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Department of Rheumatology and Inflammation Research, Gteborg University, Gteborg, Sweden
Department of Microbiology, Trinity College, Dublin, Ireland
Staphylococcus aureus is a commonly encountered pathogen in humans, and it has the potential to cause destructive and life-threatening conditions, including septic arthritis. The pathogenicity of staphylococci depends on the expression of virulence factors. Among these, staphylococcal cell-surface proteins with tissue-adhesive functions have been suggested to mediate the colonization of host tissues, a crucial step in the establishment of infection. We investigated the relative contribution of the fibronectin-binding proteins (FnBPs) and fibrinogen-binding clumping factors (Clfs) to staphylococcal virulence in an experimental model of septic arthritis. The results show that these 2 sets of proteins play distinctly different roles in the development and progression of septic arthritis. Although Clfs significantly contributed to the arthritogenicity of S. aureus, FnBPs had no effect on the development of arthritis. Conversely, FnBPs played an important role in the induction of systemic inflammation, characterized by interleukin-6 secretion, severe weight loss, and mortality.
Staphylococcus aureus is an important human pathogenit most frequently causes superficial infections of the skin, but it also has the potential to cause severe systemic infections, such as endocarditis, osteomyelitis, and septic arthritis [1]. The increasing ability of S. aureus to withstand antibiotic treatment is alarming [2] and calls for the development of alternative treatment strategies. An improved knowledge of the bacterium-host interactions taking place during the establishment and course of infection might provide new targets for antistaphylococcal prophylaxis and intervention in the future.
The pathogenicity of S. aureus depends on the expression of a variety of virulence factorsbacterial products that, by different means, enable the establishment of an infection. Tissue-adhesive functions exerted by cell wallassociated proteins of S. aureus seem to be of crucial importance in this context [3].
Clumping factors (Clfs) A and B are 2 structurally related fibrinogen-binding proteins that are expressed on the surface of S. aureus. Both proteins mediate the fibrinogen-dependent adhesion and clumping of S. aureus cells [47]. However, the ligand-binding A regions of ClfA and ClfB interact with different parts of the fibrinogen molecule (the -chain for ClfA and the -chain for ClfB) [68].
Fibronectin-binding proteins (FnBPs) A and B [911] enable staphylococcal adherence to and invasion of a range of cell types, including epithelial cells, endothelial cells, fibroblasts, and osteoblasts [1223]. Through the formation of a fibronectin bridge to the fibronectin-binding integrin 51 expressed on the host cell surface, FnBPs trigger bacterial invasion [12, 20, 21]. Such an invasion might provide a means by which the staphylococci evade host defenses and resist antibiotic killing. Although most studies have focused on the fibronectin-binding activity of the C-terminal D repeats of FnBPs [24], other regions of these proteins also mediate fibronectin binding [20, 25]. Moreover, the A region of FnBPA has recently been shown to interact with the C terminal of the fibrinogen -chain with an affinity similar to that of ClfA [26], which suggests that Clfs and FnBPs may have additive functions with respect to fibrinogen/fibrin-binding during in vivo infection.
The relevance of studying the role that FnBPs and Clfs play as virulence determinants is underscored by previous reports that have shown that genes encoding these proteins are present in virtually all clinical isolates [27, 28]. Interestingly, Proctor et al. [29] observed that S. aureus strains isolated from patients with invasive infection were more readily agglutinated by fibronectin, compared with noninvasive isolates, which possibly indicates that FnBPs contribute to the development of invasive infections.
The objective of the present study was to investigate the relative contributions of FnBPs and Clfs to the development of septic arthritis and systemic inflammation during S. aureus infection. For this purpose, we used a well-established murine model of staphylococcal infection [30, 31] to study gene knockout mutants derived from S. aureus LS-1, a strain isolated from a mouse that spontaneously developed septic arthritis [32].
MATERIALS AND METHODS
Mice and bacterial strains.
Female NMRI mice were obtained from B&K Universal and were maintained in the animal facility at Gteborg University, Gteborg, Sweden, under standard conditions of light and temperature, with free access to standard laboratory chow and water. The mice were used for experiments as approved by the local ethical board.
