The colonization of the mammalian cell's extracellular matrix proteins (fibrinogen, fibronectin, elastin, collagen, laminin, and vitronectin) occurs covalently or non-covalently, mediated by several molecules of S. aureus that are collectively termed as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs; Table 1). Teichoic acids (TAs), which are common components of Gram-positive bacterial cell wall, also contribute significantly to the adherence to host cells (Qin et al., 2007). Approximately 15% of individuals (persistent carriers) have been reported to have continuous S. aureus colonization, whereas 70% of individuals have intermittent colonization (frequent S. aureus infection but immediate eradication), and the remaining 15% of individuals are non-carriers (Eriksen et al., 1995).

Apart from the host- and pathogen-associated factors, various other factors also play roles in S. aureus colonization. The nasal cavity is one of the most important sites of colonization for S. aureus as nose-picking can lead to the spread of bacteria to other body parts as well as to other hosts (Von Eiff et al., 2001; Wertheim et al., 2004, 2006). The nasal microbiota (Corynebacterium spp., Propionibacterium acnes, Staphylococcus epidermis, and Staphylococcus lugdunensis) and the invading pathogen S. aureus compete among themselves in various ways for colonization. Nutrition is one of the major limiting factors for colonization in the human nose and therefore S. aureus has been found to better adapt than coagulase-negative staphylococci due to the ability of the former to thrive in the low-nutrient environment (Lemon et al., 2010; Krismer et al., 2014; Liu et al., 2015; Lee et al., 2018).

The growth of S. aureus including MRSA has been found to be inhibited by the indigenous nasal bacterium Staphylococcus lugdunensis both in vitro and in vivo due to the production of an antimicrobial compound called lugdunin that can rapidly breakdown bacterial energy resources (Zipperer et al., 2016). The risk of colonization of S. aureus has been reported to be 6-fold lower in humans with Staphylococcus lugdunensis in their nasal cavities. However, nasal colonization with Staphylococcus lugdunensis has been reported in only 9–26% of the general population (Kaspar et al., 2016; Zipperer et al., 2016; Lee et al., 2018). S. aureus has been reported to compete with other commensal bacteria by the activation of host defense via upregulation of the production of antimicrobial proteins that are less harmful to it than to others (Krismer et al., 2017; Lee et al., 2018). Apart from the nasal cavity, other sites in the human body where S. aureus colonizes are armpits (8%), abdomen (15%), intestine (17–31%), perineum (22%), vagina (5%), and pharynx (4–64%; González-García et al., 2023). The virulence genes responsible for toxin production in S. aureus are downregulated during the colonization process.

The multiplication of bacteria after colonization on both biotic and abiotic surfaces leads to the formation of a three-dimensional complex community of bacteria within a layer of exopolysaccharide (EPS) termed as “biofilm” (Figure 1; Costerton et al., 1999; Sedarat and Taylor-Robinson, 2022). The accumulation of biofilm is facilitated by the formation of microcolonies due to the production of exopolysaccharide (a major component of biofilm), which comprises 97% water and 3% of structural and functional molecules (Guzmán-Soto et al., 2021). EPS of biofilm contains both positively and negatively charged groups as well as hydrophobic groups. The negatively charged groups of EPS include phosphates, sulfates, carboxyl groups, glutamic acid, and aspartic acid, whereas the positively charged groups include amino sugars such as polysaccharide intercellular adhesin (PIA). Despite positively charged PIA being a major component, the overall charge on the EPS surface is negative, serving as a better target site for positively charged drug candidates (Idrees et al., 2021).

The major EPS produced by S. aureus is polysaccharide intercellular adhesin (PIA), also known as poly-N-acetyl-glucosamine (PNAG; Mack et al., 1996). PIA/PNAG has a net positive charge and it promotes intercellular interactions by binding to the negatively charged surfaces of bacterial cells such as teichoic acids (O'Gara, 2007; Vergara-Irigaray et al., 2008). The multivalent electrostatic interaction of the cationic PIA polymer with the negatively charged wall teichoic acids on staphylococcal cells as revealed by single-cell force spectroscopy confirmed that the cationic nature of PIA is crucial for its attachment to the cell surface and intercellular adhesion (Formosa-Dague et al., 2016). PIA mutants exhibited a decreased ability to adhere to each other (Peng et al., 2022). There is no evidence for a covalent linkage of PIA to the cell surface (Cue et al., 2012). PIA is evident for biofilm formation under high-shear flow conditions such as those found inside catheters as compared to low-shear conditions such as those in subcutaneously implanted tissue, ocular infections, or platelet concentrate (Nguyen et al., 2020).

In addition to PIA/PNAG, biofilms contain bacterial proteins, eDNA, ions, and carbohydrates (Guzmán-Soto et al., 2021) as essential components with their ratios being variable. A number of staphylococcal strains exhibit PIA/PNAG-independent biofilm formation where the secreted proteins and extracellular DNA substitute for PIA/PNAG (Cue et al., 2012).

Extracellular DNA (eDNA) is another important structural component of biofilm matrix serving as a “glue” for the community due to its adhesive property. The destabilization of a matured biofilm by using DNAase I in Pseudomonas aerugionosa reported by Whitchurch et al. (2002) suggested the role of eDNA in biofilm formation in pathogenic bacteria. In a matured biofilm when there is a scarcity of nutrients, a subsequent population of damaged cells is eliminated in order to release nutrients for healthy cells. This self-destructive or suicidal act of cells is termed as autolysis or programmed cell death (PCD; Lewis, 2000). Along with the nutrients, the lysed cells also release genomic DNA in the form of eDNA. Rice et al. (2007) reported that only a small fraction of the bacterial population (< 1%) within the biofilm undergoes cell lysis to release a sufficient amount of eDNA required for biofilm stability (Bayles, 2007). eDNA can also confer antibiotic resistance mainly due to the horizontal gene transfer of eDNA to the healthy cells of biofilm (Molin and Tolker-Nielsen, 2003; Tetz et al., 2009).

