ABSTRACT
Staphylococcus aureus is a formidable pathogen capable of causing infections in different sites of the body in a variety of vertebrate animals, including humans and livestock. A major contribution to the success of S. aureus as a pathogen is the plethora of virulence factors that manipulate the host’s innate and adaptive immune responses. Many of these immune modulating virulence factors are secreted toxins, cofactors for activating host zymogens, and exoenzymes. Secreted toxins such as pore-forming toxins and superantigens are highly inflammatory and can cause leukocyte cell death by cytolysis and clonal deletion, respectively. Coagulases and staphylokinases are cofactors that hijack the host’s coagulation system. Exoenzymes, including nucleases and proteases, cleave and inactivate various immune defense and surveillance molecules, such as complement factors, antimicrobial peptides, and surface receptors that are important for leukocyte chemotaxis. Additionally, some of these secreted toxins and exoenzymes can cause disruption of endothelial and epithelial barriers through cell lysis and cleavage of junction proteins. A unique feature when examining the repertoire of S. aureus secreted virulence factors is the apparent functional redundancy exhibited by the majority of the toxins and exoenzymes. However, closer examination of each virulence factor revealed that each has unique properties that have important functional consequences. This chapter provides a brief overview of our current understanding of the major secreted virulence factors critical for S. aureus pathogenesis.
EXOTOXINS
Introduction
Staphylococcus aureus is a highly successful pathogen that colonizes ∼30% of the population asymptomatically, but it is also capable of causing infections ranging from mild skin and soft tissue infections to invasive infections, such as sepsis and pneumonia (1). When S. aureus infects the host, it produces many virulence factors that promote the manipulation of the host’s immune responses while ensuring bacterial survival. These virulence factors include secreted toxins (exotoxins), which represent approximately 10% of the total secretome (2). While there are over 40 known exotoxins produced by these bacteria, many of them have similar functions and have high structural similarities. Closer examination of these seemingly redundant exotoxins revealed that each has unique properties. Exotoxins fall into three broad groups based on their known functions: cytotoxins, superantigens (SAgs), and cytotoxic enzymes (Table 1). Cytotoxins act on the host cell membranes, resulting in lysis of target cells and inflammation. Superantigens mediate massive cytokine production and trigger T and B cell proliferation. Secreted cytotoxic enzymes damage mammalian cells. Collectively, these exotoxins modulate the host immune system and are critical for S. aureus infections.
TABLE 1.
Major exotoxins produced by S. aureus
| Exotoxin(s) | Gene(s) | Function(s) |
|---|---|---|
| α-Toxin | hla | Pore-forming toxin |
| PVL (LukSF-PV) | lukS, lukF | Pore-forming toxin |
| HlgAB | hlgA, hlgB | Pore-forming toxin |
| HlgCB | hlgC, hlgB | Pore-forming toxin |
| LukED | lukE, lukD | Pore-forming toxin |
| LukAB/HG | lukA/H, lukB/G | Pore-forming toxin |
| LukMF′ | lukM, lukF′ | Pore-forming toxin |
| LukPQ | lukP, lukQ | Pore-forming toxin |
| PSMα1 to PSMα4 | psmα1 to psmα 4 | Phenol soluble modulins |
| PSMβ1, PSMβ2 | psmβ1, psmβ2 | Phenol soluble modulins |
| δ-Toxin | hld | Phenol soluble modulins |
| PSM-mec | psm-mec | Phenol soluble modulins |
| ε-Toxin | cytE | Cytotoxin |
| SEA to SEE, SEG | sea to see, seg | Enterotoxins, T cell superantigens |
| SE-l H to SE-l Y | selh to sely | T cell superantigens |
| TSST-1 | tst | T cell superantigen |
| SpA | spa | B cell superantigen |
| β-Toxin | hlb | Sphingomyelinase, biofilm ligase |
| Exfoliative toxin A | eta | Serine protease |
| Exfoliative toxin B | etb | Serine protease |
Cytotoxins
β-barrel pore-forming toxins
α-Toxin: the prototypic pore forming toxin (PFT)
α-Toxin (also known as α-hemolysin or Hla) is encoded by the gene hla as part of a monocistronic operon in the core genome of S. aureus. Like all conventionally secreted proteins, α-toxin is synthesized with an N-terminal signal peptide. Water-soluble, α-toxin monomers form heptameric β-barrel pores in target cell membranes, resulting in cell lysis (Fig. 1A) (3). The α-toxin heptamer resembles a mushroom and has three major domains: an extracellular cap domain, a stem domain that forms the β-barrel pore, and a rim domain that confers receptor specificity (Fig. 2A to C) (4). The β-barrel pore is formed from a prepore by a conformational change in a toxin substructure known as the amino latch (5). The critical role of the amino latch in the β-barrel pore formation is exemplified by a single amino acid mutation at His-35, which disrupts interprotomer stabilization, thus preventing pore formation and inactivating the toxin (6–8).
FIGURE 1.
Current models for PFT pore formation for (A) α-toxin and (B) the bicomponent PFTs. (A) α-toxin is secreted as a monomer. Upon binding to the host receptor, ADAM-10, the toxin monomers oligomerize to form a heptameric prepore on the target cell surface. The prestem domains of the prepore then extend to form a β-barrel pore that punctures the target cell membrane. (B) The bicomponent PFTs are also secreted as monomers (except LukAB, which is secreted as dimers). The S-subunit recognizes the target cell by binding to cell surface receptors (LukPQ is an exception; the F-subunit LukQ is the receptor recognition subunit). These receptors are typically G-protein-coupled receptors (except for LukAB, which binds to the integrin CD11b). Upon receptor binding, the S-subunit dimerizes with the F-subunit, followed by oligomerization of three additional leukocidin dimers, resulting in an octameric prepore. Similar to the α-toxin pore formation model, the prestem domains of the prepore extend to form a β-barrel pore, thus disrupting the target cell membrane.
FIGURE 2.
Structures of (A to C) α-toxin and (D to G) the bicomponent PFT, HlgAB. (A) The α-toxin monomer (PDB:4U6V) (351). The amino latch is colored blue, the cap domain red, the rim domain pink, and the prestem domain green. (B, C) The α-toxin heptamer (7AHL) (4). Each α-toxin is colored a different shade of pink to denote individual protomers. The amino latches are highlighted in blue, and the β-barrel pore is green. The monomers of (D) HlgA (2QK7) (352) and (E) HlgB (1LKF) (353). The amino latch of HlgB is blue; the cap domain for HlgA is cyan and HlgB is beige; the rim domains are yellow for HlgA and pink for HlgB; and the prestem domains are green. (F, G) The HlgAB octamer (3B07) (33). The HlgA protomers are cyan, the HlgB protomers are beige, and the β-barrel pore is green.
The role of α-toxin in disease has been studied extensively. α-Toxin causes lysis of many cell types: erythrocytes, platelets, endothelial cells, epithelial cells, and certain leukocytes (9–12). For many years, α-toxin was thought to mediate cytolysis through nonspecific binding to the lipid bilayer of cells. However, this model did not explain the species specificity exhibited by the toxin (i.e., lysis of rabbit but not human erythrocytes). In 2010, Wilke et al. identified the protein ADAM-10 (a disintegrin and metalloprotease 10), as the cellular receptor for α-toxin, thus providing an explanation for the observed species and cell type specificities (Fig. 3) (13). Recently, the mammalian junction protein, PLEKHA7 (plekstrin-homology domain-containing protein A7), was also demonstrated to be involved in α-toxin cytotoxicity and is thought to contribute to α-toxin-mediated tissue injuries in murine skin infection and pneumonia models (14).
FIGURE 3.
S. aureus PFTs and their receptor, species, and cell type specificities. Currently, S. aureus is known to produce eight β-barrel PFTs. Each of these PFTs targets different cell surface receptors. While some PFTs share the same receptors, they can differ in their species specificity. Collectively, the PFTs exert their sublytic and lytic effects on a variety of cells, including erythrocytes, endothelial cells, epithelial cells, neutrophils, monocytes, macrophages, dendritic cells, and T cells.
α-Toxin is not only lethal, but can also modulate cellular responses at sublytic concentrations, including the release of nitric oxide from endothelial and epithelial cells, extracellular Ca2+ influx, production of proinflammatory cytokines, and pyroptosis of monocytes through the activation of caspase-1 and the nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome (10, 15–19). Additionally, sublytic levels of α-toxin upregulate the expression of ADAM10 and activate the ADAM10 protease to cleave the junction protein E-cadherin, resulting in disruption of the epithelial barrier (11). Nanogram to microgram amounts of α-toxin can cause severe dermonecrosis when administered subcutaneously in rabbits and mice (20, 21). Moreover, intravenous administration of this toxin also results in rapid lethality of the animals (20, 21). S. aureus Δhla strains are severely attenuated in several infection models, resulting in enhanced host survival, decreased bacterial burden, inflammation, and tissue injuries (22–27).
The bicomponent PFTs
The bicomponent PFTs are relatives of α-toxin (Fig. 4), share structural homology with α-toxin, and have a similar pore formation mechanism (Fig. 1 and 2). However, in contrast to α-toxin, the bicomponent PFTs require two subunits: the fast-eluting subunit, F-subunit, and the slow-eluting subunit, S-subunit, named on the basis of their liquid chromatography behavior (28, 29). The current model for leukocidin pore formation suggests that the S-subunit recognizes and binds to a surface receptor on the target cell and then recruits the F-subunit for dimerization (30–32). This is followed by oligomerization with three additional dimers to form an octameric prepore on the target cell membrane (33). Next, the stem domains of the prepore extend in the center of the structure, forming a β-barrel pore that inserts into the target cell membrane, resulting in cell lysis (Fig. 1B) (33, 34). Similar to the α-toxin heptamer, the bicomponent PFT octamer also resembles a mushroom, consisting of the cap, the rim, and the stem domains (Fig. 2).
