Staphylococcus aureus is a human pathogen known for its ability to cause both community- and nosocomial-acquired diseases ranging from mild skin infections, such as impetigo to severe diseases, such as endocarditis, pneumonia, sepsis and toxic shock syndrome (David and Daum, 2010). Treatment of S. aureus infections with antibiotics is often ineffective due to the development of antibiotic-resistance strains, such as methicillin-resistant S. aureus (MRSA). Therefore, alternative treatment options and vaccination are now being explored (Bagnoli et al., 2012; Pozzi et al., 2015). The success of S. aureus as a pathogen depends on the production of several virulence factors. S. aureus can express up to 24 cell wall-anchored proteins, which promote adhesion to extracellular matrices, invasion of non-phagocytic cells, biofilm formation (Foster et al., 2014) and interference with neutralization of the innate immune system (Sjodahl, 1977; Cary et al., 1999; Kang et al., 2013).

S. aureus also produces a wide variety of peptides that inhibit specific steps of the innate immune system, which represents the first line of defense of the host (Rooijakkers et al., 2005a; Itoh et al., 2010; Thammavongsa et al., 2015) (For more details see below).

Potentiation of S. aureus pathogenesis is determined by secretion of proteases that cleave specific components of the host immune system or disrupt the integrity of extracellular matrix and intercellular connections, compromising the stability of the host tissues and contributing to the dissemination of the infection (Koziel and Potempa, 2013). S. aureus also secretes proteins that can bind and modulate host protease precursors which, in turn, can target specific defense components, providing the bacterium with additional tools to establish colonization of the tissues (McAdow et al., 2012). Lastly, some S. aureus secreted molecules can bind and inhibit neutrophil serine proteases which are important for several functions including the regulation of extracellular trap formation (Hu, 2012; Kolaczkowska et al., 2015). Altogether, these findings highlight the relevance of these compounds as important virulence agents of S. aureus infections.

In this review, we focus on recent advances in the characterization of S. aureus proteases and modulators of host proteases, and their ability to avoid innate immunity. We also discuss how understanding the mechanisms of these immune evasive factors can have an impact in the development of therapeutics against S. aureus diseases.

The innate immune system is the collection of tissues, cells and molecules that protect the body from a variety of pathogenic microbes and toxins present in our environment. The innate immune system has numerous functions, including:

Intact epithelial surfaces form physical barriers between microbes in the external environment and host tissue. The main interfaces between the environment and the host are the skin and the mucosal surfaces of the gastrointestinal and respiratory tracts. Tight junctions between neighboring cells prevent easy entry by potential pathogens, such as S. aureus. The interior epithelial surfaces are also covered with a mucus layer that protects these surfaces against microbial, mechanical, and chemical insults. The slimy mucus coating, made primarily of secreted mucin and other glycoproteins, physically helps prevent pathogens from adhering to the epithelium. Epithelia also produce peptides that kill or inhibit the growth of pathogens. Those that are most abundant include antimicrobial peptides called defensins. They are generally short and positively charged, and have hydrophobic or amphipathic domains in their folded structure. Defensins are also the most abundant protein type in neutrophils, which use them to kill phagocytosed pathogens (Zhao and Lu, 2014). Besides the defensins, cathelicidins represent another family of antibacterial peptides in mammals (Boman, 2003; Brogden et al., 2003). The human cathelicidin hCAP-18, constitutively expressed by neutrophils and squamous epithelia in response to inflammatory challenge is processed by proteinase 3 to generate the active peptide LL-37 (Sørensen et al., 1997) that possesses considerable anti-staphylococcal activity (Tkalcevic et al., 2000; Travis et al., 2000). S. aureus uses several mechanisms to counteract the epithelia defense actions. Adhesion to epithelia is a multifactorial process that involves the host as well as bacterial factors. One key factor is the glycopolymer cell wall teichoic acid of S. aureus, which directly interacts with nasal epithelial surface through a type F scavenger receptor named SREC-I (Baur et al., 2014). Another important surface factor with a role in nasal and possibly skin epithelia colonization is the cell wall-anchored protein clumping factor B, which binds to fibrinogen, cytokeratin, the dominant component of the interior of squamous cells and loricrin, the most abundant protein of the cornified envelop of squames (Lacey et al., 2016). Iron-regulated surface determinant A protein also promotes the adhesion of S. aureus to squames cooperating in binding to cornified cell envelop loricrin, involucrin, and cytokeratin. Other cell wall-anchored proteins such serine-aspartate dipeptide repeat proteins SdrC, SdrD, and SasG promote adhesion to squames but their ligands are unknown (Foster et al., 2014; Figure 1).

