Immune System Evasion Mechanisms in Staphylococcus aureus: Current Understanding

Staphylococcus aureus is a major human pathogen that may cause a wide range of infections and is a frequent cause of soft tissue and bloodstream infections. It is a successful pathogen due to its collective virulence factors and its ability to evade the host immune systems. the review aims to highlight how S. aureus destroys and damage the host cells and explains how immune cells can respond to this pathogen. this review may also provide new insights that may be useful for developing new strategy for combating MRSA and its emerging clones such as community-associated methicillin-resistant S.

these infections complicated and more challenging to achieve 7 . This review aims to provide and updated literature survey on epidemiology, virulence and pathogenicity as well as interaction mechanisms with host immune system by S. aureus.

Staphylococcus aureus Epidemiology
S. aureus infections may not be all reported, given the wide variety of community and hospital-acquired of infection, however, the number of reported cases is increasing worldwide, for instance, in the USA around 80461 cases of MRSA infections in 2011, resulting in a 11000 deaths 6 . Reported MRSA infections in Australia increased from 10.3% in 2000 to 16% in six years 8 . In Taiwan, dramatic increase of MRSA infection rose from 9.8% in 2000 to 56% in 2005, similar increases of S. aureus infections were also reported in Saudi Arabia, Lebanon, England, India 6,8,9 . It is important to mention that these figures represent over all infections, the epidemiological data of every type of Tong et al. 5 has well documented S. aureus infections concluding that there is an increase of hospital-acquired infections particularly in the cases of infective endocarditis and infections related to prosthetic devices, moreover, an epidemic of community-associated skin and soft tissue infections controlled by certain virulence factors and resistance to antibiotics particularly β-lactam group 5 . Molecular epidemiology of S. aureus infections showed diversity of clones responsible for infections in every content, with the existence of predominant clones such as ST-80 which was found in North and South America and Europe. In Asia, high heterogenecity in terms of the clones associated with infection was highlighted. In general, it was suggested that low restrictions in the sales of antibiotics and/or prescribing antibiotics with proper laboratory analysis in some countries aided the increase of S. aureus infections as well the diversity of clones responsible for these infections 8 . Staphylococcus aureus virulence factors S. aureus infections occur when the mucosal or skin barriers are breached, following the successful invasion of the bacterium into the host tissues and its ability to escape the defensive barriers of the immune system to enter the bloodstream. S. aureus have an arsenal of virulence factors that play significant roles in the wide variety of infections and diseases in humans including animals. These factors can also support and provide protection for S. aureus to evade the host immune system recognition and their actions. One of the most important virulence factors is surface proteins which promote and encourage the binding and the attachment processes of this bacterium to the host cells surfaces. To avoid recognition from the host immune cells, the surface proteins combined with the blood proteins that lead to aid the bacterium to survive and cause damage to host tissues 10 . Another vital factor is protein A, which is located in the cell wall of S. aureus which accurately anchored to the peptidoglycan pentaglycine bridges of the bacteria. It is also known as an IgG-binding protein which combines with the Fragment crystallisable (Fc) region of the antibody in order to cover the surface of the bacterium with IgG antibody to make the recognition of this organism by the immune cells difficult and hard to detect 11,12 .
The clumping factor A (ClfA) is another S. aureus virulence factor that is expressed by the surface of the bacteria cells. Also, it is known as fibrinogen binding protein that promotes the clotting process of blood cells and the damage process of tissues 13 . In addition, the polysaccharide capsule is considered as one of the most essential virulence factor which can contribute to assist the S. aureus surviving within the host cells by inhibiting the phagocytosis process by macrophage and dendritic cells 10 , moreover, S. aureus cells are capable of secreting several important toxins and enzymes such as; coagulase; DNAase; leukocidin; hemolysins; exfoliative toxin in order to promote bacterial penetration and help the bacterium to evade in to the host tissues (Table 2) 10 .
Moreover, S. aurues can generate another harmful type of toxins such as Panton-Valentine leukocidin (PVL) which cause pneumonia in children and Toxic Shock Syndrome Toxin-1 (TSST-1) which is associated with some cases of  10,11,12,13 septicaemia due to the use of particular types of tampons (Table 2) 13,10 . A number of S. aureus strains have the ability to produce a pigment that known as staphyloxanthin which acts as an important virulence factor. This strain has an antioxidant role against reactive oxygen species used by the host immune cells, in order to help the bacterium to escape from killing action of immune cells 14 . Furthermore, S. aureus are capable of forming biofilms of various surfaces which associated generally with most indwelling medical devices problems such as heart valves and knee replacements and via this biofilm community resistance can be acquired to antibacterial agents through horizontal gene transfer 12 .