FIGURE 4.
Phylogenic tree of S. aureus PFTs. The tree is constructed based on the mature protein sequences using the DNASTAR MegAlign ClustalW method for multiple sequence alignment.
The bicomponent PFTs primarily target leukocytes, and thus they are known as leukocidins (Luk). Currently, five of the leukocidins are known to be associated with human infections: LukSF-PV (originally known as the Panton-Valentine leukocidin [PVL]), γ-hemolysins AB and CB (HlgAB, HlgCB), LukED, and LukAB (also known as LukHG) (30–32). Two other bicomponent PFTs, LukMF′ and LukPQ, are associated with animal infections (35–37).
All the leukocidins share structural homology and sequence identity, ranging from 40 to 90% within each S-subunit and F-subunit family (Fig. 4) (38). The only exception is LukAB, which shares only ∼30% sequence similarity with the others (38). LukA has an ∼33-amino acid sequence at the N-terminus and a 10-amino acid C-terminal tail that are absent from other S-subunits, contributing to its divergence. Nevertheless, the structure of LukAB remains homologous to the other bicomponent PFTs (39).
In additional to mediating cell lysis, many of the leukocidins have sublytic effects, causing extracellular Ca2+ influx on host cells (40–42) and production of proinflammatory cytokines (40, 43–47). Several of the leukocidins, PVL, HlgAB, and LukAB, stimulate K+ efflux, which is involved in the activation of the NLRP3 inflammasomes and caspase-1, resulting in a form of inflammatory cell death known as pyroptosis (43, 45–47).
Although the bicomponent PFTs exhibit many similarities, there are subtle differences that confer unique properties on each of them, which are briefly summarized below (see references 30–32 for in-depth summaries).
LukSF-PV (PVL)
The pvl locus is encoded within the genomes of at least six prophages (48–51). Although less than 40% of clinical isolates from the United States carry a pvl-encoding prophage, over 90% of strains associated with severe necrotizing pneumonia and community-acquired infections carry pvl (1, 52). The relationship between pvl-encoding S. aureus and severe infections in humans is supported by strong epidemiological data and recent data using animal models of infection (53–57).
PVL exhibits species specificity, killing only rabbit and human leukocytes. The species specificity is due to the targeting of the human and rabbit G-protein-coupled receptors, C5aR1 and C5aR2, but not the murine counterparts (Fig. 3) (58, 59). Consequently, regular mice are inappropriate for the study of this toxin; the availability of rabbit models and human ex vivo models has provided insight into the complexity of PVL-mediated pathology. PVL is a critical factor in invasive diseases such as osteomyelitis and pneumonia in rabbits. Deletion of pvl results in less inflammation, reduces tissue injuries and bacterial burden, and promotes host survival (57, 60). Remarkably, sublytic levels of PVL can enhance phagocytosis and killing of the bacteria by primary human neutrophils (61). In contrast, the role of PVL during skin and soft tissue infections is unclear. While one study demonstrated that PVL did not contribute to lesion sizes and bacterial burden (62), another study showed that infections caused by Δpvl strains have smaller lesions compared to the wild type and complemented controls (63). These studies seem to suggest that the pathogenic effect of PVL could be dependent on the site of infection, but more work must be done to test this hypothesis.
γ-Hemolysin (HlgAB, HlgCB)
The γ-hemolysin locus is part of the core S. aureus genome, present in ∼99% of sequenced S. aureus genomes (64, 65). This locus comprises three genes: the hlgA gene, transcribed by its own promoter, followed by an operon containing hlgC and hlgB, transcribed by a different promoter (66). HlgA and HlgC are S-subunits that share the same F-subunit, HlgB, to form two leukocidins, HlgAB and HlgCB, each possessing its own unique properties.
HlgAB binds to the human receptors CXCR1, CXCR2, CCR2, and DARC (the Duffy antigen receptor for chemokines), lysing human erythrocytes, neutrophils, monocytes, and macrophages (Fig. 3) (44, 67). HlgAB can also target murine monocytes and macrophages but cannot target murine neutrophils, because it cannot bind to murine CXCR2 (44). In contrast, HlgCB is a human-specific toxin that targets cells expressing the receptors C5aR1 and C5aR2 (the same receptors targeted by PVL) (Fig. 3) (44).
γ-Hemolysins cause acute tissue injury and inflammation and contribute to S. aureus disease in various animal models. Retroorbital administration of microgram amounts of HlgAB is lethal to mice (68). Intravitreal injection of γ-hemolysins in rabbits is highly toxic, resulting in destruction of the eye and tissue injury in surrounding areas (69). The tissue damage could be the result of a combination of toxin-mediated cell lysis and pyroptosis caused by sublytic concentration of the toxins (47). The contribution of HlgAB to disease has been further demonstrated in several infection models with strains that do not produce HlgAB. With such strains, there is reduced neutrophil lysis, less inflammation, reduced bacterial burden, and enhanced host survival (44, 70–72).
LukED
The lukED locus is in the νSaβ gene cluster. The lukED locus is present in ∼70% of S. aureus isolates and is conserved in a lineage-specific manner (64, 65). The two genes in the locus, lukE and lukD, are cotranscribed during the late exponential phase (73).
Early studies of LukED demonstrated the lytic activity of the toxin in rabbit and human erythrocytes and neutrophils (74). Subsequently, LukED was demonstrated to also mediate lysis of many different human and murine bone-marrow-derived cells (75, 76). LukED targets the G-protein-coupled receptors (CXCR1, CXCR2, CCR5, and DARC) on neutrophils, monocytes, macrophages, dendritic cells, NK cells, T-cells, and red blood cells, conferring on the toxin broad leukocidal activity (Fig. 3) (67, 76, 77). Moreover, the pathogenic effects of LukED are receptor-dependent (75–77).
LukED is an important contributor to the virulence of S. aureus. Initial studies of LukED demonstrated toxin-induced dermonecrosis of rabbit skin (73). Retroorbital administration of microgram amounts of the toxin leads to acute lethality in mice (68). Corroborating the intoxication studies, ΔlukED strains are severely attenuated, resulting in lowered inflammatory responses, reduced bacterial burden, and enhanced host survival in murine systemic infections (75–77).
LukAB (also known as LukHG)
The lukAB locus is part of the core S. aureus genome found in 99% of S. aureus. The toxin is found in abundance in the secreted proteome during the late exponential growth phase, which led to its discovery (78, 79). The C-terminal region of LukA is critical for toxin activity, because its deletion or mutation within this region (i.e., the E323A mutation) renders the toxin inactive (80). Unlike the other leukocidins, which are secreted as monomers, LukAB is secreted as heterodimers (80).
LukAB exhibits sharp species specificity, with its greatest potency being for human and primate cells, followed by rabbit, and it is ∼1,000-fold less active in mice (81). The selectivity of LukAB was explained when it was discovered that it mediates cytotoxicity through targeting the I-domain of the CD11b receptor present on leukocytes, including neutrophils, monocytes, macrophages, dendritic cells, and NK cells (Fig. 3) (80, 82).
Since LukAB is only weakly active toward murine leukocytes and mildly active with rabbit cells, the role of LukAB during infection remains to be fully elucidated. Although the field currently lacks robust in vivo models to directly assess the pathogenicity of the toxin, in vitro and ex vivo studies have provided insights into the role of LukAB in disease, revealing that lukAB-defective mutants are greatly attenuated for virulence. For example, primary human neutrophils infected with ΔlukAB strains exhibit enhanced survival compared to wild type (78). Additionally, LukAB promotes the escape of phagocytosed S. aureus from neutrophils and monocytes (46, 83). Anti-LukAB antibodies were observed in the serum of patients with invasive S. aureus disease, thus demonstrating production of the toxin during infection (84, 85).
LukMF′
The lukMF′ locus is encoded by the temperate phage ΦSa1 (37). While lukMF′ is infrequently found among human isolates, it is commonly found among animal isolates (86). LukMF′ exhibits high cytolytic activity toward bovine neutrophils and macrophages through targeting the surface receptor bovine CCR1, CCR2, and CCR5 (Fig. 3) (87). LukMF′ can be isolated from bovine mastitis tissue samples, indicating a role of this toxin in disease progression (36). However, unlike the other leukocidins, purified LukMF′ does not elicit a strong proinflammatory response when incubated with primary bovine macrophages (88).
LukPQ
LukPQ is encoded by the temperate phage ΦSaeq1 (35). Like other bicomponent PFTs, LukPQ demonstrates species and cell type specificities. Equine neutrophils are most sensitive to the lytic effect of LukPQ, followed by bovine neutrophils; human neutrophils are relatively insensitive to the toxin (Fig. 3) (35). LukPQ targets the equine G-protein-coupled receptors, CXCRA and CXCR2, to initiate cytolysis (35). At a high concentration, the toxin also targets equine CCR5. However, toxin binding can be inhibited by cytokines binding to these receptors, suggesting that LukP and the receptor ligands may share a common binding site (35). Uniquely, the F-subunit, LukQ, is responsible for conferring species specificity of LukPQ, whereas species specificity is conferred by the S-subunit in the other leukocidins (35).
