielded >97% PMNL with >95% viability, as determined by trypan blue exclusion. All reagents used in PMNL isolation were certified endotoxin-free.

Phagocytosis of bacteria by human PMNL

Whole bacterial cells grown to stationary phase were washed twice in PBS and labelled with 30 μg mL−1 fluorescein isothiocyanate (FITC) in PBS for 1 h at 37°C with shaking. Cells were washed three times in PBS and enumerated in a Neubauer haemocytometer (Brand GmbH, Wertheim, Germany), adjusted to 1 × 109 CFU mL−1 in RPMI and stored frozen at −20°C. FITC-labelling of bacteria did not affect their ability to bind fibrinogen (data not shown). Bacteria were thawed on ice and diluted to 5 × 107 CFU mL−1 in RPMI. Pooled human serum was diluted in RPMI and fibrinogen (Calbiochem, Schwalbach, Germany) was added to serum where necessary. The final concentration of fibrinogen, where present, was the same percentage as the serum concentration, calculated as 3 mg mL−1=100%. Bacteria (50 μL) were opsonized in 10 μL diluted serum (with or without fibrinogen) for 10 min at 37°C, followed by addition of 50 μL prewarmed PMNL and incubation at 37°C with vigorous shaking. The final bacteria : PMNL ratio was 10 : 1. Reactions were stopped after 5–15 min by addition of 100 μL ice-cold 1% (w/v) paraformaldehyde in PBS. The percentage of PMNL bearing FITC-labelled bacteria (% phagocytosis) was determined by flow cytometric analysis of 5000 cells with manual gating using a FACScan flow cytometer (Becton Dickinson, Oxford, UK). The percentage of internalized bacteria was determined by trypan blue quenching of extracellular fluorescence (Benne et al., 1997; Su et al., 2001). Addition of 20 μg mL−1 (final concentration) trypan blue (Merck, Schwalbach, Germany) demonstrated that >85% bacteria associated with PMNL were internalized (data not shown). The absence of bacterial clumping during the assay was verified microscopically. Samples were prepared in triplicate and all experiments were repeated a minimum of three times using the blood of different donors. Statistical analyses were performed using the Student's t-test for paired data in Kaleidagraph (Synergy software). P values <0.01 were considered significant.

Whole cell dot immunoblotting

Quantitation of the relative amounts of protein expressed by L. lactis strains was carried out by whole cell dot immunoblotting, as described previously (Loughman et al., 2005).

Results

In order to investigate the antiphagocytic effects of protein A and ClfA, strains Newman, Newman clfA, Newman spa and Newman spa clfA were tested for their uptake by fresh human PMNL in the presence of human serum opsonins. Increasing concentrations of serum resulted in increased phagocytic uptake of all strains, indicating that the process was dependent on serum opsonins. At 1% normal human serum (NHS) strain Newman was phagocytosed by approximately 46% of PMNL (Fig. 1). Newman spa and Newman clfA were taken up by approximately 45% of PMNL at this serum concentration, whereas the Newman spa clfA double mutant was taken up by 72% of PMNL (P<0.001). This demonstrated that both protein A and ClfA can contribute to the inhibition of phagocytosis of Staphylococcus aureus Newman, and that the absence of one factor was compensated for by the presence of the other. The absence of both factors led to a significant increase in phagocytic uptake.

1

Phagocytosis of Newman strains by human polymorphonuclear leucocytes (PMNL). Freshly isolated human PMNL were incubated with Staphylococcus aureus Newman strains grown to stationary phase and opsonized with varying concentrations of normal human serum in the absence (solid lines) or presence (dashed lines) of physiologically relevant concentrations of fibrinogen. Wild-type Newman (squares) expresses both clumping factor A (ClfA) and protein A, Newman spa (diamonds) is protein A-deficient, Newman clfA (circles) is ClfA-deficient and Newman spa clfA (triangles) is deficient in both protein A and ClfA. The percentage of PMNL containing bacteria is expressed as % phagocytosis. All samples were prepared in triplicate. Data are representative of at least three independent experiments using three different donors.

