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Virulence Potential of the Staphylococcal Adhesin CNA in Experimental Arthritis Is Determined by Its Affinity for Collagen

  1. Yi Xua,
  2. Jorge M. Rivasa,
  3. Eric L. Brown,
  4. Xiaowen Liang and
  5. Magnus Höök
  1. The Center for Extracellular Matrix Biology, Texas A & M University System Health Science Center, Institute of Biosciences and Technology, Houston
  1. Reprints or correspondence: Dr. Magnus Höök, The Center for Extracellular Matrix Biology, Texas A & University System Health Science Center, Institute of Biosciences and Technology, 2121 W. Holcombe Blvd., Ste. 603, Houston, TX 77030 (mhook{at}ibt.tamushsc.edu)

Background. Staphylococcus aureusis a major cause of bacterial arthritis, which often results in severe joint damage. CNA, a collagen adhesin of S. aureus,was shown to be a virulence factor in several animal models. However, the precise molecular mechanism by which CNA contributes to virulence remains unclear.

Methods. We examined the role of the collagen-binding function of CNA in a mouse model of septic arthritis by comparing the virulence of isogenic strains of S. aureusexpressing (1) wild-type CNA, (2) a truncated form of CNA (CNA35) with a higher affinity for collagen than the wild type, (3) CNA35 containing a single point mutation resulting in loss of collagen binding, (4) CNA lacking the collagen-binding domain, and (5) the collagen-binding domain of ACE (adhesin of collagen from Enterocoecus faecalis).

Results and conclusions. The results provide, for the first time, direct evidence that the virulence of CNA depends on its collagen-binding ability. Collagen binding facilitated early colonization of the joints of mice. Furthermore, the virulence potential of the adhesin is determined by the adhesin's affinity for its ligand, as well as its binding kinetics.

Staphylococcus aureus is the bacterium most commonly associated with septic arthritis [1], which often results in severe and irreversible joint damage and, occasionally, death. The development of S. aureus-medmtedarthritis is likely to be initiated by the successful adherence to and colonization of joint tissues by staphylococci. Earlier studies from our group demonstrated that the collagen adhesin CNA was necessary and sufficient for S. aureusto attach to cartilage in vitro [2]. Mice infected with CNA-deficient S. aureuspresented with a reduced incidence of arthritis, compared with mice infected with CNA-expressing S. aureus[3]. Furthermore, vaccination with a recombinant fragment of CNA protected mice from septic death after an infection with a lethal dose of S. aureus[4]. Studies from other groups also indicated that CNA-deficient S. aureusstrains were less virulent in their capacity to induce endocarditis [5], osteomyelitis [6], and keratitis [7]. Two recent reports indicated that the cnagene was associated more frequently with isolates from patients than with isolates from healthy adults [8, 9]. Taken together, these studies demonstrate that CNA is a virulence factor of importance in the pathogenesis of S. aureusinfections.

CNA belongs to the MSCRAMM (microbial surface component recognizing adhesive matrix molecule) family of adhesins [10]. It contains from the N-terminus, a signal peptide, an A domain followed by 1–4 repeats, the cell-wall anchoring region, a transmembrane segment, and a short cytoplasmic tail. The A domain is responsible for binding to several types of collagen, whereas the repeats have no known effect on binding [10-12]. The function of the repeats is unknown, but this region is thought to form a stalk that displays the A domain on the cell surface [11, 13, 14].

Recently, CNA-like collagen-binding MSCRAMMs have been identified in other bacteria, among which are ACE (adhesin of collagen from Entero coccus faecalis)[15], Acm (adhesin of collagen from Enterococcus faecium)[16], and CNE (collagen-binding protein from Streptococcus equi)[17]. These proteins all share a domain organization similar to that of CNA, in which the A domain is responsible for their collagen-binding activities; however, the specific binding mechanisms for each collagen adhesin may be somewhat different. ACE40, a His-tag fusion protein containing the ACE A domain, bound collagen with an affinity similar to, but with kinetics different from, that of the CNA A domain [15]. Specifically, ACE40 displayed a much faster association and dissociation rate and recognized fewer binding sites in type I collagen, compared with the CNA A domain. Through genome sequencing, an open-reading frame encoding a CNA homolog was found in Clostridium perfringens[18], a gram-positive anaerobic bacterium that causes gas gangrene. In addition, a collagen-binding domain from Clostridium histolyti-cumclass I collagenase had an X-ray crystal structure [19] that was strikingly similar to that of the minimum collagen-binding domain of CNA [20], although the 2 molecules do not share significant sequence homology. It is likely that, with the elucidation of more bacterial genomes, the family of CNA-like collagen adhesins will continue to expand.

Although there is a wealth of information demonstrating the importance of CNA in the pathogenesis of S. aureus, the molecular mechanisms by which CNA acts as a virulence factor are unclear. Several questions need to be addressed. Is the collagen-binding activity and affinity of CNA responsible for its role in disease? Do the domains contribute to the potential of the MSCRAMM as a virulence factor? Can other collagen-binding MSCRAMMs replace CNA on staphylococci without loss of virulence?

In the present study, we have taken advantage of S. aureus strain CYL316, which does not contain the cnagene but can be used as a host for the expression of functional wild-type (wt)and mutated CNA. In this system, we expressed 2 truncations of CNA: 1 that shows an increased affinity for collagen, and 1 that contains the non-collagen-binding domain only. We also expressed a form of CNA that did not bind collagen, because of a point mutation. Finally, we replaced the collagen-binding domain of CNA with the corresponding domain of ACE. These strains were examined in vitro for their surface display of each recombinant fragment, as well as for their collagen-binding capabilities, before they were analyzed in a mouse model of septic arthritis. The results provided, for the first time, direct evidence that CNA contributed to S. aureusvirulence by binding to collagen and that the contribution correlates with its affinity for collagen (i.e., the higher the affinity, the higher the virulence).