S. aureus strain LS-1, which was originally isolated after a spontaneous outbreak of S. aureus arthritis in a mouse colony [30, 32], was used as the control (wild-type [wt]) strain in the study. The following mutants of LS-1 were also used: a clfA-clfB- double-mutant strain (DU6013), an fnbA-fnbB- double-mutant strain (DU6012), and a clfA-clfB-fnbA-fnbB- quadruple-mutant strain (DU6014) (table 1). Briefly, the strains were constructed by the transduction of previously isolated insertion mutations, with the exception of a frameshift mutation clfA5 constructed in LS-1 by allele replacement. Insertion mutations were transduced from 8325-4 or Newman backgrounds into LS-1 by use of bacteriophage 85 [33]. Each mutation was validated by phenotypic analysis and Southern hybridization. Phenotypically, the clfA-clfB- and fnbA-fnbB- double-mutant strains were clearly deficient in their abilities to adhere to immobilized fibrinogen and fibronectin, respectively. The clfA-clfB-fnbA-fnbB- quadruple-mutant strain was completely deficient with respect to both binding activities. Furthermore, the mutant strains were not affected in their ability to secrete hemolysins, as verified by culturing on blood-agar plates.
Infection procedure.
Before inoculation, the bacterial strains were cultured on tryptic soy-agar plates for 48 h at 37°C, after which the collected colonies were suspended in PBS supplemented with 10% dimethyl sulfoxide and 5% human albumin (crystallized; ICN Biomedicals). Bacterial suspensions were kept in aliquots at -20°C until they were used. The number of colony-forming units in the suspensions was determined by culturing of several diluted aliquots on horse bloodagar plates. Before inoculation, bacterial suspensions were thawed and washed in PBS. Bacterial pellets were diluted to the desired cell density. Two hundred microliters of PBS that contained 107 cfu of S. aureus was intravenously administered to 8-week-old mice. Viable counts of injection suspensions were assessed in all experiments, and the variations (0.81.3 × 107 cfu/mouse) were judged to be unlikely to affect the course of infection.
Clinical examination of infected mice.
The S. aureusinfected mice were examined individually in a blinded manner. To evaluate the severity of arthritis, an arthritic index was constructed for each mouse at each time point. For this purpose each paw was evaluated and given an estimated score based on a 03 point scale (0, no swelling or erythema; 1, mild swelling and/or erythema; 2, moderate swelling and erythema; 3, marked swelling and erythema). The scores for the 4 paws were thereafter added to form the arthritic index. The overall condition of each mouse was also examined by assessing any change in weight and signs of systemic inflammation (e.g., reduced alertness and ruffled coat). In cases of severe systemic infection, mice were killed by cervical dislocation and considered to have died from sepsis.
Histopathological examination.
Histopathological examination of the joints was performed after routine fixation, decalcification, and paraffin embedding of specimens. Tissue sections from upper and lower extremities were prepared and stained with hematoxylin-eosin. All slides were examined in a blinded manner and scored with respect to severity of synovitis and cartilage/bone destruction as follows. Synovitis scores were 0, no synovitis; 1, mild synovial hypertrophy (synovial membrane thickness of >2 cell layers); 2, moderate synovial hypertrophy and infiltration of inflammatory cells; and 3, marked synovial hypertrophy and infiltration of inflammatory cells. Cartilage and bone-destruction scores were 0, no destruction; 1, mild destruction; and 2, marked destruction. Histopathological scores for synovitis and erosivity, respectively, were constructed for each mouse by summing the scores for the examined joints (elbow and wrist/carpal joints [front extremities] and knee and ankle/tarsal joints [hind extremities]).
Determination of bacterial load in kidneys.
Kidneys were homogenized and mixed with cold PBS. The suspension was serially diluted and thereafter spread onto horse bloodagar plates. The number of colony-forming units formed after 24 h of incubation at 37°C was used to assess the bacterial load.
Interleukin (IL)6 analysis.
For measurement of IL-6 levels in serum samples, a bioassay based on the murine hybridoma cell line B13.29, subclone B9, was used as described elsewhere [34]. This cell line is dependent on exogenously supplied IL-6 for its growth [35, 36].
Statistical analyses.
The Mann-Whitney U test or Kruskal-Wallis test (with post hoc analysis) was used to compare severity of arthritis, weight changes, histopathological scores, and serum IL-6 levels between groups. The incidences of arthritis and mortality during the experimental period were analyzed with Fisher's exact test. P .05 was considered to be statistically significant.