Other cytotoxins
Phenol soluble modulins
Phenol soluble modulins (PSMs) belong to a family of amphipathic peptides uniquely found in staphylococci. In S. aureus, PSMs are encoded in three loci in the core genome: (i) the psmα operon, encoding PSMα1-4, (ii) the psmβ operon, encoding PSMβ1-2, and (iii) hld, encoding δ-toxin (89, 90). hld is also part of the coding sequence of RNA III, the master regulatory RNA in staphylococci. Additionally, certain methicillin-resistant staphylococci carry PSM-mec, encoded by psm-mec in the staphylococcal cassette chromosome mec (SSCmec) (91). Like hld, psm-mec is encoded within a regulatory RNA (92).
Unlike the other cytotoxins described thus far, PSM peptides are often secreted without a signal peptide (90). Therefore, most PSM peptides isolated from staphylococci contain an N-terminal formylmethionine. However, some PSM peptides lack this N-formylmethionine due to the cytoplasmic enzyme N-deformylase (93). PSMs are secreted by the ABC transporter phenol-soluble modulin transporter (Pmt). The absence of Pmt causes accumulation of PSM in the cytosol, resulting in cell death (94).
PSMs are classified based on length (95). The α-type PSMs are typically 20 to 25 amino acids long, most having a neutral or positive net charge. PSMα and δ-toxin are α-type PSMs, the entire protein forming one α-helix (96, 97). In contrast, the β-type PSMs are longer, typically 43 to 45 amino acids in length, most having a negative net charge. The structure of the β-type PSM, PSMβ2, consists of three α-helices that fold to a “v”-like shape (97).
PSMs have multiple roles in S. aureus pathogenesis, including cell lysis, biofilm formation, and immune modulation. α-type PSM peptides have high potency in lysing eukaryotic cells in a receptor-independent manner through targeting the cell membranes (90, 98–100). However, lipoproteins present in the serum can inhibit the cytolytic activity of PSMs (101). Therefore, the role of PSMs in extracellular cytolysis in vivo is unclear. In contrast, phagocytosed S. aureus produces PSMs to lyse neutrophils and osteoblasts intracellularly (99, 100). As such, the role of PSMs could be to mediate intracellular escape of S. aureus.
Sublytic concentrations of PSMs have immune modulatory effects on host cells. In humans, PSMs are detected by the pattern recognition receptor formyl peptide receptor 2 (FPR2) (102). FPR2 is a member of the G-protein-coupled receptor family that specializes in recognizing pathogen-associated molecular patterns produced by bacteria. FPR2 is predominately expressed on innate immune cell types, including neutrophils, monocytes, macrophages, and immature dendritic cells. Upon activation by PSMs, FPR2 induces a series of proinflammatory responses, including cytokine production, neutrophil chemotaxis, and leukocyte activation (102).
PSMs can shape biofilms by forming channels needed for nutrient delivery and dissemination (103). Additionally, the α-type can cause a leaky S. aureus membrane, resulting in the release of cytoplasmic proteins (104).
ε-toxin
Merriman et al. identified ε-toxin in 2015. The gene encoding ε-toxin, cytE, is conserved in the core genome of S. aureus (105). Since this is a recently identified toxin, the regulation of ε-toxin expression and the mode of action of the toxin are unknown. However, ε-toxin lyses rabbit erythrocytes and human keratinocytes. Lytic concentration of ε-toxin in keratinocytes promotes the secretion proinflammatory cytokine, interleukin 8 (105). In contrast, sublytic concentration of ε-toxin slows the rate of keratinocyte proliferation, suggesting a role for the toxin in impairing normal wound healing (105). A microgram amount of ε-toxin can result in neutrophil recruitment to the injection site when administered subcutaneously in rabbits (105). Moreover, the same dosage of ε-toxin can cause rabbits to develop fever after intravenous administration of the toxin (105). The role of this toxin in S. aureus infections remains to be fully elucidated.
Staphylococcal Superantigens
T cell Superantigens
T cell superantigens (SAgs) represent the largest family of exotoxins produced by S. aureus. Their molecular weights range from 19 to 30 kDa. SAgs are unique because they are resistant to heat, proteolysis, and desiccation (106). Due to their extreme stability and high toxicity in humans, some of them are classified as select agents for bioterrorism (i.e., staphylococcal enterotoxin B).
The genes encoding SAgs are found in various components of the S. aureus genome. The recently discovered selX and selW are part of the core genome (107, 354). The other SAgs are encoded in different mobile genetic elements, such as bacteriophages, plasmids, and pathogenicity islands (108–111). However, the distribution of SAgs is highly variable in the same mobile genetic element found in different strains (112).
SAgs share structural homology with another family of closely related proteins, the SSLs (staphylococcal superantigen-like proteins). The ssl genes are encoded in the gene cluster νSaα (113). This family of proteins was originally called SETs for staphylococcal enterotoxin-like proteins (113). However, the International Nomenclature Committee for Staphylococcal Superantigens renamed them in 2004 to reflect their lack of emetic and mitogenic properties (114). The primary role of these proteins seems to be immune evasion (see reference 111 for a review on the SAg-like proteins).
SAgs exhibit tremendous sequence diversity (Fig. 5), but their overall structures are similar. SAgs have two primary domains: an N-terminal oligosaccharide/oligonucleotide binding fold that is shaped like a β-barrel and a C-terminal β-grasp domain composed of antiparallel β-sheets. The two domains are connected by an α-helix (115). Additionally, all SAgs have a dodecapeptide binding site, a Vβ T cell receptor (TCR) binding site, and up to two major histocompatibility complex (MHC) binding sites (106).
FIGURE 5.
Phylogenic tree of S. aureus SAgs. The tree is constructed based on the mature protein sequences using the DNASTAR MegAlign ClustalW method for multiple sequence alignment.
An SAg exerts its mitogenic property by cross-linking the Vβ TCR on a T cell with the MHC class II molecule (MHCII) on an antigen presenting cell, resulting in polyclonal T cell proliferation (Fig. 6) (116). SAgs are highly effective T cell mitogens that can stimulate up to 50% of T cells (106). SAg-induced T cell proliferation is followed by a state of T cell anergy, when activated T cells failed to proliferate and/or undergo apoptosis. SAgs are one of the many ways S. aureus manipulates the host immune system to prevent the generation of functional adaptive immunity.
FIGURE 6.
Crystal structures of S. aureus superantigens in complex with their cellular targets. (A) The T cell superantigen, SEB, in complex with the TCR and MHC II molecule (4C56) (116). SEB (blue) cross-links the α-chain of MHC (dark green) to the Vβ TCR (orange) to induce T cell proliferation that results in T cell anergy and/or apoptosis. (B) The B cell superantigen, SpA (teal), in complex with the Fab fragment (pink/magenta) (1DEE) (138). Conventional antigens bind to B cell receptors at the complementarity-determining region (blue), a hypervariable region that confers antigen specificities. SpA binds at a constant region of the receptor to activate B cells for supraclonal expansion, which leads to clonal deletion of SpA-activated B cells.
SAgs can be broadly divided into three groups: staphylococcal enterotoxins (SEs), staphylococcal enterotoxin-like (SE-l) SAgs, and toxic shock syndrome toxin-1 (TSST-1). Each group of SAgs is briefly summarized below (see references 106, 355).
Staphylococcal enterotoxins
There are six distinct SEs: SEA to SEE and SEG. Additionally, several variants of SEB and SEC have been identified. They were originally named for their ability to induce emesis, a key characteristic of staphylococcal food poisoning (114). Ingestion of SEs causes vomiting and diarrhea. However, the disease is usually self-limiting. The emetic activity of SEs is correlated with the presence of a 9- to 19-amino acid-long disulfide loop in the protein.
SEs can have up to two MHC binding sites. SEBs and SECs contain only one MHC binding site, while the other SEs have two MHC binding sites (106, 117, 118). The low-affinity binding site targeting the MHCII α-chain is common to all SAgs. The second site is a Zn2+-dependent high-affinity binding site that targets the β-chain of MHCII (119). SAgs that have two MHC binding sites are 10- to 1,000-fold more potent than SAgs that have only one. However, they are produced at a much lower abundance compared to some other SAgs that contain one MHC binding site, such as TSST-1 and SEB (106).
Staphylococcal enterotoxin-like SAgs
The other SAgs are SE-l H to SE-l Y (106, 114, 355). SE-ls include all newly identified SAgs that are T cell mitogens but have unproven emetic activity (114). However, this group also contains SE-ls that lack the emesis-associated disulfide loop and are proven to not induce emesis (106). SE-ls can have up to two MHC binding sites and a 15-amino acid extension for specific TCR interactions (106, 120).
Toxic shock syndrome toxin-1
The gene tst is encoded in several pathogenicity islands, including SaPI1, SaPI2, and SaPIbov1 (121). TSST-1 has only one low-affinity MHC binding site that targets the MHCII α-chain, a Vβ TCR binding site, and a dodecapeptide binding site (122). This dodecapeptide binding site is proposed to be important for the interaction of TSST-1 with epithelial cells and the immune stimulatory molecules, CD40 and CD28 (123–125).
TSST-1 originally was known as SEF. In 1984, it was renamed to reflect the lack of emetic activity and to emphasize its association to toxic shock syndrome (TSS). TSS is an acute systemic illness characterized by hypotension, fever, rash, and desquamation 1 to 2 weeks after onset. As defined by the Centers for Disease Control, TSS involves at least three of the following organ systems—gastrointestinal, muscular, mucous membrane, renal, hepatic, hematologic, or central nervous system (126). TSS can be further classified as menstrual and nonmenstrual TSS. Menstrual TSS is usually associated with vaginal/cervical mucosae colonization of TSST-1-producing S. aureus and tampon use (127, 128). Approximately 50% of nonmenstrual TSS cases are caused by TSST-1-producing strains, and the remaining are caused by strains that produce SEB or SEC (129).