 
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MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. Strains and plasmids used in the present study are listed in table 1. Escherichia colistrains were grown at 37° overnight in Lennox L broth (LB; Sigma) or on LB agar with ampicillin (50 jOtg /mL) or spectinomycin (50 jOtg /mL), when appropriate. Staphylococci were grown at 37° in tryptic soy broth (TSB; Difco) or on tryptic soy agar (TSA), with tetracycline (Tc; 3 jOtg /mL), when necessary.

Table 1.
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Table 1.

Plasmids and strains used in the present study.

Cloning and expression of CNA35 and the CNA35 mutant. The sequence encoding amino acid residues 30–344 of CNA was amplified by use of polymerase chain reaction (PCR) from a construct containing the entire cnagene, using primer pairs CNA55 5' and CNAN2 3' (table 2). The PCR product was cloned into pQE30, as described elsewhere [21].

Table 2.
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Table 2.

Oligonucleotides used in the present study.

A 2-step PCR method was used to construct the CNA35 mutant, as described elsewhere [21]. Primers CNAYKL and CNAYKR contain sequences for lysine instead of the tyrosine residue at position 175 of CNA. Primer pairs CNA55 57CNAYKR and CNAYKL/CNA35R were used to amplify regions encoding amino acid residues 30–176 and 174–344 of CNA, respectively. The PCR products were used as templates for the second round of PCR amplification with primer pairs CNA55 5; and CNA35R. Large-scale expression and purification of recombinant proteins were performed as described elsewhere [22].

Analysis of the collagen binding of various CNA fragments. Surface plasmon resonance in a BIAcore 1000 system (BIAcore AB) and solid-phase binding assays were used for the analysis, as described elsewhere [22].

Construction of an S. aureus surface-display expression vector. The integration vector pCL84 [23] was modified as follows: pCL84 was digested with SaRand Pstl,and the ends were filled in with Klenow fragment and religated. A 121-bp sequence 5'to the start of the cnagene was previously shown to support the expression of cnain a heterologous host [3]. Primer pairs CNAI and CNAS were used to amplify, by use of PCR, this region plus the region encoding the signal peptide of CNA (table 2). The PCR product was cloned into pCL84, resulting in plasmid pYX102. To introduce the cell wall-anchoring motif into pYX102, the sequence encoding the last repeat to 51 nucleotides 3'to the end of the cnagene was amplified by use of PCR using primer pairs CNAB57 and PSTCNA37. The product was cloned into pYX102, resulting in plasmid pYX104. The inserted regions were confirmed by use of DNA sequence analysis.

Generation of heterologous strains. Primers CNA55 5' and CNA35R were used to amplify, by use of PCR, regions encoding CNA35 and CNA35Y175K from pQE30CNA35 and pQE30CNA35Y175K, respectively. Primers ACE40L and ACE40R were used to amplify regions encoding ACE40 from an ACE40 construct [15]. The PCR products were cloned into pYX104. The ligation mixture was transformed into E. coliJM101. Trans-formants were verified by examining the DNA banding patterns by use of agarose gel electrophoresis of restriction digestions of plasmid preparations.

The correct constructs were introduced into S. aureus CYL316, as described elsewhere [24]. The integration should occur in the promoter region of the lipase gene on the chromosome of CYL316, which would lead to the loss of lipase activity. This was verified by assaying the lipase activity of the transformants [3]. The presence of the integrated gene was verified by use of PCR.

Extraction of S. aureus cell wall-anchored proteins and Western blot analysis. Lysostaphin was used to extract cell wall-anchored proteins from S. aureusstrains, as described elsewhere [25]. Extracts from equal amounts of S. aureuscells were analyzed by use of Western blot analysis.

Attachment of S. aureus strains to collagen. Various concentrations of log-phase S. aureuscells were incubated with immobilized bovine type I collagen (10 ji ¿g/well), as described elsewhere [25]. Attached bacteria were fixed with 25% formaldehyde and stained with 0.5% crystal violet. After washing, 100 jOtL of 10% acetic acid was added, and absorbance at 590 nm was recorded.

Determination of the growth kinetics of S. aureus CYL strains. Individual colonies were used to inoculate 2 mL of fresh TSB-Tc and were cultured overnight at 37°. The cell cultures were adjusted to an optical density at 600 nm (OD600) of ∼ 1.0, and 1 mL of each suspension was then used to inoculate 20 mL of fresh TSB-Tc. OD600 was measured at every hour after inoculation.

Examination of S. aureus recombinant strains in a septic arthritis mouse model. Pathogen-free, 4–6-week-old BALB/ c female mice (Harlan Sprague-Dawley) were used. Single colonies of S. aureusstrains were used to inoculate 2 mL of TSB. After shaking overnight at 37°, 1 mL of the overnight culture was inoculated into 20 mL of fresh TSB. Bacteria were harvested when the culture reached an OD600 of ∼0.4. The cell suspensions were further quantified by use of a BBL CrystalSpec Nephe-lometer (BD Biosciences) and adjusted to the corresponding colony-forming units per milliliter. Each mouse was injected intravenously in the tail vein with 0.5 mL of bacterial suspension. TSB alone was injected into mice as a control and did not show any effects.

Mice were assessed over the course of 4 weeks, at days 3, 7, 14, and 28 after infection. Individual weights, appearance, and pathogenic lesions were documented. Mouse limbs were visually assessed for symptoms of arthritis by an observer with no knowledge of the infection status of the mice.

Histopathological examination. BALB/c female mice were injected with the different S. aureus strains at a dose of 2 X 107cfu/mouse (15 mice/group). At day 28 after injection, both hind limbs of mice were removed and histologically examined and scored for arthritis by an observer with no knowledge of the infection status of the mice, as described elsewhere [26].