RESULTS
To further investigate the contribution of these 4 proteins to S. aureus virulence, the impact of clfA-clfB- or fnbA-fnbB- was compared with that of wt LS-1 and that of clfA-clfB-fnbA-fnbB-. Interestingly, throughout the experiment, the mice infected with clfA-clfB- displayed significantly less-severe arthritis than did the mice infected with the wt strain (figure 2a; P < .001.01). This was partly due to a higher incidence of arthritis induced by the wt strain (83% vs. 50% induced by clfA-clfB-; P = .03). Notably, the carriage of intact clfA and clfB genes by the fnbA-fnbB- double-mutant strain significantly contributed to arthritogenicity, because it gave rise to more-severe (figure 2a; P < .001.02) and frequent (92% vs. 59%; P = .05) arthritis than the quadruple-mutant strain.
In contrast, the fnbA-fnbB- strain was not attenuated in its ability to cause arthritis, compared with the wt strain (figure 2a; incidence of arthritis, 83% and 92% for LS-1 and fnbA-fnbB--infected groups, respectively). Furthermore, the arthritogenicity of the quadruple-mutant strain was not reduced, compared with that of the clfA-clfB- double-mutant strain (which carries the genes for fnbA and fnbB) (figure 2a; incidence of arthritis, 50% and 59%, respectively, for the clfA-clfB-- and quadruple-mutantinfected groups). These findings indicate that Clfs are important mediators of arthritis and that FnBPs are less important in this regard.
After inoculation, LS-1infected mice lost significantly more weight than did mice infected with the clfA-clfB- double-mutant strain (figure 2b; P < .001.05, days 27). This difference was most pronounced early after inoculation. Notably, the fnbA-fnbB- double-mutant strain (which carries the clfA and clfB genes) gave rise to more weight reduction than did the quadruple-mutant strain only transiently, on day 2 (figure 2b, *3; P < .05). Furthermore, clfA and clfB gene carriage did not significantly affect mortality (48% and 23% mortality for LS-1 and clfA-clfB--infected mice, respectively [P, not significant], and 8% and 0% mortality for fnbA-fnbB- and quadruple-mutantinfected mice, respectively [P, not significant]).
With respect to infection-induced weight loss at all time points, the fnbA-fnbB- strain was significantly less virulent than was the LS-1 (figure 2b; P < .001.01). Also, the clfA-clfB- double-mutant strain was significantly more virulent than the quadruple-mutant strain in this respect, from days 4 to 10 (figure 2b, *4, *5, and *6; P < .001). Furthermore, the incidence of mortality was significantly higher after inoculation with strains carrying fnbA and fnbB genes, compared with that after infection with their counterparts (48% and 8% mortality for LS-1 and fnbA-fnbB--infected mice, respectively [P = .03], and 23% and 0% mortality for clfA-clfB-- and quadruple-mutantinfected mice, respectively [P = .05]).
Accordingly, both Clfs and FnBPs affect weight loss after staphylococcal inoculation, although they act during different stages of the infection. The expression of FnBPs by staphylococci clearly contributes to mortality, whereas our results provide no evidence that Clfs play a role in this respect. Notably, we did not observe any significant differences between any of the staphylococcal strains with respect to the number of bacteria retrieved from the kidneys 910 days after inoculation (data not shown).
In summary, our findings indicate that Clfs are important arthritogenic factors that, by mediating the rapid onset of localized joint inflammation, also contribute to slight weight loss, whereas FnBPs contribute mainly to the induction of systemic inflammation, which is characterized by severe weight loss and mortality, without affecting arthritis development.
Contribution to synovitis and erosive damage of cartilage and bone by Clf expression.
Histopathological examination of joint sections revealed that inoculation with the wt strain LS-1 caused significantly more synovitis (figure 3; P < .001) and cartilage and bone erosion (figure 3; P < .001) than did inoculation with the clfA-clfB- double-mutant strain. Furthermore, the fnbA-fnbB- double-mutant strain gave rise to significantly more synovitis (figure 3; P < .05) and erosive damage (figure 3; P < .01) than did the quadruple-mutant strain (clfA-clfB-fnbA-fnbB-). These findings indicate that the expression of Clfs contributes to synovitis as well as to persistent damage of cartilage and bone after staphylococcal infection. In contrast, the expression of FnBPs does not contribute to synovitis or erosive damage, as judged by histopathological analysis (figure 3).