B cell Superantigen
Staphylococcal protein A (SpA) is the only known B cell superantigen produced by S. aureus. A majority of clinical isolates contain spa in the core genome (130). The SpA precursor has an N-terminal signal peptide that is cleaved prior to the secretion of the mature protein. Mature SpA has four to five highly conserved immunoglobulin (Ig) binding domains connected by short linkers at the N-terminus (131). These are followed by a hypervariable region called region X, which comprises subregions Xr and Xc (132). The highly variable and repetitive octapeptide in Xr is the basis of SpA-typing, a high-throughput method of grouping S. aureus isolates (130). Region X is followed by the C-terminal LPXTG motif for covalent anchoring of the protein to the cell wall (131). However, SpA proteins can be released from the cell wall by the cell wall hydrolase, LytM (133).
The Ig binding domains confer upon SpA the ability to bind the Fcγ portion of Igs to prevent opsonization (134). These Ig binding domains also mediate SpA binding to B cells by cross-linking VH3-expresssing B cell receptor, which results in B cell activation; however, activation without costimulatory signals results in death and subsequent clonal deletion of B cells (135–137).
Conventional antigen recognition by a B cell receptor requires antigen recognition at the complementarity-determining region. In contrast, SpA exerts its mitogenic activity by binding to the variable region of the heavy chain, away from the complementarity-determining region, thus bypassing the antigen specificity requirement for B cell activation (Fig. 6) (138, 139). SpA-mediated clonal deletion of VH3-expressing B cells can lead to the impairment of the B cell repertoire that is important for mounting effective antimicrobial defenses against the pathogen (140, 141).
During intravenous infection, SpA prevents opsonophagocytosis of the bacteria by sequestering Igs and impedes the development of specific anti-S. aureus antibodies (142). In contrast, isogenic strains that lack spa or express variants that cannot bind to Ig exhibited reduced kidney abscess formation and elicited specific anti-S. aureus antibodies (142). Mice immunized with the Ig binding-deficient SpA variant, SpAKKAA, acquired protective immunity and could mount a more effective humoral response against S. aureus antigens (143).
Cytotoxic Enzymes
β-toxin (also known as β-hemolysin)
The β-toxin encoding gene, hlb, is part of the core S. aureus genome. However, due to the presence of the hlb-converting prophage (i.e., ΦSa3, Φ13), which disrupts the gene, only a limited number of human clinical isolates produce β-toxin (144, 145). The prophage carries the immune evasion gene cluster encoding for immune evasion factors, such as the staphylococcal complement inhibitor proteins, chemotaxis-inhibitory proteins, and staphylokinase (145). These virulence factors are thought to be involved in S. aureus immune evasion and survival in the human host. The hlb-converting prophage is prevalent in strains associated with human infections (∼90%), but it is less frequently found in animal isolates (∼30%) (146). However, chronic infections or environmental pressures (i.e., oxidative stress, antibiotics, temperature) can promote the excision of the prophage and the production of β-toxin (147–150). Thus, the contribution of β-toxin during human infection is unclear.
β-toxin is a Mg2+-dependent neutral sphingomyelinase (SMase), a phospholipase that specifically cleaves sphingomyelin to produce ceramide and phosphocholine (151). This toxin was first identified in 1935 by Glenny and Stevens based on several unique observations: hemolysis of erythrocytes in the presence of α-toxin neutralizing serum, lysis of sheep but not rabbit erythrocytes, and enhanced hemolysis caused by the temperature shifting from 37°C to a lower temperature (152). As such, β-toxin is also known as a hot-cold hemolysin. This unique phenomenon is the result of ceramide hydrolysis products at 37°C being held together by cohesive forces in the membrane. When the temperature decreases (i.e., to 4°C), this causes a phase separation that condenses ceramide into pools and collapses the lipid bilayer, resulting in the invaginations observed on erythrocyte membranes by electron microscopy (153).
The crystal structure of β-toxin reveals structural homology to members of the DNase I superfamily (154). β-toxin is a single domain protein consisting of four layers: two layers of β-sheets at the center and two outer layers that comprise α-helices and β-strands (154). Based on the structural homology to the DNase I superfamily, a secondary function for β-toxin was hypothesized. Later, β-toxin was shown to enhance biofilm formation through catalyzing the formation of nucleoprotein matrix in biofilms; therefore, β-toxin is also a biofilm ligase (155).
β-toxin exhibits species-dependent hemolytic activity that correlates with the amount of sphingomyelin content in erythrocytes: sheep, cow, and goat erythrocytes are highly sensitive to the toxin, rabbit and human erythrocytes exhibit intermediate sensitivity, and murine and canine erythrocytes are resistant (156). The SMase activity of β-toxin also causes the lysis of human keratinocytes, monocytes, T cells, and bovine epithelial cells (154, 157–159). β-toxin stimulates the production of proinflammatory cytokines in human monocytes (158) but suppresses IL8 production and cell adhesion molecule expression in human endothelial cells, and therefore the toxin can prevent leukocyte migration across the endothelium (160).
Infection with β-toxin-producing S. aureus results in larger lesions in the organs without affecting the overall bacterial burden in the rabbit endocarditis and pneumonia models (147). The presence of β-toxin enhances S. aureus colonization of the skin (159) and induces injuries to the scleral epithelial cells during ocular keratitis in mice (27). In an infective endocarditis model, rabbits infected with S. aureus that produce β-toxin mutants that lack SMase activity have enhanced survival and smaller lesions in the heart, but there were no differences in bacterial burden compared to the isogenic strain that produces active β-toxin (161). Intranasal administration of β-toxin induces the shedding of syndecan-1, a major heparan sulfate proteoglycan molecule on lung epithelial cells, and causes neutrophil infiltration into the lungs in mice (162). The shedding of sydecan-1 is caused by the SMase activity of β-toxin, because intranasal intoxication with SMase mutants reduced shedding of this protein in vivo, resulting in reduced lung pathology (162).
Exfoliative toxins
Exfoliative toxins (ETs) are also known as epidermolytic toxins. There are four antigenically distinct forms found in S. aureus: ETA, ETB, ETC, and ETD. Each ET is encoded on a different mobile genetic element: eta is encoded in the genome of a temperate phage that has been shown to convert eta-negative strains to toxin producers (163, 164). etb is found on the plasmid pETB (165), and etd is encoded as part of a pathogenicity island (166). ETC was purified from an S. aureus isolate from equine infection; however, the genetic locus of ETC has not been described (167).
Most of what is currently known about the ETs is based on ETA and ETB. ETs are the causative agents for staphylococcal scalded skin syndrome (SSSS), including Ritter’s disease, toxic epidermal necrosis, bullous impetigo, and certain erythema cases. SSSS predominantly affects neonates, infants, and immunocompromised adult patients (168). Symptoms of SSSS are characterized by formation of blisters and superficial desquamation, involving only the skin layer (168). Although SSSS was initially described in 1878, its association with S. aureus infections was first suggested in 1967, and the contribution of ETs to the blistering symptoms was not identified until in the 1970s (163, 169–171). This delay was in part contributed by the ETs’ unique mode of action. The lesions characteristic of SSSS are often sterile, because the ETs can be distributed through the bloodstream from a distant site to cause symptoms (171).
ETs are glutamate-specific serine proteases of the chymotrypsin family. The catalytic triad (histidine, aspartate, serine) is conserved in all ETs (168). ETA and ETB are similar in structure and share homology with other staphylococcal serine proteases: SspA and the serine protease-like proteins (Fig. 7) (172–174). The N-terminal α-helical extension is required for enzyme activity (172, 174). The crystal structures of both ETA and ETB show that the key residues in loop D occupy the oxyanion hole required for enzymatic activity of all serine proteases, and thus the crystal structures represent the inactive forms of the enzymes (172, 174). These findings suggest that the protease activity of ET may require a specific cellular target and occur under specific conditions.
FIGURE 7.
Overlay of the crystal structures of ETA and ETB. ETA (1EXF, green) (174) and ETB (1QTF, blue) (173) share high structural identity. ETs cause SSSS by cleaving Dsg1 at the epithelial cell junctions. Both ETs are serine proteases. Loop D and the catalytic triad are highlighted in pink for ETA and red for ETB.
In the early 2000s, ETs were shown to interact with human and mouse desmoglein 1 (Dsg1), causing blistering of the superficial skin (175, 176). ET recognition of Dsg1 is conformational, requiring the presence of Ca2+. Lack of Ca2+ results in the unfolding of Dsg1 and thus the inability of ETs to cleave the protein (177). Through domain swapping and site mutagenesis studies, it was determined that five amino acids (Q271, Y274, T275, I276, E277) belonging to the extracellular domain 3 of Dsg1 are critical for ETA to exert its protease activity (178).
With the identification of Dsg1 as the substrate for ETs, the pathophysiology of the superficial skin blistering in SSSS was explained. In humans, there are four isoforms of desmogleins, Dsg1 to Dsg4 (179). Desmogleins are cadherins that are required for desmosome cell-to-cell adhesion to maintain the integrity of the epidermis. ETs target only Dsg1, which is expressed throughout the human epidermis. Cleavage of Dsg1 disrupts the cell-to-cell adhesion of the epidermis, resulting in blistering and desquamation of the superficial skin. The other strata of the epidermis are unaffected because of the presence of Dsg2 to Dsg4, which are not targeted by ETs and thus compensate for the destruction of Dsg1 (175, 180).