How does S. aureus subvert immune responses?
When S. aureus invade the host tissues, the innate immune system responds rapidly as an early defense against the bacterial invasion. This system consists of three important parts; (i) the complement system, (ii) phagocytes and (iii) antimicrobial peptides. It also play many significant roles against the pathogen for instance; it summons the immune cells to the infection sites by producing chemical signaling molecules called cytokines, activates the complement system to recognize the microbe to encourage the clearance process of dead cells, and it activates the second line of the host defense which is known as the adaptive (specific) immune system through antigen presentation process, in order to help the body to eliminate the microbe and to generate long lasting immunity against it to prevent any challenge in the future by the same organism 15,16 . the Complement System The complement system is a group of proteins and proteolytic molecules that are found and circulate in the blood (Table 3). With S. aureus, complement employs some significant molecules to: (1) mark the microbe with C3b and iC3b to make its parts ready for phagocytosis through important immune cells such as; neutrophil and macrophage, and also to (2) seize the attention of phagocytes by small chemoattractant molecules such as: C3a and C5a which are produced during the activation of complement to facilitate phagocytosis as well 15,17 . This system is initiated and activated by three significant pathways: (a) the classical, (b) the alternative and (c) and the lectin pathways ( Fig.  1). All the three pathways come together at the formation of a surface bound biomolecular enzyme known as C3 convertases. This enzyme stimulates and enhances the activation of complement system by cleaving the complement protein C3 to form C3a and C3b which are required to promote and facilitate any additional essential activation events such as; opsonization and phagocytosis [15][16][17] .