Tissue colonization of infected mice. Mice (3-7/group) that were injected with 2 X 107 cfu of S. aureuswere killed at various time points (8 h and 1, 2, and 7 days after infection). Samples were obtained from tibiotarsal joints, as described elsewhere [27]. Bacteria were grown on TSA-Tc plates at 37°, and the number of colonies was counted 24–36 h later.

Statistical analysis. The differences in frequency of arthritis and mortality were analyzed by use of the Fisher's exact test. Differences between means for bacterial load and arthritis severity were assessed by use of the Mann-Whitney Utest. Differences in weight changes were assessed by use of the Student's ttest. Multiple comparisons were conducted for non-parametric data by use of the Kruskal-Wallis test.

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RESULTS

Generation of CNA35 and a CNA35 mutant and collagen-binding analysis. Several recombinant proteins containing different segments of the CNA A domain were expressed in E. coli.One of the proteins, CNA35, which includes the N-ter-minal two-thirds of the collagen-binding A domain of CNA (figure 1A), showed a higher affinity for collagen than did the full-length A domain (CNA55), by both surface plasmon resonance (SPR) (figure 2A)and ELISA (figure 2B).The apparent Kd(Kd(app)) for CNA35 was 20.7 ± 0.6 nmol/L, ∼ 100-fold greater than that for CNA55 (2.1 ± 0.3 /L). In a parallel study, CNA35 and CNA55 could inhibit each other for binding to collagen, indicating that the binding specificity was not changed (authors' unpublished data). Therefore, the increased affinity for collagen by CNA35 was probably due to easier access of the collagen molecules and/or tighter interactions between specific residues.

Figure 1.
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Figure 1.

A,Domain organization of CNA and its recombinant fragments. CNA contains a signal peptide (black box),a collagen-binding A region (gray box),3 repeats (B1, B2, and B3), and WMC (the cell wall-anchoring region, a transmembrane segment, and a cytoplasmic tail). CNA55, CNA35, CNA35Y 175K, and CNA19 are N-terminus His-tag fusion recombinant proteins. The asterisk in CNA35Y 175K indicates the point mutation. B,Constructs for the heterologous expression and display of CNA fragments on the surface of Staphylococcus aureusCYL316. Pena,the promoter region of cna. Black box,signal peptide sequences of cna.MCS, multiple cloning site.

Figure 2.
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Figure 2.

Binding of CNA55, CNA35, and CNA35Y 175K to bovine type I collagen. A,Surface plasmon resonance (SPR) response of 5 /L CNA55, CNA35, and CNA35Y 175K passed over a collagen-coated surface. B,Dose-dependent saturation curves for CNA35 and CNA55 over immobilized collagen. Immobilized bovine type I collagen (1 ⩾g/well) were incubated with increasing concentrations of CNA35 and CNA55, then with a mouse anti-His monoclonal antibody and a goat anti-mouse IgG (H+L)-AP conjugate. Apparent Kd(ATd(app)) values were calculated by use of the nonlinear regression method with the -binding class model [22]. The Kdiapp)values for CNA35 and CNA55 were 20.7 ± 0.6 nmol/L and 2.1 ± 0.3 /L, respectively. C,Comparison of collagen-binding capabilities between CNA35 (●) and CNA35Y 175K (■), by use of ELISA.

Amino acid residue Y175 in CNA was previously shown to be critical for collagen binding of the minimal collagen-binding domain of CNA (CNA19) [20]. In the present study, we mutated Y175 to a lysine residue in the context of CNA35, to generate CNA35Y 175K. The mutated recombinant protein lost collagen-binding capability, by both SPR (figure 2A) and ELISA (figure 2C). The i⩾d(app) for CNA35Y 175K was not calculated.

Generation and characterization of S. aureus strains expressing wt and mutated CNA. S. aureus strain CYL316 does not contain the gene for cnaand does not bind collagen. This strain was used as a host to display various CNA constructs, which were then examined in a mouse model of S. aureusseptic arthritis [27]. CYL316CNA, which was previously constructed, contains the intact cnagene (previously called CYL574) [3]. Corresponding regions in CNA35 and CNA35Y 175K were cloned into the surface-display integration vector pYX104 (table 1 and figure 1B).The resulting constructs, as well as the vectors pYX102 (the integration vector without the surface-display features) and pYX104, were introduced into CYL316, to generate strains CYL316CNA35, CYL316CNA35Y175K, CYL316PYX102, and CYL316PYX104 (table 1). Western blot analysis of lysostaphin extracts of equal amounts of log-phase cells of the above 4 strains, as well as CYL316CNA, indicated that the different segments of CNA were displayed on the surface of these strains and were approximately equal in amount (figure 3A). The bands in CYL316PYX104 and CYL316PYX102 (data not shown) extracts bound secondary antibodies and presumably represent different forms of protein A that bound IgG nonspecifically (figure 3B).

Figure 3.
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Figure 3.

Western blot analysis of cell-wall extracts of recombinant Staphylococcus aureusstrains. Exponential phase S. aureuscells were treated with lysostaphin to extract cell wall-anchored proteins. Arrows indicate the recombinant fragments. A,Probed with an anti-CNA19 monoclonal antibody 9G7 (1:500 dilution) and goat anti-Mouse IgG (H+L)-AP conjugate. B,Probed with goat-Anti-Rabbit IgG (H+L)-AP (1:2000 dilutions). C,Probed with a rabbit anti-ACE40 polyclonal antibody (1:5000 dilution) and goat anti-Rabbit IgG (H+L)-AP (1:2000 dilutions).

In attachment assays, CYL316CNA35, which displays the region corresponding to CNA35 and a repeat region, showed the highest capability for collagen attachment (figure 4A). CYL316CNA, which displays the CNA, attached to collagen at an intermediate level. CYL316CNA35Y175K, which contains the inactivating point mutation, did not attach to collagen, indicating that the point mutation was sufficient to disrupt collagen binding and attachment of S. aureuscells. Negative control strains CYL316PYX102 and CYL316PYX104 did not attach to collagen. These results are consistent with data obtained from direct-binding experiments using the corresponding recombinant proteins.