EXOENZYMES
Introduction
S. aureus devotes a significant amount of its resources to produce virulence factors to evade the host immune system and to acquire nutrients for its survival. The previous section discussed the mechanisms of toxin-mediated host immune evasion and their roles in S. aureus virulence. In addition to the toxins, S. aureus also produces a large number of virulence factors that have enzymatic properties. They can be broadly categorized into two groups: cofactors that activate host zymogens and enzymes for degradation of tissue components (Table 2). While these cofactors and secreted enzymes (exoenzymes) have different substrates and mechanisms of action, they function to break down bacterial and host molecules for nutrient acquisition, bacterial survival, and dissemination.
TABLE 2.
Major secreted cofactors and enzymes produced by S. aureus
| Cofactor/enzyme | Gene | Function(s) |
|---|---|---|
| Coagulase | coa | Cofactor, activates prothrombin |
| vWbp | vwb | Cofactor, activates prothrombin |
| Staphylokinase | sak | Cofactor, activates plasminogen |
| Nuc (thermonuclease) | nuc | Nuclease |
| Aureolysin | aur | Metalloprotease |
| ScpA (V8 protease) | sspA | Serine protease |
| SplA | splA | Serine protease |
| SplB | splB | Serine protease |
| SplC | splC | Serine protease |
| SplD | splD | Serine protease |
| SplE | splE | Serine protease |
| SplF | splF | Serine protease |
| Exfoliative toxin A | eta | Serine protease |
| Exfoliative toxin B | etb | Serine protease |
| Staphopain A | scpA | Cysteine protease |
| Staphopain B | sspB | Cysteine protease |
| Hyaluronidase | hysA | Lyase |
| β-Toxin | hlb | Sphingomyelinase, biofilm ligase |
| PI-PLC | plc | Phospholipase |
| SAL1 | lip1 | Lipase |
| SAL2 | geh | Lipase |
| FAME | Unknown | Detoxify free fatty acids |
Cofactors for Host Enzyme Activation
Coagulase (Coa), von Willebrand factor binding protein (vWbp), and staphylokinase (Sak) are cofactors produced by S. aureus that have no enzymatic activities by themselves, but they can activate host zymogens. These three proteins hijack different aspects of the host coagulation system, thereby manipulating the host innate defenses to promote bacterial survival and dissemination.
Staphylococcal coagulases: Coa and vWbp
The ability to induce coagulation is one of the key criteria used in modern medical microbiology for species classification in the genus Staphylococcus—separating coagulase-positive and coagulase-negative species. A majority of staphylococci are coagulase negative, but a few are coagulase positive species, including S. aureus and S. intermedius; however, S. schleiferi has both coagulase-positive and coagulase-negative subspecies (181).
S. aureus-induced coagulation of human plasma was initially documented in 1903 (182). The causative agents, Coa and vWbp, are highly active in coagulating human and rabbit plasma (183).
Both coa and vwb are chromosomally encoded. There are 12 isoforms of coa that have been identified thus far; the majority of the variability is attributed to the high sequence variability (>50%) of the N-terminus coding region among different strains (184, 185). In contrast, vwb, which encodes vWbp, is relatively conserved, with only two known alleles (184). However, a recent report identified several vwb paralogues carried by SaPIs that produce vWbps that coagulate ruminant and equine plasma (186).
Coa and vWbp share ∼30% protein sequence homology at the N-terminus (187). They both have a D1D2 domain for prothrombin binding (188, 189). However, they differ significantly at the C-terminus. The C-terminus of Coa has a 188-residue linker region followed by a repeat region composed of tandem repeats of 27 residues responsible for fibrinogen binding (188, 189). In contrast, the C-terminus of vWbp has a von Willebrand factor binding domain and a fibrinogen binding domain (188, 190).
Coa or vWbp bind to prothrombin at a ratio of 1:1 to form staphylothrombin. Insertion of the N-terminus of Coa into the Ile16 pocket of prothrombin causes a conformational shift resulting in the activation of the zymogen (189, 191). Staphylothrombin is highly efficient in converting fibrinogen to fibrin (Fig. 8).
FIGURE 8.
S. aureus produces cofactors that hijack the host’s coagulation system. Coa and vWbp bind to prothrombin and alter the conformation of the protein to form the complex, staphylothrombin. This complex is highly active and cleaves fibrinogens to fibrins, promoting the formation of fibrinous clots. Sak binds to plasmin to form the Sak-plasmin complex. Sak stabilizes plasmin to enhance enzymatic activity. Sak-plasmin cleaves plasminogen to form plasmin, which breaks down fibrin clots.
The activity of staphylothrombin cannot be inhibited by common anticoagulants (i.e., EDTA, heparin) or thrombin inhibitors, such as hirudin and bivalirudin (189, 191–193). However, two recently discovered small molecules, argatroband and dabigatran, can inhibit the activity of staphylothrombin (194, 195).
The ΔcoaΔvwb strain is less virulent than its wild-type parent, demonstrating a role of the coagulases during infection (187, 196, 197). However, coagulases must be present concurrently with the infecting strain to promote virulence. Ekstedt and Yotis demonstrated that while intracerebral coinjection of purified coagulase with coagulase-negative S. aureus enhances the virulence of coagulase-negative S. aureus; preinjection of purified coagulase before infection has no effect (198). Additionally, Coa is suggested to have a role in the formation of device-associated biofilm formation (199).
In an abscess, the coagulases generate a fibrin shield to protect S. aureus from immune cell infiltration. Coa is required for the formation of pseudocapsule immediately surrounding the abscess, and both vWbp and Coa are required for fibrin formation around the pseudocapsule (187, 200).
Staphylokinase
Staphylokinase (Sak) is a cofactor that hijacks host plasmin to activate plasminogen for the breakdown of fibrin clots and promotes bacterial dissemination (Fig. 8). Sak is produced by lysogenic strains of staphylococci; the prophage encoding Sak typically carries other genes that encode virulence factors such as enterotoxin A and chemotaxis inhibitory proteins (201, 202). There are three groups of phages that carry the sak gene (203). Serotype B phages (i.e., ΦC) cause positive conversion of Sak without disrupting other genes (204, 205). Positive conversion of sak can also be mediated by some serotype F phages (i.e., ΦSa3, Φ42D), but the phage integration disrupts the hlb gene (144, 202, 206). The phage carrying sak has also been reported to disrupt the coding sequences of N-acetylmuramyl-l-alanine amidase and peptidoglycan hydrolase (207, 208).
Sak is a single-domain protein that consists of a central α-helix, a five-strand β-sheet, and two shorter β-strands (Fig. 9) (209). Sak forms a 1:1 complex with plasmin in the serum to form Sak-plasmin (210, 211). This complex is highly efficient in converting plasminogen to plasmin. Sak can also bind to plasminogen, but this complex is inactive and must be converted to Sak-plasmin to have enzymatic activity (212). In an active Sak-plasmin complex, the first 10 residues at the N-terminus of mature Sak are removed to expose the charged residue, Lys11 (213). Deletion of Lys11 inactivates Sak (214). The binding of Sak to plasmin directs the active site of plasmin to favor cleavage of the activation loop in plasminogen and promotes the conversion of plasminogen to plasmin by enhancing substrate presentation to plasmin. (Fig. 9) (215).
FIGURE 9.
The crystal structure of Sak in complex with two plasminogen molecules (1BUI) (215). While Sak binding to plasminogen does not have enzymatic activity, the trimeric complex captures how Sak may bind to plasmin to cleave plasminogen. Sak (orange) is in complex with plasminogen (blue), exposing the catalytic site (red). Sak facilitates the docking of the substrate plasminogen (pink) to promote cleavage by plasmin.
Circulating Sak-plasmin complexes are sensitive to dissociation by α2-antiplasmin, but fibrin-bound complexes are protected from inactivation (216). The fibrin-bound complexes cleave IgG and human C3b, thus preventing opsonization of the bacteria by the complement system (217). Additionally, Sak-plasmin complexes can activate the matrix metalloprotease 1, which is important for leukocyte migration and activation (218). Importantly, Sak neutralizes the bactericidal activities of α-defensins and LL-37, two major human antimicrobial peptides (219, 220).
Sak is highly species specific. It is active for human, dog, goat, rabbit, and sheep plasminogen but is inactive for mouse, pig, cow, and buffalo plasminogen (221). Using transgenic mice that produce human plasminogen, studies demonstrated that Sak facilitates S. aureus invasion of the skin barrier to generate large and open lesions (222, 223). However, plasmin activation is known to promote wound healing and to reduce inflammation. Thus, during skin infection, Sak may function as a vanguard to establish the primary infection, but after the infection is established, Sak limits the severity of infections to promote dissemination (223).
Furthermore, Sak reduces biofilm formation and facilitates the detachment of mature biofilm by activating plasminogen (221). Corroborating these observations, high Sak-producing strains are often associated with less biofilm formation in vitro and noninvasive infections in humans (221, 223).
Enzymes that Degrade Host Tissue Components
Nucleases
Staphylococcal nuclease, originally known as micrococcal DNase, was identified in the culture supernatants of S. aureus by Cunningham et al. in 1956 (224). Nuclease requires Ca2+ ions for activity, but not other divalent cations (224, 225). Staphylococcal nuclease is also known as thermonuclease, named after its resistance to heat inactivation (224, 225). Staphylococcal nuclease functions as both an endo- and exo-nuclease that breaks down DNA and RNA substrates through the cleavage of the 5′-phosphoryl ester bond (224, 225).
With the availability of whole-genome sequencing in the late 1990s, the sequence of the S. aureus genome became available, which led to the identification of two staphylococcal nuclease genes, nuc (SA0746) and nuc2 (SA1160) (226, 227). The two genes are located at disparate regions in the genome, under the control of separate promoters. The two nucleases share 34% amino acid similarity overall and 42% similarity within the catalytic domain (228). Both nucleases are Ca2+ dependent and heat resistant and are able to use DNA and RNA as substrates (226, 228). A major difference between Nuc and Nuc2 is their cellular localization. Nuc is a secreted enzyme with two isoforms, NucB and NucA (229, 230). In contrast, Nuc2 is surface bound (228).