Although S. aureus is not considered an intracellular pathogen, it can govern its uptake into non-phagocytic cells. Bacterial internalization is promoted by fibronectin-binding proteins A and B. Binding of fibronectin to fibronectin-binding proteins and its subsequent recognition by integrin a₅b₁ leads to internalization of the bacterium into epithelial and endothelial cells (Foster et al., 2014). Recently, it has been shown that clumping factor A binds annexin A2, a calcium-regulated membrane-binding protein, and it has been proposed that this interaction could also mediate S. aureus invasion into bovine mammary epithelial cells (Bonora et al., 2015; Figure 1). In lung epithelial cells S. aureus internalization also involves the efflux pump Tet38 via interaction with CD36 (Truong-Bolduc et al., 2015, 2017).

To disturb the defensive barrier function of the airway epithelium, S. aureus α-hemolysin disrupts cell-matrix adhesion by activating Fak signaling with the consequent acceleration of focal contact turnover (Hermann et al., 2015). Additionally, treatment of airway epithelial cells with recombinant α-hemolysin results in plasma membrane depolarization, and increased phosphorylation of paxillin and p38 MAP kinase, a signal transduction module involved in host defensive actions (Eiffler et al., 2016). Lastly, staphylococcal EsxA protein interferes with epithelial cell apoptotic pathways and, together with EsxB, mediates the release of intracellular staphylococci from the host cells (Truong-Bolduc et al., 2015).

During colonization and in the infection stage, S. aureus is faced with the host's innate immune defense, and one of the first barriers it encounters is the complement system. Several complement effector molecules can indeed sense and opsonize S. aureus cells and promote their phagocytic killing by neutrophils in blood and macrophages in tissues. The complement system is a proteolytic cascade of plasma proteins, which is crucial to the host's defense against invading bacteria (Figure 2). Complement fixation by bacteria can occur through three activation routes, the classic pathway (CP), the lectin pathway (LP), and the alternative pathway (AP). Activation of the CP starts after C1q molecules are deposited on the bacterial surface via direct binding, immunoglobulin recruitment, or pentraxins bridging and interacting with C1r and C1s proteases to form the C1 proteolytic complex. Through the LP pathway, collectins, such as mannose-binding lectin (MLB), mannan-binding lectin or ficolin, bind to microbial surface polysaccharides, resulting in activation of mannan-binding lectin-associated serine protease (MASP). Both CP and LP proteolytic complexes can split surface-bound C4 into C4a plus C4b, and C2 into C2b plus C2a protease. C4b and C2a directly combine and form the C3 convertase C4bC2a, which cleaves native C3 into C3b and C3a. C3b molecules effectively opsonize the bacterium and facilitate activation of C3bBb convertase, the AP convertase that promotes transformation of new C3 molecules into C3b and C3a, thus amplifying the number of C3b molecules that opsonize bacteria and promote phagocytic killing. Surface-tethered C3b also plays a central role in the formation of the two C5 convertases (C4bC2aC3b and C3bBbC3b), which cleave C5 into C5a and C5b. C5b initiates the assembly of the membrane attack complex (MAC), a pore made up of components C5b, C6, C7, C8, and multiple units of C9 and ultimately leading to cell lysis. Important host regulators controlling complement homeostasis are C3b-cleaving factor I, factor H, a cofactor of factor I and a displacer of Bb from the AP C3bBb convertase, and C4b-binding protein, which interferes with the assembly of the CP/LP C4bC2a convertase.

