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Triosephosphate isomerase (TPI; EC 5. 3. 1. 1) displayed on the cell surface of Staphylococcus aureus acts as an adhesion molecule that binds to the capsule of Cryptococcus neoformans, a fungal pathogen. This study investigated the function of TPI on the cell surface of S. aureus and its interactions with biological substances such as fibronectin, fibrinogen, plasminogen, and thrombin were investigated. Binding of TPI to plasminogen was demonstrated by both surface plasmon resonance analysis and Far-Western blotting. It is suggested that lysine residues contribute to this binding because the interaction was inhibited by ɛ-aminocaproic acid. Activation of plasminogen to plasmin by staphylokinase or tissue plasminogen activator decreased in the presence of TPI, whereas TPI was degraded by plasmin. In other experiments, intact S. aureus cells had the ability to both increase and decrease plasminogen activation depending on the number of cells. Several molecules expressed on the surface of S. aureus were predicted to interact with plasminogen, resulting in its increased or decreased activation. These findings indicate that S. aureus sometimes localizes and sometimes disseminates in the host, depending on the molecules expressed under various conditions.
Triosephosphate isomerase displayed on the cell surface of S. aureus acts as an adhesion molecule that binds to the capsule of C. neoformans, a fungal pathogen (1, 2). Previously, we have shown that TPI recognizes α-1,3-linked mannooligosaccharides of a size equal to or greater than that of triose. This structure is present in the backbone of GXM, which is the major component of the cryptococcal capsule.
Staphylococci, streptococci, and enterococci target extracellular matrix components such as collagen, fibronectin, laminin, and fibrinogen for adherence to, and colonization of, host tissues. Microbial surface components recognizing adhesive matrix molecules have common structural properties: a C-terminal domain anchoring the cell wall and an N-terminal signal peptide (3). Furthermore, several anchorless proteins exposed on the bacterial cell surface may act as adhesion molecules. Housekeeping proteins such as GAPDH and enolase, which are glycolytic enzymes, act as anchorless proteins interacting with the extracellular matrix of the host (4–6). For example, GAPDH, which is found in Streptococcus species (7, 8), Escherichia coli (9), and Candida albicans (10), binds fibrinogen, fibronectin, plasminogen, transferrin, laminin, and cytoskeletal proteins. Enolase on S. aureus, Streptococcus species, and Mycobacterium fermentans binds to plasminogen and fibronectin (6, 11–17).
Several studies have investigated TPI, although these studies are fewer in number than those for GAPDH and enolase. TPI on Paracoccidioides brasiliensis interacts with laminin and fibronectin. Thus, TPI may be important in adherence to, and invasion of, host cells, indicating that TPI may be a virulence factor for the pathogen (18). TPI of Schistosoma mansoni, a causative agent of parasitic infection, is a protective antigen in mice, suggesting that it may have potential as a vaccine candidate (19).
The presence of TPI on the cell surface of S. aureus has been suggested by proteomics analysis (20) and the results of our previous studies (1, 21). The secretory pathways of the glycolytic protein to the cell surface are unknown. However, the TPI gene is located in the same operon as GAPDH and enolase. The amino acid residue lysine common is common to the C-terminal of GAPDH, enolase, and TPI. The contribution of the C-terminal lysine residue of enolase to plasminogen activity has been demonstrated (12). To elucidate the function of TPI on the cell surface of S. aureus during infection, we performed a binding assay for TPI with several bioreactive host proteins using SPR analysis, which has been widely used to determine interactions of molecules, including GAPDH and enolase (7, 12).
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Proteins localized on the surfaces of microbes play important roles in interactions with other cells and molecules. In the process of infection, these proteins can be key substances for adherence to, and invasion of, the host (20, 24, 25). Glycolytic enzymes act in the cytoplasm of microbes, but accumulating evidence indicates that some glycolytic enzyme proteins, such as GAPDH, enolase, and TPI, are present on the cell surface, where they may have multiple functions (5, 6). Several of these enzymes interact with proteins in the plasma. For example, in Gram-positive bacteria, GAPDH of Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus equisimilis is a plasminogen-binding protein (7, 26, 27), suggesting the possibility of immunogenic GAPDH as a candidate for a pneumococcal vaccine (8). In Gram-negative bacteria, GAPDH is thought to act as a virulence factor and may contribute to the pathogenesis of enterohemorrhagic E. coli and enteropathogenic E. coli by binding to human plasminogen, fibrinogen, or intestinal epithelial cells (9). In the fungus Candida albicans, GAPDH on the outer surface of the cell wall is able to adhere to fibronectin and laminin (28). Enolase from Streptococcus, Staphylococcus, and Lactobacillus species interacts with plasminogen and laminin (6, 29, 30).