Much of what is currently known about nucleases is gathered from studies performed on Nuc. During infections, Nuc regulates biofilm formation and mediates bacterial escape from neutrophil extracellular traps (NETs).
Nuc disperses biofilm by breaking down extracellular DNA. Biofilm formation is enhanced in strains that do not produce Nuc (230, 231). Expression of nuc is repressed during biofilm formation, providing evidence that S. aureus controls nuclease expression to regulate biofilm formation (230, 232). Furthermore, the nuc mutant has decreased fitness during intraperitoneal infection in vivo (232).
The second role of Nuc is to mediate bacterial escape from NETs. NET is an innate immune defense mechanism by which DNA released from dying neutrophils immobilizes and facilitates killing of invading pathogens (233). Nuc degrades NETs to allow S. aureus to escape (234).
Moreover, when Nuc degrades DNA in the abscess or NETs, the degradation products, monophosphate nucleotides, become a substrate for another enzyme, adenosine synthase A (235). Adenosine synthase A converts the degraded DNA to deoxyadenosine, which induces caspase-3 activation, leading to apoptosis of macrophages surrounding the abscess or the NET, thus promoting S. aureus survival (235).
The contribution of Nuc2 to S. aureus virulence is less clear due to its low expression level compared to Nuc (228). Purified Nuc2 has been demonstrated to disperse biofilms in vitro (228). A mutant expressing only Nuc2 but not Nuc showed that the nuclease is produced during intramuscular infections in mice, albeit at a much lower level (228). The identification of Nuc2 in vivo suggests that it may have a role in S. aureus virulence, possibly performing functions similar to those of the secreted Nuc but on the bacterial surface.
Proteases
Staphylococci encode three families of secreted proteases: metalloproteases, cysteine proteases, and serine proteases. Collectively, these proteases have roles in nutrient acquisition, bacterial dissemination, and immune evasion. Currently, S. aureus is known to produce 12 proteases: one metalloprotease (aureolysin [Aur]), two cysteine proteases (staphopain A [ScpA] and staphopain B [SspB]), and nine serine proteases. These serine proteases include V8 protease (SspA), serine protease-like proteins A to F (SplA to SplF), and ETA and ETB. Although Spls, ETA, and ETB are secreted as active enzymes, all the other proteases are secreted as zymogens, requiring proteolytic cleavage of the propeptide for activation (Fig. 10). The roles of ETA and ETB in S. aureus virulence are described in the exotoxin section, above.
FIGURE 10.
Staphylococcal protease cascade. The metalloprotease, Aur, is activated by autoproteolysis after protein secretion. Aur is required to activate the serine protease, SspA. SspA processes one of the staphopains, SspB, from zymogen to active enzyme. The other staphopain, ScpA, is activated by autoproteolysis. Both staphopains are inhibited by staphostatins prior to secretion. SspB is inhibited by SspC, and ScpA is inhibited by ScpB.
In the following sections, we will discuss the mode of action of each protease family and its proposed role in S. aureus virulence.
Metalloprotease: Aureolysin
The S. aureus metalloprotease, aureolysin (Aur), also known as protease III, was identified in the culture supernatant of strain V8 by Arvidson et al. in 1972 (236, 237). The structure of Aur comprises two conserved domains common to bacterial metalloproteases of the thermolysin family: the N-terminal β-pleated domain and the C-terminal α-helical domain (238). The mechanisms of substrate binding and protein catalysis are also common among the proteases in this family. However, unlike other bacterial metalloproteases in the thermolysin family, Aur does not have elastase activity (238, 239).
Aur self-activates by autoproteolysis through the cleavage of the N-terminal propeptide (Fig. 10) (240). The active enzyme prefers to cleave peptide bonds at the N-terminal side of bulky hydrophobic residues, such as alanine, isoleucine, and tyrosine (241). The presence of Zn2+ is required for enzyme activity, but Co2+ can act as a substitute and increases enzyme activity (242). Additionally, binding to Ca2+ ions stabilizes Aur, whereas chelating agents, such as EDTA, irreversibly denature the protein (236, 238).
The broad substrate specificity of Aur allows the metalloprotease to target a variety of substrates, including other S. aureus proteins that are important for virulence and host proteins that are important for immune defense. Aur activates SspA, the second protease in the staphylococcal protease activation cascade (Fig. 10) (243). Additionally, Aur can degrade clumping factor B (ClfB) and the PSMα peptides (244, 245). Collectively, Aur can shape the secreted and surface proteome of S. aureus (246).
Aur contributes directly to S. aureus immune evasion and dissemination through the cleavage and inactivation of the antimicrobial peptide LL-37, thus promoting S. aureus survival (247). Aur can also degrade the human plasma protease inhibitors—α1-proteinase inhibitor and α1-antichymotrypsin present in the serum—albeit not as efficiently as SspA (248, 249). As such, Aur and SspA are proposed to work synergistically to achieve immune evasion.
Aur can affect complement activation by cleaving the complement proteins C3 to C3a and C3b in serum (250). The anaphylatoxin, C3a, is further degraded by Aur, preventing leukocyte activation (250). The soluble C3b fragment is inhibited and degraded by factor H and factor I in the serum (250, 251). Degradation of C3 by Aur results in the depletion of C3 proteins, thus preventing the formation of the membrane attack complex on the bacteria, and promotes bacterial survival. Furthermore, Aur activates prothrombin and prourokinases and inactivates plasminogen inhibitors, thereby manipulating the host coagulation system (252, 253). The various roles of Aur in modulating S. aureus proteome and host innate defense molecules suggest that Aur has an important role in promoting survival and dissemination of the bacteria in vivo. This is corroborated by the detection of Aur in phagocytosed S. aureus, suggesting that the protease may have a role during intracellular infection (254).
Serine proteases: SspA
The serine protease, SspA is also known as the V8 protease or GluV8. The gene encoding SspA (sspA) is part of the staphylococcal serine protease operon (ssp), which consists of three genes: sspA, sspB, and sspC (255). The functions of SspB and SspC are discussed later in this section.
SspA was identified in the culture supernatants of strain V8 by Drapeau et al. in 1972 (256). Around the same time, Arvidson et al. identified protease I, which exhibited properties similar to those of the SspA identified by Drapeau et al.; however, whether these two reports describe the same enzyme was difficult to decipher because there were differences in molecular weight and protease inhibitor sensitivity between the two reports (241, 257). Based on the report by Drapeau et al., the enzymatic activity of SspA can be inhibited by the serine protease inhibitor disopropyl flurophosphate (256).
SspA is a glutamyl endopeptidase, part of a small group of serine proteases that preferentially cleave substrates at the C-terminal side of glutamate and aspartate (256). The preference for negatively charged residues as substrates at neutral pH is due to the protein’s positively charged N-terminus (258). The crystal structure of SspA showed that the protein lacks the disulfide bonds commonly found in other proteins of this family (258). However, SspA shares high structural homology to the serine proteases—staphylococcal ETs and bovine trypsin—despite having limited protein sequence similarity (258). The conserved trypsin-like serine protease catalytic triad, consisting of histidine, asparagine, and serine, is found in SspA. The C-terminal repeat domain consists of tandem repeats of Pro-Asp or Asn-Asn that range from 9 to 19 repeats (259–261); however, because this C-terminal repeat domain is not required for activity (262), its role in the function of the protein is unclear.
SspA is secreted by S. aureus as a zymogen. However, pro-SspA can undergo autoproteolysis to generate a shorter version of pro-SspA (243). Aur processing is required for both forms of pro-SspA to become active enzymes (Fig. 10) (243).
SspA contributes to S. aureus immune evasion and dissemination by breaking down self and host proteins. SspA cleaves fibrinogen binding factors on the S. aureus cell surface and thus reduces bacterial adhesion and enhances bacterial dissemination and ultimately results in the breakdown of biofilms (263, 264). SspA can also degrade host proteins such as α1-proteinase inhibitor (248), the interleukin 6 cytokine (265), and immunoglobulins (266, 267); thus, SspA directly modulates immune activation and opsonization. SspA can cleave LL-37, but the cleavage does not inactivate the antimicrobial peptide (247).
SspA is produced upon S. aureus phagocytosis by neutrophils, suggesting its role in facilitating S. aureus intracellular escape, potentially through activating the cysteine protease, staphopain B (254). SspA can disrupt epithelial barriers, compromising cell junction integrity (265, 268). Skin infection models of sspA mutants suggest a slight decrease in bacterial fitness in vivo (269). However, since the activity of SspA can be inhibited by an α2-macrogobulin present in the serum, its role as a soluble virulence factor in serum during S. aureus pathogenesis remains unclear (263).
Cysteine proteases: Staphopains
Staphopain A (ScpA) was the first cysteine protease identified in S. aureus by Arvidson et al. (described as protease II) in 1973 (257). Subsequently, staphopain B (SspB) was identified as ORFX in 1998 by Chan et al. (270). A third staphopain, staphopain, C was described in avian-associated S. aureus isolates (271, 272). Most of what is known about staphopains is derived from studies of staphopains A and B.
ScpA and its intracellular inhibitor staphostatin A are encoded in the staphylococcal cysteine protease operon (scp) by the genes scpA and scpB, respectively (273). Staphopain B (SspB) and it intracellular inhibitor staphostatin B (SspC) are encoded in the staphylcoccal serine protease (ssp) operon by the genes sspB and sspC, respectively (255).