DISCUSSION
Septic arthritis caused by S. aureus is a severe joint infection that is highly associated with irreversible joint damage and death in humans [37]. The staphylococci are believed to reach the joint area via hematogenous spread from an initial nidus of infection, such as an infected wound [38]. The bacterium-host interactions that take place during the establishment of a joint infection remain largely unknown. However, tissue-adhesive proteins expressed on the staphylococcal cell surface likely contribute to the pathogenicity of staphylococci. Indeed, staphylococcal cell-surface proteins that mediate attachment to collagen, fibronectin, and fibrinogen/fibrin have all been shown to promote the colonization of catheter-damaged heart valves [3944]. In the present study, we investigated the relative impact of FnBPA, FnBPB, ClfA, and ClfB on the development and progression of murine septic arthritis and systemic inflammation. For this purpose, we used S. aureus LS-1, a murine septic arthritis isolate, and gene knockout mutants derived from this strain.
Our results clearly indicate distinct roles of Clfs and FnBPs in the development and progression of staphylococcal infection. Although the expression of Clfs potentiated the induction of localized joint inflammation and contributed to erosive lesions of cartilage and bone, FnBPs did not affect arthritis development at all. On the other hand, FnBPs significantly contributed to systemic inflammation, characterized by weight loss, IL-6 secretion, and mortality. Notably, Clfs also contributed to weight loss. However, this effect was most pronounced during the early stage of infection, and the Clfs did not significantly affect mortality or serum levels of IL-6. Thus, we believe that the mild weight loss triggered by Clf expression is primarily due to the development of localized joint inflammation, whereas the severe FnBP-dependent weight loss seen later on is characteristic of a systemic proinflammatory response.
The functional overlap between Clfs and FnBPs that has been suggested in the colonization of damaged heart valves [3943] does not hold true with respect to the induction of arthritis. Our findings indicate that the staphylococcal fibronectin-binding phenotype is insufficient for the induction of septic arthritis. However, staphylococcal interaction with fibronectin is likely involved in the systemic inflammation triggered by FnBP expression.
Interestingly, fibronectin binding mediated by FnBPs is required for staphylococcal invasion of several different cell types, including endothelial cells [12, 19, 20]. The internalization of S. aureus by endothelial cells in vitro resulted in the production of proinflammatory chemokines and cytokines, such as IL-8, monocyte chemotactic protein1, IL-6, and IL-1 [4548]. Furthermore, endothelial adhesiveness for monocytes and granulocytes increased on staphylococcal internalization, because of the up-regulation of adhesion molecule expression [49]. Because the vascular endothelium constitutes a substantial surface structure in close contact with the blood, the invasion of endothelial cells by fibronectin-coated bloodborne staphylococci could be an important trigger for the severe systemic inflammation associated with FnBP expression.
As for the contribution of Clfs to staphylococcal arthritogenicity, we question that their fibrinogen-binding functions play an important role. Indeed, we have recently demonstrated that ClfA-mediated arthritogenicity is retained despite the in vivo depletion of fibrinogen [50]. In addition, FnBPA has recently been shown to interact with the C terminal of the fibrinogen -chain with an affinity similar to that of ClfA [26]. Thus, if an interaction between ClfA and the fibrinogen -chain C terminal were a prerequisite for ClfA-mediated arthritogenicity, FnBPA would also be expected to contribute to the induction of arthritis, at least to some extent. However, as has been shown in the present article, this is not the case. Rather, we find it likely that one or both of the Clfs promote the establishment of joint infection by mediating interaction(s) with as yet unknown host ligand(s).
Virtually all clinical isolates of S. aureus carry genes that encode the virulence factors identified in the present study [27, 28], which further suggests their importance in the pathogenesis of staphylococcal infections. Because of the frequent occurrence of these proteins, they are strong candidate target structures for antistaphylococcal prophylaxis and intervention in humans. Indeed, findings in animal models of infection have supported this idea [5158].
To summarize our findings, Clfs were identified as important arthritogenic factors, whereas FnBPs contributed to infection-induced weight loss and mortality without affecting the development of arthritis. Thus, these virulence factors play distinct roles in the pathogenesis of staphylococcal infections.
Acknowledgments
We gratefully acknowledge the skillful technical assistance of Berit Ericsson, Ing-Marie Jonsson, and Margareta Verdrengh.
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