1

Phagocytosis of Newman strains by human polymorphonuclear leucocytes (PMNL). Freshly isolated human PMNL were incubated with Staphylococcus aureus Newman strains grown to stationary phase and opsonized with varying concentrations of normal human serum in the absence (solid lines) or presence (dashed lines) of physiologically relevant concentrations of fibrinogen. Wild-type Newman (squares) expresses both clumping factor A (ClfA) and protein A, Newman spa (diamonds) is protein A-deficient, Newman clfA (circles) is ClfA-deficient and Newman spa clfA (triangles) is deficient in both protein A and ClfA. The percentage of PMNL containing bacteria is expressed as % phagocytosis. All samples were prepared in triplicate. Data are representative of at least three independent experiments using three different donors.

The addition of a physiologically relevant concentration of fibrinogen had no significant impact on the level of uptake of the ClfA-deficient strains Newman clfA and Newman spa clfA (Fig. 1). However, the levels of uptake of both the wild-type strain and of Newman spa in 1% NHS were significantly reduced in the presence of fibrinogen (P<0.005 and P<0.01, respectively). This indicated that strains bearing ClfA on their cell surface had enhanced antiphagocytic properties in the presence of soluble fibrinogen. The presence of fibrinogen had no effect on the phagocytic uptake of cells lacking ClfA.

Bacterial cells used in this assay were grown to stationary phase to minimize any possible contribution of the fibrinogen-binding protein clumping factor B, which is absent in stationary phase due to proteolysis and cessation of clfB transcription (McAleese et al., 2001). The fibronectin-binding proteins are also predominantly expressed during the exponential phase of growth and can also bind fibrinogen. However, the fnbA and fnbB genes of strain Newman each have a frameshift mutation that results in secretion of truncated forms of the proteins (Grundmeier et al., 2004). Thus, ClfA was the only known fibrinogen-binding surface protein expressed by the cells in this assay.

In order to investigate further the antiphagocytic properties of ClfA, the protein was expressed on the surface of the Gram-positive surrogate host Lactococcus lactis (O'Brien et al., 2002). Increasing concentrations of serum led to increased uptake of L. lactis cells (Fig. 2), indicating that efficient opsonization of the bacteria was required to facilitate phagocytosis. Lactococcus lactis cells bearing an empty plasmid vector (pKS80) were taken up by 50% of PMNL at 1% NHS. The addition of fibrinogen had no significant impact on the level of uptake of these bacteria. L. lactis expressing ClfA (pKS80 clfA) were taken up by significantly fewer PMNL (25% at 1% NHS, P=0.002 compared with L. lactis pKS80). The addition of fibrinogen further reduced this level to 2% (P=0.001). Lactococcus lactis expressing protein A were taken up by only 9% of PMNL at 1% NHS (P=0.003 compared with L. lactis pKS80), however the addition of fibrinogen did not affect the level of uptake of this strain. Protein A showed very potent inhibition of phagocytosis, but showed no fibrinogen-dependent reduction of uptake. ClfA was capable of inhibition of phagocytosis in the absence of fibrinogen, and showed increased inhibition in the presence of fibrinogen.

2

Phagocytosis of Lactococcus lactis strains expressing clumping factor A (ClfA) or protein A. Freshly isolated human polymorphonuclear leucocytes (PMNL) were incubated with L. lactis MG1363 strains expressing no protein (pKS80, squares), ClfA (pKS80 clfA, diamonds) or protein A (pKS80 spa, circles) grown to stationary phase and opsonized with varying concentrations of normal human serum in the absence (solid lines) or presence (dashed lines) of physiologically relevant concentrations of fibrinogen. The percentage of PMNL containing bacteria is expressed as % phagocytosis. All samples were prepared in triplicate. Data are representative of at least three independent experiments using three different donors.

2

Phagocytosis of Lactococcus lactis strains expressing clumping factor A (ClfA) or protein A. Freshly isolated human polymorphonuclear leucocytes (PMNL) were incubated with L. lactis MG1363 strains expressing no protein (pKS80, squares), ClfA (pKS80 clfA, diamonds) or protein A (pKS80 spa, circles) grown to stationary phase and opsonized with varying concentrations of normal human serum in the absence (solid lines) or presence (dashed lines) of physiologically relevant concentrations of fibrinogen. The percentage of PMNL containing bacteria is expressed as % phagocytosis. All samples were prepared in triplicate. Data are representative of at least three independent experiments using three different donors.