A central role in the innate immune response and protection against staphylococcal infection is played by the central molecule of complement C3 but not factor B. In fact, mice with C3 deficiency show susceptibility to S. aureus septic arthritis and display impaired host clearance, presumably due to reduced opsonization and phagocytosis of bacteria (Na et al., 2016).

In turn, S. aureus secretes several peptides that interfere with the deposition of the complement on the bacterial surface. Indeed, the Staphylococcal binder of immunoglobulin Sbi helps to protect S. aureus from innate immune defense of the host and this effect is based on the ability to bind to the Fc region of IgG and the complement factor C3 in serum, promoting its futile consumption (Zhang et al., 1999; Burman et al., 2008). Furthermore, a secreted chemotaxis inhibitory protein of S. aureus (CHIPS) blocks function of the C5a and formylated peptide receptors needed for neutrophil chemotaxis (de Haas et al., 2004; Postma et al., 2004).

If microorganisms cross an epithelial barrier and start to replicate in the tissues of the host, they are promptly recognized, ingested and killed by the mononuclear phagocytes or macrophages that reside in the tissues. Another important family of phagocytes, neutrophils, are short-lived cells that are abundantly present in the blood but not in the tissues. Both macrophages and neutrophils play a key role in innate immunity because they can efficiently destroy many pathogens without the aid of adaptive immunity. In particular, neutrophils are a central player in the interaction between host and S. aureus (Newsom, 2008; Spaan et al., 2013). During infection, neutrophils leave the blood and migrate to the focus of infection in a multistep process mediated through adhesive interactions that are regulated by cytokines and chemokines (Spaan et al., 2013).

Cytokines are small proteins (~25 kDa) that are released by various cells in the body, in response to an activating stimulus and that induce responses through binding to specific receptors. They can act in an autocrine or in paracrine manner. Chemokines are a class of cytokines that have chemoattractant properties, inducing cells with the appropriate receptors to migrate toward the source of the chemokine. Chemokines mainly recruit leukocytes, in particular monocytes and neutrophils, and other effector cells from the blood to sites of infection. All the chemokines are related in amino acid sequence and their receptors are all integral membrane proteins containing seven membrane-spanning helices (Allen et al., 2007). Members of the chemokine family include CXC motif, in which two cysteine residues are separated by another amino acid. CXC chemokines bind to at least seven different CXC receptors (CXCR1-7) expressed on different cell types (Murdoch and Finn, 2000). The CXC chemokine receptor 2 (CXCR2), highly expressed on neutrophils, recognizes chemokines produced at the site of infection and plays an important role in antimicrobial host defenses, such as neutrophil activation and chemotaxis (Sekido et al., 1993; Chuntharapai et al., 1994; Eisele et al., 2011).

Staphylococcus aureus secretes a number of proteases, including two cysteine proteases (staphopain A, ScpA, and staphopain B, SspB), a serine protease (V8 or SspA), serine protease–like proteins (Spls) and a metalloproteinase (aureolysin, Aur). The protease genes are positively regulated by agr (accessory gene regulator) and negatively regulated by sarA (staphylococcal accessory regulator) (Shaw et al., 2004) and are organized into four distinct operons, encoding seven serine proteases (SspA and SplA-F), two cysteine proteases (ScpA and SspB), and Aur. The Aur, V8, SspB, and ScpA proteases are produced as zimogen, while the six Spl enzymes are active upon secretion. The Aur and ScpA precursors self-activate outside the cell, and SspA and SspB activation relies on a proteolytic cascade in which Aur processes V8 (Drapeau, 1978) and V8 cleaves and activates SspB (Massimi et al., 2002). Proteases of S. aureus were initially thought to play a role only in nutrient acquisition, however, evidence is emerging that they are crucially involved in the evasion of host immunity by interacting with neutrophils, Smagur et al. (2009) plasma proteins (Prokesová et al., 1992) and antimicrobial peptides (Sieprawska-Lupa et al., 2004; Table 1).