S. aureus Complement avoidance mechanisms
It has been shown that S. aureus developed different mechanisms to avoid the action of the complement system by producing a number of proteins that can change and affect the stages of the complement cascade, these mechanisms include; preventing complement identification; cleavage of complement proteins; and/or inhibit the interaction of complement receptors on phagocytes 18, 19 .
Although, the classical pathway C1 complex has the ability to recognize the microbe bound IgG and IgM antibodies and the lectin pathway (mannose-binding lectins and ficolins) bind the saccharide elements of microbe, S.  C4bC2a acts as a C3 convertase and cleave C3 which results in products that bind to, and cause the destruction of, invading bacteria. (B) The lectin pathway is initiated by the binding of either mannose binding lectin (MBL) or ficolin -associated with MBL-associated serine protease1 (MASP2), MASP2, MASP3 and small MBL-associated protein (sMAP) -to an array of carbohydrate groups on the surface of a bacteria cell. Similar to C1s, MASP2 is responsible for the activation of C4 and C2, which leads to the generation of the same C3 convertase (C4bC2a). As in the classical pathway, C3 convertase cleaves C3 to C3b and the chemo attractant peptide C3a. The C3b-C2a-C4b complex then cleaves C5 to C5a and the chemo attractant peptide C5b, which stimulates assembly of factors C6, C7, C8 and C9. MASP1 is able to cleave C3 directly.
(C) The alternative pathway is initiated by the low grade activation of the C3 by hydrolysed C3 (C3(H2O)) and activated factor B (Bb). The activated C3b binds factor B (B), which is then cleaved into Bb by factor D (D) to form the alternative pathway C3 convertase, C3bBb. Once C3b is attached to the cell surface, the amplification loop consisting of the alternative pathway components is activated, and the C3 convertase enzyme cleaves many molecules of C3 to C3b, which bind covalently around the site of complement activation [15,17,48,49] .
aureus produce two surface proteins that can damage and harm the IgG function (Fig 2.a), the first protein is the Staphylococcal protein A (SpA) and the second protein is known as the Staphylococcal immunoglobulin binding protein (Sbi). Staphyloccocal protein (SpA) is a protein located on the surface of the bacterium that consists of four or five immunoglobulin binding domains. Every domain is able to combine with the Fc parts of IgG antibody, thus inhibiting the interaction with Fc receptors on neutrophils in vitro [17][18][19] .
Staphylococcal Sbi is made of four parts of Sbi-I and Sbi-II which are also able to bind with IgG. Besides blocking Fc-receptor-mediated phagocytosis, Sbi has been shown to play some roles in blocking the binding of C1q and subsequent activation of the complement classical pathway 18 . Furthermore, S. aureus uses another approach to evade the recognition of the complement system by removing and degrading the opsonic molecules (the molecules that are able to bind with both antigen and receptors of phagocytic cells like C3b component of complement system) from it surface through proteolytic process. In addition, S. aureus produce a staphylokinase (SAK) which is an anti-opsonic protein, in order to stimulate and encourage surface bound plasminogen into plasmin, which make the bacteria able to invade and infect the host tissues 17 . In fact, the presence of the C3 convertases is significant for the activation of complement system and for the response of host immune cells. S. aureus uses three ways to affect and change this vital step in the complement cascade ( Fig. 2.b) 17,18 . the Cleavage of C3 convertase The S. aureus clumping factor A (ClfA) is a surface protein which can join the human C3b protease factor I (fI), thereby enhancing cleavage of surface-bound C3b into iC3b in vitro 20 .  [17,18,19] . Leukocytes move through the gradient toward the higher concentrations, a process called chemotaxis [25,26,17] .

Direct inactivation of C3 convertases
Convertases are the major complement target among S.aureus immune evasion strategies.
S. aureus produces five unlike molecules which work directly to prevent the action of these important enzyme mixtures. Staphylococcal complement inhibitor (SCIN) and its homologues Journal of Pure and Applied Microbiology SCIN-B and SCIN-C are extremely efficient C3 convertase inhibitors which inhibit the alteration of C3, later phagocytosis and C5a formation in vitro at low concentrations 18 .
Besides that, the alternative pathway C3 convertase made of a vital cofactor called C3b which connected to the protease subunit (Bb) loosely 18 . The action of SCIN on the classical pathway convertase remains to be resolved but seems to be caused by a stabilizing mechanism as well 21 . In addition, the alternative pathway convertase can be changed via the extracellular fibrinogen-binding protein (Efb) and the extracellular complementbinding protein (Ecb) by binding the convertase to the C3b particle directly 20 . The crystal structures of both molecules in complex with the C3d domain of C3 have revealed their exact binding sites 21 .

Modulating human convertase regulators
In order to protect from unnecessary action of the complement system, humans produce complement regulators which reduce and decrease the activity of the convertase 22 . On the other hand, a large number of microbes generate molecules which interfere with the function of theses regulators. In addition to its two IgG binding fragments, Sbi-III and IV that can also bind to the C3, the staphylococcal IgG-binding molecule Sbi plays several important roles in the alteration of the complement 23 . Moreover, Sbi has the ability to bind the human complement regulators such as; factor H (FH) and factor H-related proteins in order to make a constant tripartite compound with FH and C3. In general, these actions lead to inhibit the activity of the alternative pathway in vitro 24 .