Previously, we reported that TPI of S. aureus is a lectin and that it binds to the fungal pathogen C. neoformans via a capsular polysaccharide, GXM. The attachment of S. aureus to C. neoformans induces apoptosis-like cell death of the fungal pathogen (1, 31, 32). TPI recognizes at least three mannose residues in the α-1,3-linked mannan backbone of GXM. Furthermore, TPI may have two binding pockets for mannooligosaccharides (2). As TPI appeared to be a multifunctional protein, we investigated the interaction of TPI with proteins in the plasma.
Here, we have shown that TPI is a plasminogen-binding protein. Moreover, Far-Western blotting analysis revealed a plasminogen-binding protein in addition to TPI in the crude extract. Based on size, we speculate that the band at about 47 kDa is enolase. Enolase in S. aureus binds plasminogen and enhances its activation to plasmin (33). Recently, enolase was proposed as a candidate for a vaccine against S. aureus infection (34).
An interaction between TPI expressed on the cell surface of the fungus Paracoccidioides brasiliensis and laminin has been suggested (18). Our preliminary experiments indicated binding between staphylococcal TPI and laminin by SPR. However, further study would help to clarify the multiple functions of TPI.
In the kinetic analysis of the TPI and plasminogen interaction, two equilibrium constants, KD1 (3.18 × 10−10) and KD2 (3.12 × 10−7), were calculated from the heterogeneous ligand-parallel reaction model. Bergmann and Hammerschmidt reported the equilibrium constants of S. pneumoniae GAPDH for plasminogen (KD1= 4.3 × 10−7 M and KD2= 1.6 × 10−10 M) and for plasmin (KD1= 2.8 × 10−8 M and KD2= 5.2 × 10−8 M) (7). The binding affinities of S. pneumoniae enolase for plasminogen have been calculated as 8.6 × 10−8 M and 5.5 × 10−10 M (12). Thus, GAPDH, enolase, and TPI show multiple binding sites and similar affinities for plasminogen. The multiple binding sites could facilitate higher affinity fitting between the molecules. In our experiments the interaction of TPI and plasminogen was inhibited by EACA, a lysine analog. The common amino acid residue at the C-terminus of these enzymes is lysine. For enolase from S. pneumoniae, Bergmann et al. have shown that, in addition to the C-terminal lysyl residue being the binding site for the kringle motif of plasmin(ogen), nine amino acid residues (residues 248–256) form a second binding site (12). Recently, Cork et al. reported that surface enolase group A Streptococcus lysine residues at positions 252, 255, 434, and 435 play a concerted role in plasminogen acquisition (35).
We examined whether TPI could affect the activity of plasminogen. Enolase, GAPDH, and known plasminogen-binding proteins of S. pneumoniae promote invasion of pathogens into the host tissue via binding with plasminogen to initiate its proteolytic activity (6, 16, 36–38). However, in our experiments, TPI decreased the conversion of plasminogen to plasmin, suggesting involvement of TPI in the inhibition of fibrinolysis. In contrast, staphylococcal enolase activates plasminogen (6, 39, 40). Here, we also observed that cleavage of TPI by plasmin. S. aureus produces a large number of virulence factors. Some of these factors are toxins secreted into the extracellular environment, including exoenzymes, whereas others are components of the cells. The well-known coagulase and SAK seem to have opposite functions. The former plays a role in the formation of fibrin, whereas the latter acts in fibrinolysis. The plasminogen activation experiments using intact cells instead of purified TPI suggested that the overall phenomenon is affected by the number of cells. Plasminogen was activated in the presence of many bacterial cells, whereas it was delayed in the presence of fewer cells. The activities of enolase and TPI were determined to ascertain whether they are expressed on the cell surface. At similar concentrations of cells enolase and TPI were detected, although a quantitative investigation remains to be conducted. Glycolytic enzymes may be involved in the coordinated activities of coagulation and fibrinolysis. These proteins may serve multiple functions on the cell surface in addition to glycolysis (41), although the expression and posttranscriptional processing of each enzyme's actions on the cell wall are unknown.