Staphylococcus aureus is specialized in handling host proteins that are involved in the complement system, coagulation cascade and fibrinolysis cascade (Chavakis et al., 2005; Imamura et al., 2005; Nizet, 2007). Proteins that play a role in these pathways circulate in biological fluids or are in the extracellular matrix as inactive zymogen that can be activated upon interaction with specific secreted staphylococcal proteins. Alternatively, S. aureus secretes specific proteinaceous inhibitors of host serine proteases that play key roles in immune defense. In both cases, these proteins positively affect bacterium pathogenicity in vivo (Stapels et al., 2014; Table 2).

The fact that proteases and regulators of host proteases are secreted abundantly by almost all the strains of S. aureus, and the observation that mutants lacking proteases show a decrease in abscess formation and impairment during organ invasion, indicate that they play a crucial role as virulence factors (Kolar et al., 2013). Important progress has been made over recent decades in the identification and understanding of the functional role of S. aureus proteases and secreted factors that regulate host proteolytic activities. Specific advancements include the assessment of the multiple roles of these factors in the modulation of the complement system and prevention of phagocytosis. It has been proven that S. aureus proteases cleave tissue adhesion molecules allowing transition from adhesive to invasive phenotype and the consequent dissemination and spreading of bacterial infection. Furthermore, incubation of human serum with a combination of Aur, protease V8 and cysteine proteases staphopain A and B causes complete inhibition of all complement pathways. This results in a drastic decrease in the haemolytic activity of the serum and suggests that the concerted action of the four proteases is important for pathogen-mediated evasion of the human complement system (Jusko et al., 2014).

Despite the acquisition of these important insights, many structural and functional aspects of these factors remain unknown. For example, the expression and structural analysis by X-ray crystallography of many of these factors, alone or in combination with specific targets, are still lacking. Similarly, we have just begun to explore the role of these proteins in high quality animal studies and how each factor relates to the complex sequence of events in the initiation and progression of infection. Finally, new advances in the antigenic properties of these factors represent an important basis for the development of promising approaches for managing staphylococcal disease. The acquisition of this wealth of information in the short term would provide opportunities to develop specific synthetic drugs targeting one or more proteases or inhibiting the activity of staphylococcal modulators of host proteases. On this matter, it has been suggested that Eap “might serve as a template for developing a new class of synthetic inhibitors of neutrophil serine proteases” to treat inflammatory disorders like cystic fibrosis and emphysema, where neutrophil serine proteases play a significant role (Stapels et al., 2014). Although in a different contest, this example illustrates how the understanding of staphylococcal virulence factors might contribute to the development of therapeutics in medicine.

Moreover, the consistency between the positive regulation of S. aureus proteases by the accessory gene regulator agr (Shaw et al., 2004; Novick and Geisinger, 2008) and the observation that selective chemical inhibition of agr quorum sensing promotes and strengthens the host defense immunological system (Sully et al., 2014; Tsuchikama et al., 2017) paves the way for the development of new therapeutic strategies against S. aureus diseases.

Prospectively, antibodies against such S. aureus factors targeting elements of the immune system could restore host defense mechanisms and have clinical utility as adjunctive agents enhancing antibiotic efficacy in severe invasive diseases. Approaches to S. aureus vaccine development have so far been unsuccessful (Pozzi et al., 2015). This failure can be partly explained by the broad spectrum of the bacterium's immunevasive attributes that neutralize phagocytic killing of bacteria. Therefore, development of an effective S. aureus vaccine should consider the important role of virulence factors produced by this pathogen.

PS and GP wrote the manuscript, GP, GN, and SR prepared the references, and PS finalized the manuscript.

This work was supported by Fondazione CARIPLO (Grant Vaccines 2009-3546) to PS.