Phagocytosis
Phagocytosis is a common mechanism that involved in the immune response to eliminate pathogens or foreign particle may be a toxin invading the body. It is activated through the binding to pathogen-associated molecular patterns (PAMPS), which leads to the activation of nuclear factor kappa B (NF-κB) that is important to control and regulate the response of the immune system 25 . Furthermore, there are types of the immune cells that have the ability to engulf microbial pathogens in order to remove them from the host, these cells include; neutrophils, macrophages and dendritic cells 26 . Moreover, complement system plays an important role in phagocytosis by facilitating the uptake of the pathogen by phagocyte cells. This process occurs through specific complement receptors (CRs) such as; CR1 and CR2 (Table 4), these receptors bind the microbe to make it ready for phagocytosis 25,26 .
In addition, at the early stage of the immune response to the site of infection, the pathogen and its fragments are engulfed by antigen-presenting cells and other immune cells such as macrophages and neutrophils and transferred to the lymph nodes. This leads to the activation of the B cells to discriminate and to produce antibodies in order to reduce the effects of the bacterial toxins and to encourage and promote the phagocytosis of the pathogen 26 . Furthermore, following the entry of S. aureus to the host tissue, the response of neutrophils and macrophages initiate which is critical and essential to promote and assist the body in   [19,30,48] .
removing and eliminating the pathogens during the phagocytosis 21 . (Fig. 3.a). These cells are called via an important chemoattractants such as C5a; leukotriene B4; and chemokines such as CXCL8 which also known as interleukin-8 (IL-8) in order to direct their actions to the site of infection 27 . This chemoattractants formed through activated host cells and some of them produced as an activated component of the complement system (C5a) at the time of identification of a preserved structure present on the bacterium 28 . All these chemoattractants trigger phagocytes by attaching to membrane bound receptors called G protein-coupled Journal of Pure and Applied Microbiology receptors (GPCRs). Both chemoattractants and GPCRs have an essential role which is directing the innate defence cells against the invading microbe ( Fig. 3.b). As a result S. aureus has a wide range of strategies to evade and avoid phagocytic activity 28,29 .

Inhibition of neutrophil chemotaxis
A large number of S. aureus strains (about 60%) secrete a chemotaxis inhibitory protein (CHIPS) which can combine with the C5a receptor (C5aR) and the formyl-peptide receptor (FPR) in order to block the binding of any related agonist (Fig. 4), the C5aR-and FPR-binding activities of CHIPS were separated by specific amino-acid substitutions and the specificity of blocking monoclonal antibodies 19,30 . The intercellular adhesion molecule-1 (ICAM-1) on the endothelial cell surface is one of the several ligands recognized by the extracellular adherence protein Eap (or else called the main histocompatibility class II analogue protein Map) 30 .

Resistance to phagocytosis
S. aureus produces many significant factors to avoid and evade from phagocytosis. It expresses anti-opsonic proteins bound to the surface and a capsule of polysaccharides which: (a) interfere with antibodies and with the formation of complement through classical and alternative pathways, (b) inhibit their interaction to neutrophil complement receptor and Fc receptor. Therefore, efficient phagocytosis by neutrophils that requires recognition of bound complement proteins and antibody is compromised 25 . Moreover, S. aureus has some mechanisms which help and assist the organism to evade the killing of phagocytic cells, including interference with endosome fusion and release of antimicrobial substances by factors that are dependent on the global regulator SarA 31 .

Protein A
Is a surface protein that consists of four or five domains which can bind to the Fc region of IgG antibody. Protein A interact with IgG in order to cover the cell surface with IgG molecules which (d) fibrinogen-binding protein (Efb), which binds complement factor C3 and blocks its deposition on the bacterial cell surface. Complement activation beyond C3b attachment is prevented, thereby inhibiting opsonization. (e) ClfA, which binds the γ chain of fibrinogen [19,21,30,33,34] . are in the incorrect orientation easy to recognize by the Fc receptor of neutrophil (Fig. 5).This action can clarify the anti phagocytic role of protein A and its effects on S. aureus infections 32,33 .