In order to investigate whether the effect of fibrinogen was due to direct binding to ClfA, a derivative of ClfA with substitutions in residues essential for fibrinogen binding (ClfAPY) was used (Loughman et al., 2005). Lactococcus lactis NZ9800 allows inducible expression of genes cloned into the vector pNZ8037. The relative levels of expression of ClfA and ClfAPY were monitored by whole cell dot immunoblotting and shown to be equal before cell labelling (data not shown). Lactococcus lactis NZ9800 bearing the empty vector (pNZ8037) was taken up by approximately 48% of PMNL at 1% NHS both in the presence and absence of fibrinogen (Fig. 3). Lactococcus lactis NZ9800 expressing ClfA (pNZ8037 clfA) was taken up by approximately 32% of PMNL at the same serum concentration in the absence of fibrinogen, and the level of uptake was further reduced to approximately 4% in the presence of fibrinogen (P<0.001 compared with L. lactis pNZ8037). Lactococcus lactis expressing ClfAPY (pNZ8037 clfAPY), which does not bind fibrinogen (Loughman et al., 2005) was taken up by approximately 27% of PMNL at 1% NHS. However, the addition of soluble fibrinogen did not influence the level of uptake of this strain (Fig. 3). This indicated that direct binding of fibrinogen to ClfA on the surface of the bacteria caused increased inhibition of phagocytosis by human PMNL.

3

Phagocytosis of Lactococcus lactis strains expressing clumping factor A (ClfA) or ClfAPY. Freshly isolated human PMNL were incubated with L. lactis NZ9800 strains expressing no protein (pNZ8037, squares), ClfA (pNZ8037 clfA, diamonds) or ClfAPY (pNZ8037 clfAPY, circles) grown to stationary phase and opsonized with varying concentrations of normal human serum in the absence (solid lines) or presence (dashed lines) of physiologically relevant concentrations of fibrinogen. The percentage of polymorphonuclear leucocytes (PMNL) containing bacteria is expressed as % phagocytosis. All samples were prepared in triplicate. Data are representative of at least three independent experiments using three different donors.

3

Phagocytosis of Lactococcus lactis strains expressing clumping factor A (ClfA) or ClfAPY. Freshly isolated human PMNL were incubated with L. lactis NZ9800 strains expressing no protein (pNZ8037, squares), ClfA (pNZ8037 clfA, diamonds) or ClfAPY (pNZ8037 clfAPY, circles) grown to stationary phase and opsonized with varying concentrations of normal human serum in the absence (solid lines) or presence (dashed lines) of physiologically relevant concentrations of fibrinogen. The percentage of polymorphonuclear leucocytes (PMNL) containing bacteria is expressed as % phagocytosis. All samples were prepared in triplicate. Data are representative of at least three independent experiments using three different donors.

Discussion

The importance of ClfA in the virulence of Staphylococcus aureus in a variety of animal models suggests that the protein might have an antiphagocytic effect. This study investigated a possible role for ClfA in the resistance of S. aureus to phagocytosis by human PMNL. The experiments described here show clearly that the well known antiphagocytic action of protein A (Rooijakkers et al., 2005) can be clearly demonstrated in our in vitro phagocytosis assay. Null mutation of the gene responsible in virulent staphylococci (Newman spa) resulted in increased sensitivity to phagocytosis. Moreover, introduction of the gene into the nonvirulent Lactococcus lactis (pKS80 spa) showed clear inhibition of phagocytosis. In the same system, it was also shown that ClfA is an important antiphagocytic factor for S. aureus, with both fibrinogen-dependent and fibrinogen-independent mechanisms. In S. aureus Newman, ClfA was shown to be of equal importance to protein A. Using ClfA expressed in a surrogate Gram-positive host we could also show that it possesses strong antiphagocytic properties. A ClfA mutant that was unable to bind fibrinogen had a similar antiphagocytic effect as native ClfA in the absence of fibrinogen, further establishing the existence of a fibrinogen-independent mechanism.