ScpA and SspB are secreted as zymogens. Pro-SspB is processed by SspA as the last step of the proteolytic cascade that begins with Aur (Fig. 10) (255, 274). In contrast, processing of pro-ScpA is not mediated by Aur, SspA, or SspB (269). Instead, pro-ScpA undergoes rapid autoproteolysis, but this process also leads to rapid degradation of the protease (275).
Despite limited primary sequence identity, crystal structures of both staphopains demonstrated structural similarity to papain. Classical papain-like proteases contain two domains: the helical L-domain is composed of the N-terminal part of the protein, containing the catalytic cysteine, and the R-domain is composed of the C-terminal part of the protein, which folds into antiparallel β-sheets, forming a β-barrel-like structure that contains the catalytic histidine and aspartate (Fig. 11) (276–278). The location of the catalytic triad is conserved in both staphopains (276, 277).
FIGURE 11.
Staphopain-staphostatin complex (1PXV) (293). Staphopain SspB (blue) has two domains: the L-domain is helical, and the R-domain consists of β-strands that fold into a β-barrel-like structure. The catalytic site of SspB is highlighted in red. Staphopain SspC (beige) is a single domain protein composed of eight β-strands forming a single mixed β-barrel domain. SspC is a competitive inhibitor of SspB, directly blocking substrate access to the active site.
Although ScpA and SspB share high structural similarity, subtle differences between the proteases confer different substrate specificities. ScpA cleaves elastins found in connective tissues, pulmonary surfactant protein A in the lungs, and the chemokine receptor CXCR2 on leukocytes (239, 279–281). Additionally, ScpA promotes vascular leakage by activating the plasma kallikerin/kinin system, resulting in hypotension (281). The activity of ScpA in mediating vascular leakage is enhanced by SspB; however, SspB alone does not induce vascular leakage, demonstrating substrate specificity of the two proteases (281).
In contrast, SspB degrades the antimicrobial peptide LL-37, thereby promoting bacterial survival (282). SspB also cleaves CD11b and CD31, surface proteins that are important for the activation and survival, respectively, of phagocytes (283, 284). Thus, SspB prevents S. aureus from phagocytosis while diminishing the leukocytes’ abilities to detect pathogens. Paradoxically, SspB is also a potent activator of chimerin, a chemoattractant for dendritic cells and macrophages (285). S. aureus thrives intracellularly in macrophages and dendritic cells (286, 287). Therefore, SspB may function to promote the intracellular lifestyle of S. aureus for persistent infections. In fact, S. aureus has been demonstrated to produce SspB, SspA, and Aur after neutrophil phagocytosis (254).
Both ScpA and SspB are implicated in modulating biofilm formation (288, 289). The expression of both staphopains is repressed during biofilm formation, and the production of staphopain results in the dispersal of biofilms (289).
In addition to promoting biofilm dispersal, staphopains have a direct effect on the host’s connective tissues and coagulation system. Staphopains inactivate a number of host proteins, including α1-proteinase inhibitor, collagen, and fibrinogens; however, SspB has higher activity in cleaving fibrinogen and collagen compared to ScpA (248, 290).
The activities of staphopains are inhibited by the cysteine protease inhibitor E-64; by heavy metals, such as Co2+, Ag2+, Hg+, and Zn2+; and by host-derived proteins, including α2-macroglobulin in human plasma and the epithelial serpin, SCCA1 (239, 257, 291). Additionally, S. aureus produces inhibitors against the enzymes, known as staphostatins. Staphostatins are specific reversible inhibitors of staphopains. Staphostatin A can inhibit only staphopain A but not staphopain B (273). Similarly, staphostatin B inhibits only staphopain B (273). Both staphostatins are similar in size and structure. These small proteins (∼13 kDa) each comprise eight β-strands that form a single mixed β-barrel domain (292). Staphostatin occupies the same binding site as substrate, and thus they are competitive inhibitors (Fig. 11) (276, 293). Staphostatins lack signal peptides, and thus they are proposed to inhibit intracellular staphopain activities prior to secretion of the proenzyme (273, 294).
Serine proteases: the serine protease-like proteins
Serine protease-like proteins A to F (SplA to SplF) are the newest group of secreted staphylococcal serine proteases identified. SplC (named ORF-2 in the study) was the first Spl identified from a screen of S. aureus antigens reactive to serum antibodies from endocarditis patients (295). Soon after, splC was discovered as part of the spl operon, encoding splA to SplF (296). This operon is located in the gene cluster νSaβ, which is present in over 60% of S. aureus genomes (296, 297).
Spls share 40 to 60% protein sequence identity, except for SplD and SplF, which have 95% sequence similarity with each other (Fig. 12) (296). The Spls are similar in size, ranging from 21 to 22 kDa. SplA to SplD have been characterized, and their structures were determined by X-ray crystallography (298–301). The crystal structures of the four Spls showed structural homology to the other staphylococcal serine proteases. Spls have a chymotrypsin-like fold, consisting of two β-barrel domains (Fig. 12) (298–301). The catalytic triad typical of serine proteases (His, Asp, Ser) is conserved and is present in the center between the two domains (296, 298–301).
FIGURE 12.
(A) Phylogenic tree of S. aureus Spls. The tree is constructed based on the mature protein sequences using the DNASTAR MegAlign ClustalW method for multiple sequence alignment. (B) Crystal structure of SplA (2W7S) (299). SplA has two domains connected by a linker (cyan). Domain 1 (light purple) consists of α-helices and β-strands, and domain 2 (blue) consists of of β-strands. Both domains fold into a β-barrel structure. The catalytic triad (red) is located at the center between the two domains.
Based on functional studies of the Spls, the precise cleavage of the signal peptide is critical for protease activity. An additional two amino acids (such as those resulting from a thrombin cleavage) in the N-terminus of SplA, SplB, and SplC are enough to render the enzymes inactive (296, 298, 299). Therefore, the signal peptides of Spls serve dual functions: (i) to direct the protein secretion and (ii) to serve as propeptides to prevent enzyme activation prior to secretion.
SplA, SplB, and SplD have extremely narrow substrate specificities, requiring the recognition of substrate consensus sequences that are 4 to 5 amino acids in length (Table 3) (298–300). Mucin-16, an O-glycosylated transmembrane protein present in the ocular epithelia, is a substrate for SplA (302). Additionally, searches based on the substrate consensus sequences identified many olfactory receptors as potential Spl substrates, but they remain to be verified experimentally (298–300). Nevertheless, these searches suggest that Spls may be important for nasal colonization. SplD contributes to airway inflammation and asthma by promoting IgE production and Th2 responses (303, 304). The role of SplD and SplF in asthma is supported by the identification of these proteins in nasal polyp samples from asthma patients who were also S. aureus nasal carriers (303).
TABLE 3.
Consensus cleavage sequence of Spls
| SplA | Trp/Tyr – Leu – Tyr – Tyr – Ser |
| SplB | Trp – Glu – Leu – Gln |
| SplC | To be determined |
| SplD | Arg – Trp/Tyr – Pro/Leu – The/Leu/Ile/Val |
| SplE | To be determined |
| SplF | To be determined |
However, the role of Spls in S. aureus pathogenesis remains unclear. While murine pneumonia and intraperitoneal infection models using Δspl mutants had no effect on host survival or bacterial burden, the absence of spl limited the dissemination of bacteria in vivo in a pneumonia model (296, 303). Proteomic analysis of wild-type S. aureus and Δspl mutant demonstrated significant changes in many virulence factors important for adhesion and immune evasion; thus, Spls have a role in shaping the S. aureus proteome (302).
Hyaluronidase
Hyaluronic acid (HA) is a linear polysaccharide composed of repeating units of N-acetylglucosamine and glucuronic acid linked by alternating β-1,3 and β-1,4 glycosidic bonds (305). HA is a critical component of extracellular matrices in vertebrates, providing homeostasis and structural integrity to cells and tissues; it is also important for immune regulation (306, 307). The enzymes that break down HA are collectively known as hyaluronate lyase or hyaluronidase.
In nature, hyaluronidases can be found in vertebrates, invertebrates, and bacteria. Hyaluronidases found in vertebrates and invertebrates break down HA to tetrasaccharides (308). In contrast, bacterial hyaluronidases act as endo-N-acetylhexoaminidases and cleave the β-1,4 linkage in a process known as β-elimination, breaking down HA to unsaturated disaccharides (308).
S. aureus and S. hyicus are the only staphylococci known to produce hyaluronidase (309, 310). The activity of staphylococcal hyaluronidase was initially reported by Duran-Reynals in 1933 as a “spreading factor” that increased lesion sizes in a rabbit skin infection model (311). Subsequently, this spreading factor was identified by Chain and Duthie in 1940 as hyaluronidase (312). In 1995, the gene encoding for staphylococcal hyaluronidase, hysA, was eventually cloned and the corresponding protein purified (313).
As a spreading factor, hyaluronidase is implicated in the dissemination of bacteria through breaking down HA in extracellular matrices and biofilms. The skin and the lungs are two locations where extracellular matrices are abundant. Deletion of hysA resulted in reduced skin and lung pathology and lowered bacterial burden in skin and lung infection models, respectively (314, 315). Deletion of hysA was also demonstrated to cause increased biofilm formation and reduced bacterial dissemination (316).
Lipases
Phospholipases
S. aureus can produce two phospholipases: β-toxin and phosphatidylinositol-specific phospholipase C (PI-PLC). β-toxin is a neutral sphingomyelinase with hemolytic and cytolytic activities, discussed in the exotoxin section, above. The other staphylococcal phospholipase, PI-PLC was discovered in the 1960s in S. aureus culture supernatants where PI-PLC hydrolyzed phosphatidylinositol (PI) to diglyceride and inositol phosphate (317, 318). Today, S. aureus remains the only staphylococcus known to produce PI-PLC (319). S. aureus membrane does not contain PI, and thus S. aureus is thought to have acquired PI-PLC to adapt to the host environment (320).