Figure 4.
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Figure 4.

Collagen binding of Staphylococcus aureuscells expressing various collagen-binding domains. Exponential phase S. aureuscells were incubated with immobilized bovine type I collagen for 1 h at room temperature in the wells of microtiter plates. Bound bacteria were fixed with 25% formaldehyde and stained with 0.5% crystal violet, as described in Materials and Methods. A,CYL316CNA35 (■), CYL316CNA (●), CYL316PYX102 (□), CYL316PYX104 (◆), and CYL316CNA35Y175K (◯). B,CYL316ACE40 (●) and CYL316PYX104 (◯).

The A domain of the enterococcal collagen adhesin ACE was cloned into pYX104 and introduced into S. aureus CYL316, to generate strain CYL316ACE40 (table 1 and figure 1B).Western blot analysis of the lysostaphin extracts from CYL316ACE40 showed that the ACE A domain was anchored to the cell wall (figure 3C). CYL316ACE40 also attached to collagen (figure 4B).

We measured the in vitro growth rates of the isogenic S. aureus strains. All the strains (CYL316PYX102, CYL316CNA35, CYL316CNA35Y175K, CYL316CNA, CYL316PYX104, and CYL316ACE40) grew at approximately the same rate (data not shown).

Dose effect on the infections caused by S. aureus strains CYL316CNA35 and CYL316CNA35Y175K. To compare the virulence potential between S. aureus strains CYL316CNA35 and CYL316CNA35Y175K, we injected mice with 4 X 107, 2 X 107, 1 X 107, or 5 X 106 cfu via the tail vein (15 mice/group) (figure 5). At high doses, dramatic differences were observed in both the survival and the arthritis frequencies between CYL316CNA35- and CYL316CNA35Y175K-infected mice. All the mice infected with CYL316CNA35 (4 X 107 cfu) died by day 3 after infection (figure 5A), compared with 2 mice that died in the CYL316CNA35Y175K group (P<.001) (figure 5B).Since all mice infected with 4 X 107 cfu of CYL316CNA35 died by day 3, there were no arthritis data for this group. However, after receiving the same high dose, just >30% of the mice infected with CYL316CNA35Y175K developed arthritis by day 28 (figure 5D). At lower doses (⩾2 X 107 cfu), no significant differences were observed in the survival rate. However, by day 3, 23.1% of the mice receiving 2 X 107 cfu of CYL316CNA35 developed arthritis. By day 28, 81.8% of the mice in this group had arthritis (figure 5C). In comparison, only 13.3% of the mice receiving 2X 107 cfu of CYL316CNA35Y175K developed arthritis during the same time span (P =.0031). By day 28, 50% of mice receiving 1 X 107 cfu of CYL316CNA35 had arthritis, compared with 15.4% of mice infected with the same dose of CYL316CNA35Y175K. No differences in frequency of arthritis were observed in mice infected with 0.5 X 107 cfu, regardless of the strain. These data suggest that the collagen-binding activity of CNA determines the adhesin's virulence potential in mouse models of both septic arthritis and septic death.

Figure 5.
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Figure 5.

Comparison of the virulence potentials between Staphylococcus aureusCYL316CNA35 and CYL316CNA35Y175K at different doses. BALB/c mice were infected with different doses of CYL316CNA35 and CYL316CNA35Y175K and were monitored for survival rate and signs of arthritis, for up to 28 days. Aand C,Mice infected with CYL316CNA35. Since all mice infected with CYL316CNA35 at 4 107 cfu died by day 3, there were no arthritis data for this group. and D,Mice infected with CYL316CNA35Y175K. Statistical significance was calculated by use of Fisher's exact test.

Quantitation of S. aureus in tissues of infected mice. We hypothesize that collagen-binding CNA facilitates the attachment and colonization of joint tissues by S. aureus.To test this hypothesis, bacteria were cultured from joints taken from mice infected with 2 X 107 cfu of CYL316CNA35, CYL316CNA35Y175K, or CYL316PYX104, at various time points after infection, and quantified (figure 6). At 8 h, no significant differences were observed in the numbers of bacteria recovered in mice infected with CYL316CNA35, CYL316CNA35Y175K, or CYL316PYX104 (n =3 mice/group). However, 24 and 48 h after infection, mice infected with CYL316CNA35 had more bacteria in their joints than did mice infected with CYL316CNA35Y175K or CYL316PYX104 (n =6 mice/group; P<.02). A similar trend was observed at day 7. These results suggest that the collagen-binding function of CNA is important for the colonization of the mouse joints early in the course of infection, although CNA may not be critical in the initial microbial targeting of the joints.

Figure 6.
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Figure 6.

Bacterial counts in the joints in the early stage of infection. Mice were infected with 2 X 107 cfu of Staphylococcus aureusand were killed at 8 h and 1, 2, and 7 days after infection. Joint samples were taken, homogenized, and plated for bacteria: CYL316CNA35 (◯), CYL316CNA35Y175K (◆), and CYL316PYX104 (●). Statistical significance was calculated by use of the Mann-Whitney Utest.

Analysis of arthritis frequencies in mice infected with S. aureus strains with different collagen-binding abilities. To test whether the virulence potential of CNA was regulated by the affinity of MSCRAMMs for collagen, we compared the frequency of arthritis in mice infected with CYL316CNA35 with that of mice infected with CYL316CNA, which binds collagen with lower affinity than does CYL316CNA35. By day 28, 79.2% of mice in the CYL316CNA35 group (n =48) had arthritis, 58% more than in the CYL316CNA group (50%; P =46; P<.005) (figure 7), suggesting that higher collagen-binding affinity resulted in a higher incidence of arthritis. Strain CYL316PYX104 caused the same degree of arthritis in mice as did CYL316PYX102, suggesting that the domains did not play a role in causing arthritis in this model. The incidence of arthritis among mice infected with strain CYL316CNA35Y175K (16.3%; P =43) was similar to that among mice infected with the vector control strain CYL316PYX102 (18.6%; P =43), again suggesting that it was the collagen-binding ability of CNA that led to increased virulence of S. aureus.