Clumping factor A (ClfA)
Is the dominant fibrinogen-binding protein present on the surface of S. aureus cells in the stationary phase of growth. In the murine model the ClfA is a virulence factor for sepsis and arthritis 34 . Virulence was thought to increase during the bacteremic stage of the infection as well as during the growth of infected joints, since bacterial cells were coated with fibrinogen (Fig. 5), which in turn inhibited deposition of, or accessibility to opsonins 30,34 . This notion is supported by the observation that ClfA protects S. aureus from phagocytosis by murine macrophages46 and by human neutrophils and that defense is at least partly dependent on fibrinogen 21,34 . Furthermore, ClfB and fibronectin-binding proteins can also bind fibrinogen, to shield the bacterium through the exponential phase of growth, when the expression of such proteins is larger than ClfA 34 .

Capsule
Is an important factor which expresses by S. aureus to inhibit the process of phagocytosis. The expression of type 5 and type 8 capsules is associated with increase virulence in animal infection models 35 . It has been considered that the presence of the capsule lead to reduce the ability of neutrophils to uptake the pathogen cells in the presence of normal serum opsonins (Fig. 5), meaning that capsule act as anti-opsonic 19 .  [36,38,39,41] .

toxins that kill leukocytes
S. aureus is a common pathogen that has the ability to secrete and produce toxins which contribute to the damage of host cells membranes. The expression of cytolytic toxins that damage leukocytes contributes to development of abscesses by the killing of neutrophils that are attempting to engulf and kill the bacteria 36 . Cytolytic toxins forming β-barrel pores in target cell cytoplasmic membranes cause leakage, and eventually lysis. The representative of this class is the α-toxin, which is secreted as a monomer paired with a heptamer in the membrane, with β-strands assembled in a 14-stranded β-barrel pore from each monomer 36,37 .
The two-component leukotoxins consist of two subunits, which are separately secreted and configured into hexameric or heptameric oligomers with a great leukocyte affinity. There are 4 different types of bicomponent leukotoxin, y-toxin (Hlg), Panton-Valentine leukocidin (PVL), leukocidin E / D, and leukocidin close to M / F-PV. access not only to the intact vessel wall, but also to subendothelial matrix and activated platelets and will form a bacteria-entrapped platelet clot (A). SERAM bind to various extracellular matrix components including fibrin (ogen) (Fg), fibronectin (Fn) or thrombospondin (Tsp) that are present in this vegetation and thereby augment bacterial attachment through molecular bridging mechanisms (Α). In addition, SERAM may facilitate bacterial binding to Fn expressed on endothelial cells resulting in enhanced uptake of microorganisms (Β). Through interaction with complement factor C3, the extracellular fibrinogen binding molecule (Efb) may interfere with opsono-phagocytosis (C). Among other interactions with host factors, the extracellular adhesion protein (Eap) binds to ICAM-1 and other ligands on endothelial cells thereby inhibiting leukocyte adhesion and preventing their extravasation (D). After passaging through the endothelial cell layer and/ or the extracellular matrix stroma, S. aureus may gain access to other capillaries, arterioles and venules, attach to the vessel wall and start disseminating from focal sites of infection (E) Eap may inhibit vascular cell proliferation and angiogenesis possibly via direct interference with agonist-stimulated endothelial functions (F), while Efb interacting both, with platelets and with fibrinogen, may interfere with fibrin formation resulting in altered wound healing (G). Finally, Eap interferes with T-cells function shifting towards a Th2-cell response and reducing delayed-type hypersensitivity reactions [42,43] .
The γ-toxin lyses both erythrocytes and leukocytes, whereas PVL is toxic only for leukocytes 36,38 . Other evasion mechanism of S. aureus Immunomodulatory molecules Protein A In addition to its immunoglobulin binding ability, Protein A is an important immunomodulatory molecule owing to its capacity to combine with the VH3 region that is next to the antigen-binding domain of IgM molecules located on the B lymphocyte surface (Fig. 6). Those cells carrying VH3 IgM are induced to proliferate and undergo apoptosis, resulting in depletion of a large proportion of potential antibody-secreting B cells in the spleen and bone marrow 39,40 .