Like other bacterial PI-PLCs, the staphylococcal PI-PLC has an imperfect (βα)8-barrel structure (also known as the TIM barrel) (321). The active site of PI-PLC is conserved and is located at the C-terminal end of the β-strands that form the β-barrel (321). The elucidation of the staphylococcal PI-PLC crystal structure provided explanations for many of PI-PLC’s biochemical properties (321). PI-PLC is reported to have an optimum pH between 5.5 and 6.0 (319). This property can be explained by the unrestricted substrate access to the active site under acidic conditions. In contrast, accessibility of the substrate is restricted under basic conditions (321). PI-PLC is inactivated by NaCl, HgCl2, and Cu2SO4 (319). The salt sensitivity of PI-PLC can be explained by the high electropositivity of the barrel rim region and the active site (321). The presence of phosphocholine enhances the activity of PI-PLC (321). Structural analysis of PI-PLC suggests that the presence of phosphocholine enables transient dimerization of two PI-PLC monomers, resulting in the enhancement of enzyme activity (322).
Bacterial PI-PLC hydrolyses PI in two steps: first, PI is hydrolyzed to diacylglycerol and the intermediate product myo-inositol 1,2-cyclic phosphate (cIP). This is followed by a second slower hydrolysis of cIP to myo-inositol 1-phosphate (323, 324). Diacylglycerol is an important secondary messenger for activating intracellular pathways in mammalian cells for growth and survival (325). PI-PLC can also release glycosyl-phosphatidylinositol-anchored proteins on the cell membrane (319). Two such proteins are C8 binding protein and the decay-accelerating factor (326, 327). Both proteins are complement regulators that are normally present on host cells to restrict complement activation on self (328, 329). Recently, PI-PLC was demonstrated to promote survival of S. aureus in human blood and neutrophils (330).
Glycerol ester hydrolases (lipases)
S. aureus has two lipases: S. aureus lipases 1 and 2 (SAL1 and SAL2). SAL1- and SAL2-encoding genes are sometimes annotated as gehA and gehB, respectively, for glycerol ester hydrolase (331–333). SAL1 has also been annotated as lip1 in the literature (331). The two genes are encoded in disparate regions in the S. aureus genome, but they share protein sequence similarity with each other and with other lipases found in other staphylococcal species (332, 333).
The lipases are produced as pre-pro-enzymes (334). The pre-pro-enzyme is processed by signal peptidase I, which cleaves the signal peptide for secretion. The secreted proenzyme is cleaved by aureolysin to yield the mature lipase (331). However, cleavage of the propeptide is not required and has no effect on the enzymatic activity (331, 335). Utilizing chimeric lipases of S. hyicus expressed in S. carnosus, the lipase propeptides were found to be important for the translocation of the lipases to the extracellular milieu and for stabilizing the proteins to prevent degradation (336, 337).
Enzymatic activities of the lipases are conferred by the conserved catalytic triad, formed by serine, aspartate, and histidine (331, 332). Although sharing a similar catalytic mechanism, SAL1 and SAL2 differ biochemically and have different substrate preferences. SAL1 functions optimally at pH 6.0 and is stable under acidic conditions, but it is inactivated when the pH is above 10 (338). Biochemical and molecular analyses showed that Ca2+ stabilizes the structure of SAL1 and increases its activity (334, 338). Accordingly, chelators, such as EDTA and EGTA, inhibit SAL1 activity (338). SAL1 has a strong preference for short-chain triglycerides but cannot hydrolyze long-chain triglycerides (338).
In contrast, SAL2 functions optimally around pH 8.0 and is inactive under acidic conditions (339). The presence of Ca2+ does not enhance the activity of SAL2 (339). As such, chelators have minimal effects on activity. SAL2 prefers long-chain triglycerides as substrates (331). However, SAL2 has also been shown to hydrolyze short-chain triglycerides, monoglycerides, and diglycerides with lower efficiency and with no apparent positional specificity (331).
The conservation of lipases in staphylococcal species implies their evolutionary importance. However, the contribution of lipases during disease is unclear. S. aureus clinical isolates from deep tissue infections produce more lipases than isolates from superficial infections (340). Purified lipases cause aggregation of granulocytes and decrease phagocytosis at high concentrations (341, 342). During infection, SAL2 was shown to be important for biofilm formation and contributed to the virulence of S. aureus strain RN4220 in a murine intraperitoneal infection model (343). These observations suggest that lipases are involved in the overall virulence of S. aureus and promote bacterial survival in biofilms and abscesses. Paradoxically, lipase-mediated triglyceride hydrolysis liberates bactericidal free fatty acids, which can interfere with pathogenicity (344). For most lipase-producing strains, these bactericidal fatty acids can be detoxified by fatty acid-modifying enzymes (FAMEs).
Fatty acid-modifying enzyme
The FAME was first described in 1992 by Mortensen et al., who observed that S. aureus culture filtrates inhibited the bactericidal activities of host lipids in abscesses (345). Since the initial discovery, FAME activity has been well documented in many staphylococcal species (346, 347). Approximately 80% of S. aureus and S. epidermidis organisms produce this enzyme (347, 348). Despite its prevalence, the corresponding gene for FAME is not known and the protein has not yet been identified.
FAME promotes staphylococcal survival by esterifying the bactericidal free lipids with an alcohol substrate to form alcohol esters. Although FAME can esterify free lipids with methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol, it prefers cholesterol, which is highly abundant in abscesses (345). Saturated and unsaturated fatty acids with 15 to 19 carbons are efficiently esterified by FAME; however, esterification is also observed for fatty acid chains of between 11 and 24 carbons (349). The optimal pH of the enzyme ranges between 5.0 and 5.5 and has an optimal temperature of about 40°C (345). Enzyme activity is inhibited by di- and triglycerides with unsaturated fatty acid side chains (349).
In abscesses, lipases and FAME are thought to complement each other to enhance staphylococcal survival (350). While lipases break down triglycerides that inhibit FAME activities, FAME processes the free fatty acids liberated by lipases to protect the staphylococci. This hypothesis is corroborated by the observation that most S. aureus strains that carry genes encoding lipases have FAME activities and that they are correlated with the invasiveness of the bacteria in vivo (345, 348).
CONCLUSION
S. aureus devotes a significant amount of energy to the production of virulence factors to protect the bacteria from host immune surveillance and to promote bacterial survival in hostile environments (Fig. 13). The importance of these virulence factors during infection has been demonstrated extensively in various ex vivo and in vivo infection models. Pathogenic S. aureus is usually present in hostile host environments with limited resources; thus, it follows that the production of many virulence factors that serve the same purpose can be a waste of limited resources and be disadvantageous for survival. In contrast, this redundancy can ensure protection of the bacterium if one of the virulence factors is rendered ineffective. Alternatively, the bacterium may have acquired these seemingly redundant virulence factors during its evolution to better adapt to different types of infections or colonization sites.
FIGURE 13.
S. aureus secretes many different toxins and enzymes. Superantigens are proteins that have high mitogenic properties, causing T and B cell expansions that result in clonal deletion and massive cytokine production. Cytotoxins, such as α-toxin and the leukocidins, cause cytokine production, hemolysis, and leukocyte cell death through targeting specific cell surface receptors. The amphiphilic PSM peptides mediate cytolysis by inserting into the lipid bilayer of cell membranes. Enzymes, such as β-toxin and the ETs, cause cytotoxicity on mammalian cells, resulting in cell death, inflammation, and tissue barrier disruptions. Other enzymes, including various proteases and nucleases, mediate host protein degradations, thwarting many important host immune surveillance and defense molecules. These enzymes can also act on self-proteins to degrade biofilms for bacterial dissemination. Lipases and FAME work synergistically to degrade lipids in the environment for nutrients. Cofactors, including Coa, vWbp, and Sak, bind and activate host zymogens in the coagulation system to mediate clot formation and dissolution. Altogether, these toxins and enzymes provide critical nutrients (i.e., iron and carbon) that are important for the growth and survival of the bacteria. Importantly, they target various aspects of host immune defenses, thus contributing to the overall virulence of S. aureus during infections.
Many of the exotoxins and secreted enzymes discussed in this article share structural and functional similarities. However, closer examination of these proteins reveals subtle differences that have functional significance. Minor differences in the PFTs lead to the cytolysis of various cell types that are critical for immune defense. Each of the SAgs target different Vβ TCRs, resulting in broad suppression of the T cell repertoire. Proteases, such as the serine proteases, which have structural homology have disparate substrate specificities. Other exotoxins and enzymes have similar functions but differ in when and where they are produced during growth and pathogenesis, suggesting that the complex and seemingly redundant virulence factor repertoire is critical for the success of S. aureus as a versatile pathogen. With the rise in antibiotic resistance in microbes, including S. aureus, there is an urgent need to develop novel therapeutics and vaccines to combat this deadly pathogen. Understanding the roles these important virulence factors have during diseases can provide the knowledge necessary for designing better therapeutics and identifying vaccine targets.
ACKNOWLEDGMENTS
We thank members of the Torres lab and Richard Novick for comments on this article.
The S. aureus work in the Torres lab is supported by NIH-NIAID R01-AI105129, R01-AI099394, R01-AI121244, and HHSN272201400019C. K.T. is supported in part by a Public Health Service Institutional Research Training Award, NIH-NIAID T32-AI007180. V.J.T. is an inventor listed on patents and patent applications filed by New York University which are currently under commercial license to Janssen Biotech, Inc.
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