Figure 7.
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Figure 7.

Arthritis frequencies in mice infected with isogenic Staphylococcus aureusstrains having different collagen-binding abilities. BALB/c mice were infected with 2 X 107 cfu of S. aureuscells via the tail vein and were assessed for symptoms of arthritis by an observer with no knowledge of the infection status of the mice. Data were merged from 3 independent experiments. ▲, CYL316CNA35 (n =48; P< .005, vs. CYL316CNA; P< .0001, vs. CYL316PYX102, CYL316PYX104, and CYL316CA35Y175K); □, CYL316CNA ( =46; P<.03, vs. CYL316PYX102, CYL316PYX104, and CYL316CNA35Y175K); ●, CYL316ACE40 (n =25; P<.05, vs. CYL316PYX102, CYL316PYX104, and CYL316CNA35Y175K); ■, CYL316PYX104 (n =28); ◯, CYL316PYX102 [n =43); and △ , CYL316CNA35Y175K [n =43). Statistical significance was calculated using data at day 28 by use of Fisher's exact test.

To test whether a heterologous collagen adhesin could replace CNA and contribute to the virulence of S. aureus in this model, we infected mice with CYL316ACE40. By day 28, 48% of mice developed arthritis (n25), similar to the incidence of arthritis observed in the CYL316CNA group and significantly higher than that observed for the non-collagen-binding control groups (P<.05, vs. the control strains) (figure 7). Thus, ACE40, a heterologous collagen adhesin that binds collagen with an affinity comparable to that of CNA, was able to increase the arthritogenic potential of S. aureusto a level similar to that of S. aureusexpressing the wtCNA.

Weight changes in mice infected with different S. aureus strains. The extent of weight loss during an infection usually correlates with the severity of the disease. We examined the weight changes in mice (15 mice/group) infected with the different S. aureusstrains at days 3, 7, 14, 21, and 28 (figure 8). At days 3 and 7, marked weight changes were observed in mice infected with strains CYL316CNA35 and CYL316CNA, compared with mice infected with the 2 non-collagen-binding strains, which showed, at most, marginal weight losses (P<.02). Of interest, mice infected with strain CYL316ACE40 showed significant weight loss at day 3 but began regaining their weight by day 4, considerably earlier than mice infected with strains CYL316CNA35 and CYL316CNA. Moreover, these mice returned to their original weights in <2 weeks, compared with ∼4 weeks for the latter 2 groups. Overall, mice infected with strain CYL316CNA35 showed the greatest mean weight losses, suggesting that strain CYL316CNA35 caused the most systemic damage to mice during the early stages of infection.

Figure 8 .
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Figure 8 .

Mean weight changes of mice infected with isogenic Staphylococcus aureusstrains with different collagen-binding abilities. Log-phase S. aureuscells (2 X 107 cfu) were injected into the tail veins of mice (15 mice/group). Weights were recorded at days 3, 7, 14, 21, and 28 after infection and were compared with the weights before infection. The differences were averaged for each group.▲,CYL316PYX104; △,CYL316CNA35Y175K;◊,CYL316ACE40; ◯,CYL316CNA35; and ●,CYL316CNA. The statistical significance for different weight changes was calculated by use of Student's ttest. At day 3, P<.02 for the collagen-binding strains vs. the non-collagen-binding strains. At day 7, P<.03 for CYL316CNA35 vs. the non-collagen-binding strains. At day 14, P<.04 for CYL316CNA35 vs. the non-collagen-binding strains. At day 21, P<.03 for CYL316CNA vs. the non-collagen-binding strains.

Histopathological examination of mice. Visual examination revealed that the majority (>96%) of the arthritic limbs were hind limbs. Therefore, hind limbs from a representative experiment were removed for histopathological examination (table 3 and figure 9). The arthritis frequencies observed essentially paralleled the results obtained by visual examinations, as described above. With respect to severity of arthritis, when all the limbs were taken into account, a higher mean severity score was observed in mice infected with strain CYL316CNA35 than in mice infected with strain CYL316CNA. Both groups had higher severity scores than mice infected with non-collagen-binding strains (table 3). The mean arthritis score for mice infected with CYL316ACE40 was higher than that for mice infected with non-collagen-binding strains, but this difference was not statistically significant. When only the arthritic limbs were taken into account, similar trends were observed (table 3). These results corroborated the findings from visual evaluation and further suggested that the ability of S. aureusto attach to collagens increased both the frequency and severity of arthritis.

Table 3.
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Table 3.

Histopathological examination of the hind limbs of mice infected with different Staphylococcus aureusstrains.

Figure 9.
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Figure 9.

Histopathological examination of the joints of mice. BALB/c mice were infected with 2X107 cfu of Staphylococcus aureus cells. At day 28 after infection, both hind limbs were removed and histologically examined. Examples of joint sections from mice with arthritis severity scores of 0 (A), 2 (B), 3 (C), and 4 (D) are shown. A score of 0 indicates no arthritis. No joints with a severity score of 1 were found in this experiment.

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DISCUSSION

S. aureus expresses a number of cell wall-anchored proteins [28, 29], many of which act as adhesins for extracellular matrix proteins [30–33]. The importance of these MSCRAMMs (CNA, ClfA, FnbpA, and FnbpB) as virulence factors in S. aureusinfections has been demonstrated in various studies [34–46].