When S. aureus is grown in plasma it becomes coated with soluble plasma components that interfere with bacterial adherence to immobilized ligands (Massey et al., 2002). In this study, inactivation of both clfA and spa in a microencapsulated strain of S. aureus caused a significant increase in the level of phagocytosis. Although Newman can express a type 5 capsular polysaccharide, the growth conditions used in this study would not have promoted its expression at high levels. The presence of either ClfA or protein A alone seemed to be sufficient to inhibit phagocytosis. It appears that being coated with either immunoglobulin or fibrinogen, or a mixture of both, is sufficient to impede phagocytosis. This indicates that protein A and ClfA are two major antiphagocytic factors for S. aureus.

Anti-ClfA antibodies are present both in normal and convalescent sera (Colque-Navarro et al., 2000; Dryla et al., 2005). This implies that normal exposure to S. aureus as a commensal of the skin causes the formation of anti-ClfA antibodies, but that the antibodies in normal sera are not protective against invasive S. aureus disease. These antibodies have opsonizing activity (Josefsson et al., 2001; Vernachio et al., 2003; Patti, 2004). Immunization of mice with recombinant ClfA induced anti-ClfA antibody production, whereas infection of naïve mice with S. aureus Newman did not (Josefsson et al., 2001). Perhaps the ability of ClfA on the surface of S. aureus cells to bind fibrinogen in the bloodstream prevents the development of high levels of specific antibodies directed against it. In addition, the recent observation that low levels of anti-ClfA antibodies stimulate platelet aggregation may go some way to explaining why the low levels of antibodies in normal serum are not protective against staphylococcal infection (Loughman et al., 2005).

It is likely that the pooled human serum used in this study contained low levels of antibodies against ClfA (Dryla et al., 2005; Loughman et al., 2005). They might interact with ClfA in one of two ways. They may compete with fibrinogen to bind ClfA and promote phagocytosis, either alone via Fc receptors on the PMNL surface or by stimulating complement activation. Alternatively they may promote phagocytosis by binding to ClfA along with bound fibrinogen.

However, ClfA is antiphagocytic despite the presence of low levels of anti-ClfA antibodies in normal human sera. Perhaps opsonins bound to bacteria are prevented from interacting productively with Fc receptors and complement receptors on the PMNL surface, possibly through steric hindrance by bound fibrinogen, immunoglobulins bound to protein A, or other serum or bacterial factors. These may form a protective layer of host proteins around the bacterium, preventing access by antibodies to antigens on the cell wall surface such as peptidoglycan, teichoic acids and other proteins, or preventing interactions between antibodies bound to these antigens and Fc receptors on the PMNL surface. Such a protective layer might also interfere with complement deposition.

Attempts to measure the survival of bacteria following uptake gave paradoxical results. After 30 min c. 50% of wild-type bacteria were killed, however the ClfA and protein A mutants were killed less than wild type during this time (unpublished data), despite being taken up more efficiently (Fig. 1). This may have been caused by alteration of the fidelity of the phagosome due to changes in major surface proteins on the bacterial surface, as was observed by Gresham (2000). The fluorescence-based assay used in this study allows analysis of bacterial uptake independently from the complex interactions involved in bacterial killing.

The presence of two mechanisms of resistance to phagocytic uptake, one fibrinogen-dependent and the other fibrinogen-independent may reflect the presence of different niches for the bacteria within the host during disease progression, in which fibrinogen is available or unavailable. For example, it appears likely that the ability of ClfA to inhibit phagocytosis independently of fibrinogen may be important in the murine sepsis and septic arthritis model, as depletion of free fibrinogen in the bloodstream led to aggravation of septic arthritis caused by ClfA-producing S. aureus strains (Palmqvist et al., 2004). This fibrinogen-independent inhibition may also be important in vivo in environments with an elevated Ca2+ concentration, as Ca2+ inhibits fibrinogen binding by ClfA with an IC50 of 2.5 mM (O'Connell et al., 1998).

The ability of ClfA to inhibit phagocytosis by human PMNL may explain its importance in S. aureus virulence in a variety of animal models of infection. It is likely also to be the reason that high-titre, but not low-titre anti-ClfA antibodies are protective.

Acknowledgements

This work was supported by the Health Research Board of Ireland and Science Foundation Ireland.

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