Enterotoxins and TSST-1
S. aureus produces toxins which work as superantigens such as toxic shock syndrome toxin-1(TSST-1), the expression of these antigens in the host cell prevents the development of the immune response such as activation of B and T cells or formation of antibodies (Fig. 6) 36,38 . Furthermore, Antigen-specific T cells are unable to reproduce in response to antigens that are normally presented by MHC class II due to a phenomenon called anergy 41 . Therefore, immune-suppression occurred due to the failure of the antibody response. This seems to be important to prevent the production of antibodies to superantigenic toxins themselves 36,38,41 . Secretable expanded repertoire adhesive molecules (SERAM) S. aureus generates some bacterial proteins that anchored in the cell wall which has a significant role in mediating bacterial adherence to host cells and to the components of the extracellular matrix (ECM). The general roles of SERAM (Fig. 7) can be described as: (1) to facilitate bacterial adhesion to host molecules, cells, or tissues, (2) to interact with a broad array of host ligands, thereby sharing diverse activities in that they typically interfere with host defense mechanisms 42,43 .

Other proteins involved in immune-evasions mechanisms
Furthermore, some studies has shown new functions of some bacterial proteins such as extracellular matrix binding protein (Emp) and extracellular adherence protein (Eap) which play significant roles in S. aureus pathogenesis due to its ability to bind the host components or block some steps in wound healing process. Eap also known as a MHC class II analogous protein. It has a very wide spectrum of binding interactions to components of the host. However, some strains of S. aureus loss the ability to express Eap therefore these strains cannot colonize or invade host tissues 44,45 . This protein can also combine with endothelial ICAM-1 in order to inhibit the intraction between the ICAM-1 and its integrins Mac-1 and LFA-1. Thus, Eap completely destroyed the adhesion systems of respective functional leukocyte, i.e. strong adhesion and endothelial transmigration 42 . In addition to Eap anti-adhesive and anti-migratory function, it has been considered that Eap has an anti-inflammatory role by inhibiting neutrophils and T cells to get to the site of infections to prevent their effects against the bacterium. It also has been described that Eap has an immunomodulatory activity by increasing the synthesis of interleukin (IL)-4 syntheses which is vital for the differentiation of T-cells into Th2-cells in the response of the immune cells in order to down regulate and reduce the response of T-cells to facilitate the intracellular survival of S. aureus 46-49 .

CONClUSION
It seems clear that S. aureus is a widespread organism that lives as a normal flora in the nose and on the skin of humans and animals. It is a very successful pathogen because of its ability to express and produce virulence factors to evade the recognition and the killing of the host immune cells. The bacterium infection starts through its entry to the host tissue which leads to stimulate and induce the innate immune response. The activation of this system occurred via specific pathways which lead to activate other important cells such as neutrophils and macrophages in order to eliminate and remove the pathogen from the host. The function of these cells initiated by the complement system which plays a significant role by producing chemoattarctant (chemotactic fators, chemokines, and complement factors etc.) in order to label and opsonise the pathogen for phagocytosis.
However, it has been shown that S. aureus improved many ways to avoid the effects of phagocyte cells by producing some proteins such as SpA and Sbi to inhibit and block the recognition of complement and their receptors interaction on phagocytes. Also, S.aureus can inhibit neutrophil chemotaxis by producing CHIPS that can interfere with some important receptors such as C5a receptor protein to prevent the binding of any related agonist. This pathogen can also produce super antigens toxins to make the response of the immune system difficult to achieve and hard to occur. Furthermore, it has been described that S. aureus can kill the leukocytes directly by secreting important cytolytic toxins such as PVL and leukocidin E/D. Moreover, SERAM, Eap and Emp are ubiquitous proteins that can help and assist the bacteria to survive and cause infections.