In the present study, we have confirmed that CNA, a member of a growing family of collagen adhesins in gram-positive bacteria, is a virulence factor in septic arthritis. We also have determined the mechanism of action of the MSCRAMM in infection. Somewhat surprisingly, the initial targeting of S. aureusto the joint is not dependent on the presence of a collagen-binding MSCRAMM. Similar numbers of bacteria were recovered from joints 8 h after the mice received S. aureus,whether or not the particular strain expresses a collagen-binding adhesin. However, 24 h after infection, mice infected with a collagen-binding S. aureusstrain had significantly more bacteria in their joints than did mice infected with non-collagen-binding strains. These observations were reminiscent of those shown in a rat model of endocarditis [5], as well as those from a study comparing S. aureusstrains that did or did not express ClfA [35]. Thus, it appears that S. aureusrapidly disseminates throughout the body of mice when injected intravenously. The adhesins subsequently allow the bacteria to hold onto tissue structures containing their corresponding ligand, and, as a result, these adhering bacteria appear to resist clearance.

Previous studies of S. aureus strains that did or did not express CNA did not directly link the change in virulence to the ability of the strain to bind collagen. Here, we have demonstrated, for the first time, that the virulence potential of CNA is directly related to the adhesin's affinity for collagen. Expression of a CNA domain that does not contain any known ligand-binding activity or CNA35 that contains a loss-of-func-tion point mutation resulted in S. aureusstrains with the same virulence potential as S. aureusstrains that do not contain the cnagene. In addition, comparison of the results of mice infected with CYL316CNA35 and CYL316CNA indicated that there is a correlation between the affinity for collagen and the virulence of the strainthe higher the affinity, the higher the virulence. Furthermore, the origin of the collagen adhesin does not appear to be critical, since S. aureusstrain CYL316ACE40 expressing the enterococcal collagen adhesin also increased the virulence of the staphylococci in the mouse model of septic arthritis. However, it is interesting to note that the mice infected with CYL316ACE40 initially lost weight to the same extent as mice infected with CYL316CNA and CYL316CNA35 but recovered much more rapidly than the latter 2 groups. ACE40 binds collagen with a much faster dissociation rate than does CNA, which might lead to more-efficient clearance of the bacteria in vivo. Thus, it appears that the mechanism of ligand binding affects the virulence potential of an MSCRAMM.

Expression of CNA is not enough to turn a bacterial host into a pathogen in the mouse model of septic arthritis. In preliminary experiments, we have expressed CNA in Staphylococcus carnosus,a nonpathogenic staphylococci used extensively in the food industry. These collagen adhesin-expressing bacteria did not cause septic arthritis in this model even when injected into BALB/c mice at a dose of 1 X 109 cfu/mouse. Thus, virulence factors in addition to CNA are needed to cause disease. The identity of these factors is presently unclear.

S. aureus is an evolving pathogen. Its ability to acquire genetic elements from other strains or species, as well as manipulating existing ones, allows it to be an adept survivor in changing environments. It is likely that staphylococcal cells can improve on their arsenal of virulence factors by, for example, acquiring new adhesins or mutations in the corresponding genes, thereby allowing the pathogen to colonize the host more efficiently.

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Acknowledgments

We thank Agneta Höök, Emily Reisenbichler, and Lawrence Lee for technical assistance.

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Footnotes

  • ↵a Y.X. and J.M.R. contributed equally to this work.

  • Financial support: National Institutes of Health (grant AR44415 to M.H.); Arthritis Foundation (postdoctoral fellowship award to Y.X.).

  • Received October 2, 2003.
  • Accepted December 2, 2003.
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References

  1. 1.↵
    1. Shirtliff ME,
    2. Mader JT
    . Acute septic arthritis. Clin Microbiol Rev 2002;15:527-554.
    Abstract/FREE Full Text
  2. 2.↵
    1. Switalski L,
    2. Patti JM,
    3. Butcher W,
    4. Gristina AG,
    5. Speziale P,
    6. Höök M
    . A collagen receptor on Staphylococcus aureus strains isolated from patients with septic arthritis mediates adhesion to cartilage. Molecular Microbiology 1993;7:99-107.
    MedlineWeb of Science
  3. 3.↵
    1. Patti JM,
    2. Bremell T,
    3. Krajewska-Pietrasik D,
    4. et al
    . The Staphylococcus aureus collagen adhesin is a virulence determinant in experimental septic arthritis. Infect Immun 1994;62:152-161.
    Abstract/FREE Full Text
  4. 4.↵
    1. Nilsson I-M,
    2. Patti JM,
    3. Bremell T,
    4. Höök M,
    5. Tarkowski A
    . Vaccination with a recombinant fragment of collagen adhesin provides protection against Staphylococcus awrews-mediated septic death. J Clin Invest 1998;101:2640-2649.
    MedlineWeb of Science
  5. 5.↵
    1. Hienz SA,
    2. Schennings T,
    3. Heimdahl A,
    4. Flock JL
    . Collagen binding of Staphylococcus aureus is a virulence factor in experimental endocarditis. J Infect Dis 1996;174:83-88.
    Abstract/FREE Full Text
  6. 6.↵
    1. Elasri MO,
    2. Thomas JR,
    3. Skinner RA,
    4. et al
    . Staphylococcus aureus collagen adhesin contributes to the pathogenesis of osteomyelitis. Bone 2002;30:275-280.
    CrossRefMedlineWeb of Science
  7. 7.↵
    1. Rhem MN,
    2. Lech EM,
    3. Patti JM,
    4. et al
    . The collagen-binding adhesin is a virulence factor in Staphylococcus aureus keratitis. Infect Immun 2000;68:3776-3779.
    Abstract/FREE Full Text
  8. 8.↵
    1. Peacock SJ,
    2. Moore CE,
    3. Justice A,
    4. et al
    . Virulent combinations of adhesin and toxin genes in natural populations of Staphylococcus aureus. Infect Immun 2002;70:4987-4996.
    Abstract/FREE Full Text
  9. 9.↵
    1. Booth MC,
    2. Pence LM,
    3. Mahasreshti P,
    4. Callegan MC,
    5. Gilmore MS
    . Clonal associations among Staphylococcus aureus isolates from various sites of infection. Infect Immun 2001;69:345-352.
    Abstract/FREE Full Text
  10. 10.↵
    1. Patti JM,
    2. Boles JO,
    3. Höök M
    . Identification and biochemical characterization of the ligand binding domain of the collagen adhesin from Staphylococcus aureus. Biochemistry 1993;32:11428-11435.
    CrossRefMedlineWeb of Science
  11. 11.↵
    1. Rich RL,
    2. Demeler B,
    3. Ashby K,
    4. et al
    . Domain structure of the Staphylococcus aureus collagen adhesin. Biochemistry 1998;37:15423-15433.
    CrossRefMedlineWeb of Science
  12. 12.↵
    1. Snodgrass JL,
    2. Mohamed N,
    3. Ross JM,
    4. Sau S,
    5. Lee CY,
    6. Smeltzer MS
    . Functional analysis of the Staphylococcus aureus collagen adhesin domain. Infect Immun 1999;67:3952-3959.
    Abstract/FREE Full Text
  13. 13.↵
    1. Deivanayagam CC,
    2. Rich RL,
    3. Carson M,
    4. et al
    . Novel fold and assembly of the repetitive region of the Staphylococcus aureus collagen-binding surface protein. Structure Fold Des 2000;8:67-78.
    Medline
  14. 14.↵
    1. Hartford O,
    2. McDevitt D,
    3. Foster TJ
    . Matrix-binding proteins of Staphylococcus aureus functional analysis of mutant and hybrid molecules. Microbiology 1999;145:2497-2505.
    Abstract/FREE Full Text
  15. 15.↵
    1. Rich RL,
    2. Kreikemeyer B,
    3. Owens RT,
    4. et al
    . ACE: a collagen-binding MSCRAMM from Enterococcus faecalis. J Biol Chem 1999;274:26939-26945.
    Abstract/FREE Full Text
  16. 16.↵
    1. Nallapareddy SR,
    2. Weinstock GM,
    3. Murray BE
    . Clinical isolates of Enterococcus faeciumexhibit strain-specific collagen binding mediated by Acm a new member of the MSCRAMM family. Mol Microbiol 2003;47:1733-1747.
    CrossRefMedlineWeb of Science
  17. 17.↵
    1. Lannergard J,
    2. Frykberg L,
    3. Guss B
    . CNE a collagen-binding protein of Streptococcus equi. FEMS Microbiol Lett 2003;222:69-74.
    CrossRefMedlineWeb of Science
  18. 18.↵
    1. Shimizu T,
    2. Ohtani K,
    3. Hirakawa H,
    4. et al
    . Complete genome sequence of Clostridium perfringens,an anaerobic flesh-eater. Proc Natl Acad Sci USA 2000;99:996-1001.
  19. 19.↵
    1. Wilson JJ,
    2. Matsushita O,
    3. Okabe A,
    4. Sakon J
    . A bacterial collagen-binding domain with novel calcium-binding motif controls domain orientation. Embo J 2003;22:1743-1752.
    CrossRefMedlineWeb of Science
  20. 20.↵
    1. Symersky J,
    2. Patti JM,
    3. Carson M,
    4. et al
    . Structure of the collagen-binding domain from a Staphylococcus aureus adhesin. Nat Struct Biol 1997;4:833-838.
    CrossRefMedlineWeb of Science
  21. 21.↵
    1. Visai L,
    2. Xu Y,
    3. Casolini F,
    4. Rindi S,
    5. Höök M,
    6. Speziale P
    . Monoclonal antibodies to CNA a collagen-binding microbial surface component recognizing adhesive matrix molecules detach Staphylococcus aureus from a collagen substrate. J Biol Chem 2000;275:39837-39845.
    Abstract/FREE Full Text
  22. 22.↵
    1. Xu Y,
    2. Gurusiddappa S,
    3. Rich RL,
    4. et al
    . Multiple binding sites in collagen type I for the integrins a1and b2. J Biol Chem 2000;275:38981-38989.
    Abstract/FREE Full Text
  23. 23.↵
    1. Lee CY,
    2. Buranen SL,
    3. Ye Z-H
    . Construction of single-copy integration vectors for Staphylococcus aureus. Gene 1991;103:101-105.
    CrossRefMedlineWeb of Science
  24. 24.↵
    1. Augustin J,
    2. Gotz F
    . Transformation of Staphylococcus epidermidis and other staphylococcal species with plasmid DNA by electroporation. FEMS Microbiol Lett 1990;54:203-208.
    Medline
  25. 25.↵
    1. Eidhin DN,
    2. Perkins S,
    3. Francois P,
    4. Vaudaux P,
    5. Höök M,
    6. Foster TJ
    . Clumping factor (ClfB) a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus. Mol Microbiol 1998;30:245-257.
    CrossRefMedlineWeb of Science
  26. 26.↵
    1. Brown EL,
    2. Ullrich SE,
    3. Pride M,
    4. Kripke ML
    . The effect of UV irradiation on infection of mice with Borrelia burgdorferi. Photochem Photobiol 2001;73:537-544.
    CrossRefMedlineWeb of Science
  27. 27.↵
    1. Lee LY,
    2. Miyamoto YJ,
    3. Mclntyre BW,
    4. et al
    . The Staphylococcus aureus Map protein is an immunomodulator that interferes with cell-mediated responses. J Clin Invest 2002;110:1461-1471.
    CrossRefMedlineWeb of Science
  28. 28.↵
    1. Mazmanian SK,
    2. Ton-That H,
    3. Schneewind O
    . Sortase-catalysed anchoring of surface proteins to the cell wall of Staphylococcus aureus. Mol Microbiol 2001;40:1049-1057.
    CrossRefMedlineWeb of Science
  29. 29.↵
    1. Roche FM,
    2. Massey R,
    3. Peacock SJ,
    4. et al
    . Characterization of novel LPXTG-containing proteins of Staphylococcus aureus identified from genome sequences. Microbiology 2003;149:643-654.
    Abstract/FREE Full Text
  30. 30.↵
    1. Patti JM,
    2. Höök M
    . Microbial adhesins recognizing extracellualr matrix macromolecules. Curr opin Cell Biol 1994;6:752-758.
    CrossRefMedlineWeb of Science
  31. 31.
    1. Patti JM,
    2. Allen BL,
    3. McGavin MJ,
    4. Höök M
    . MSCRAMM-mediated adherence of microorganisms to host tissues. Annu Rev Microbiol 1994;48:585-617.
    MedlineWeb of Science
  32. 32.
    1. Foster TJ,
    2. Höök M
    . Surface protein adhesins of Staphylococcus aureus. Trends Microbiol 1998;6:484-488.
    CrossRefMedlineWeb of Science
  33. 33.↵
    1. Tung H,
    2. Guss B,
    3. Hellman U,
    4. Persson L,
    5. Rubin K,
    6. Ryden C
    . A bone sialoprotein-binding protein from Staphylococcus aureus a member of the staphylococcal Sdr family. Biochem J 2000;345:611-619.
    CrossRefMedline
  34. 34.↵
    1. Siboo IR,
    2. Cheung AL,
    3. Bayer AS,
    4. Sullam PM
    . Clumping factor A mediates binding of Staphylococcus aureus to human platelets. Infect Immun 2001;69:3120-3127.
    Abstract/FREE Full Text
  35. 35.↵
    1. Josefsson E,
    2. Hartford O,
    3. O'Brien L,
    4. Patti JM,
    5. Foster T
    . Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant. J Infect Dis 2001;184:1572-1580.
    Abstract/FREE Full Text
  36. 36.
    1. Massey RC,
    2. Kantzanou MN,
    3. Fowler T,
    4. et al
    . Fibronectin-binding protein A of Staphylococcus aureus has multiple substituting binding regions that mediate adherence to fibronectin and invasion of endothelial cells. Cell Microbiol 2001;3:839-851.
    CrossRefMedlineWeb of Science
  37. 37.
    1. Fowler T,
    2. Wann ER,
    3. Joh D,
    4. Johansson S,
    5. Foster TJ,
    6. Höök M
    . Cellular invasion by Staphylococcus aureus involves a fibronectin bridge between the bacterial fibronectin-binding MSCRAMMs and host cell β1 integrins. Eur J Cell Biol 2000;79:672-679.
    CrossRefMedlineWeb of Science
  38. 38.
    1. Dziewanowska K,
    2. Patti JM,
    3. Deobald CF,
    4. Bayles KW,
    5. Trumble WR,
    6. Bohach GA
    . Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells. Infect Immun 1999;67:4673-4678.
    Abstract/FREE Full Text
  39. 39.
    1. Sinha B,
    2. Francois PP,
    3. Nusse O,
    4. et al
    . Fibronectin-binding protein acts as Staphylococcus aureus invasin via fibronectin bridging to integrin αβ5. Cell Microbiol 1999;1:101-117.
    CrossRefMedlineWeb of Science
  40. 40.
    1. Lammers A,
    2. Nuijten PJ,
    3. Smith HE
    . The fibronectin binding proteins of Staphylococcus aureus are required for adhesion to and invasion of bovine mammary gland cells. FEMS Microbiol Lett 1999;180:103-109.
    CrossRefMedlineWeb of Science
  41. 41.
    1. Peacock SJ,
    2. Foster TJ,
    3. Cameron BJ,
    4. Berendt AR
    . Bacterial fibronectin-binding proteins and endothelial cell surface fibronectin mediate adherence of Staphylococcus aureus to resting human endothelial cells. Microbiology 1999;145:3477-3486.
    Abstract/FREE Full Text
  42. 42.
    1. Dziewanowska K,
    2. Carson AR,
    3. Patti JM,
    4. Deobald CF,
    5. Bayles KW,
    6. Bohach GA
    . Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins: role in internalization by epithelial cells. Infect Immun 2000;68:6321-6328.
    Abstract/FREE Full Text
  43. 43.
    1. Sinha B,
    2. Francois P,
    3. Que YA,
    4. et al
    . Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells. Infect Immun 2000;68:6871-6878.
    Abstract/FREE Full Text
  44. 44.
    1. Miyamoto YJ,
    2. Wann ER,
    3. Fowler T,
    4. Duffield E,
    5. Höök M,
    6. Mclntyre BW
    . Fibronectin binding protein A of Staphylococcus aureus can mediate human lymphocyte adhesion and coactivation. J Immunol 1900;166:5129-5138.
  45. 45.
    1. Mamo W,
    2. Jonsson P,
    3. Flock JL,
    4. et al
    . Vaccination against Staphylococcus aureusmastitis immunological response of mice vaccinated with fibronectin-binding protein (FnBP-A) to challenge with S. aureus. Vaccine 1994;12:988-992.
    CrossRefMedlineWeb of Science
  46. 46.↵
    1. Rennermalm A,
    2. Li Y-H,
    3. Bohaufs L,
    4. et al
    . Antibodies against a truncated Staphylococcus aureus fibronectin-binding protein protect against dissemination of infection in rat. Vaccine 2001;19:3376-3383.
    CrossRefMedline
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  1. J Infect Dis. (2004) 189 (12): 2323-2333. doi: 10.1086/420851
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