Capsular polysaccharides (CPs) are long-chain polysaccharides attached covalently to the peptidoglycan layer of the cell wall. They aid in the colonization of the host as well as in biofilm formation, hence are indirectly involved in the progression of invasive diseases. However, the contrasting effect on the virulence of S. aureus is observed by the presence or absence of a capsule depending upon the type of infection (O'Riordan and Lee, 2004; Tuchscherr et al., 2010). CP enhances virulence in murine models of bacteremia (Thakker et al., 1998; Watts et al., 2005), septic arthritis (Nilsson et al., 1997), abscess formation (Portols et al., 2001), and surgical wound infection (McLoughlin et al., 2006). On the contrary, in mammary gland infections (Tuchscherr et al., 2005) and in catheter-induced endocarditis (Baddour et al., 1992; Nemeth and Lee, 1995), CP mutants are more virulent. This is likely because CP also inhibits the adherence of the underlying adhesins to their specific target molecule (Phlmann-Dietze et al., 2000; Risley et al., 2007). CP-negative S. aureus strains are frequently isolated from patients with osteomyelitis, mastitis, or cystic fibrosis, providing evidence that the loss of CP expression (due to mutations in any of the genes essential for CP biosynthesis or in the promoter region; Cocchiaro et al., 2006; Tuchscherr et al., 2010) may be advantageous for S. aureus during chronic infection (Herbert et al., 1997; Lattar et al., 2009; Tuchscherr et al., 2010). CPs also help bacteria in evading the phagocytic uptake by the host immune system and also protect the important bacterial cell wall constituents (Berni et al., 2020). Out of 13 serotypes of S. aureus (Berni et al., 2020), serotypes 1 and 2 (rarely reported among clinical isolates) produce mucoid colonies while the remaining serotypes produce non-mucoid colonies on a solid medium. Serotypes 5 and 8 (prevalent among clinical isolates as well as commensal sources) constitute ~25 and 50%, respectively, of the isolates recovered from humans from various geographic locations of the world (O'Riordan and Lee, 2004).

Teichoic acids are one of the major components of Gram-positive bacterial cell wall. These diverse anionic carbohydrate-based polymers are categorized into two classes: (1) lipoteichoic acids (LTAs), which are embedded in the lipid bilayer with the help of a diacylglycerol lipid anchor that can extend from the cell surface to the peptidoglycan layer, and (2) wall teichoic acids (WTAs), which are covalently attached to peptidoglycan matrix via a phosphodiester linkage to the C6 hydroxyl of the N-acetyl muramic acid sugars and can extend through and beyond the cell wall (Swoboda et al., 2010; Berni et al., 2020). WTAs comprise ~60% of the total cell mass of Gram-positive bacteria. S. aureus WTAs are essential for adhesion to host tissues as well as to artificial surfaces including glass and polystyrene (Gross et al., 2001). WTAs null mutants are defective in their ability to produce biofilm; however, the reduced production of PNAG (an inevitable component of biofilm) was not reported, suggesting an independent role of WTAs in biofilm formation (Vergara-Irigaray et al., 2008). WTA mutant S. aureus strains were also unable to adhere and colonize the nasal epithelial cells as well as endothelial tissues of the kidney and the spleen derived from cotton rats. D-alanylation, which takes place on LTA, was reported to be intact on these strains, implying that WTAs were independently involved in cell adhesion and eventually biofilm formation (Weidenmaier et al., 2005). The involvement of WTAs in host colonization, infection, and biofilm formation makes it an important virulence factor and the enzymes involved in its biosynthesis can be novel targets for the discovery of new antimicrobials (Weidenmaier and Peschel, 2008).

Signaling molecules such as cyclic AMP (cAMP) are not only linked with the regulation of carbon metabolism and stringent response but also with the expression of virulence genes and biofilm formation in Gram-positive bacteria (Schilcher and Horswill, 2020). S. aureus requires two molecules of ATP and the enzyme diadenylyl cyclase DacA to synthesize c-di-AMP. Phosphodiesterase GdpP is involved in the degradation of c-di-AMP (Corrigan et al., 2011). Screening for essential genes of S. aureus revealed that dacA disruption was not possible, indicating the importance of c-di-AMP formation for the viability of the bacterium (Chaudhuri et al., 2009). On the other hand, gdpP deletion in the S. aureus SEJ1 strain yielded a 3-fold increase in biofilm formation, highlighting the fact that high levels of c-di-AMP induced biofilm formation in this strain. However, similar experimental conditions did not replicate the results in S. aureus USA300 LAC or its corresponding gdpP mutant (Corrigan et al., 2011). Single nucleotide polymorphisms (SNPs) or deletions in gdpP in various homogenous oxacillin-resistant (HoR) S. aureus isolates exhibited a decrease in the expression of icaADBC and agr and an increase in the expression of penicillin-binding protein 2 (PBP2). This resulted in an absence of polysaccharide content in the biofilm and the formation of a more proteinaceous biofilm (Pozzi et al., 2012). Apart from this, gdpP mutants were also impaired in their ability to form eDNA suggesting that c-di-AMP is essential for eDNA release as well (DeFrancesco et al., 2017). The role of c-di-AMP in biofilm formation may be strain dependent as different studies yielded different biofilm phenotypes in gdpP mutants of S. aureus (Schilcher and Horswill, 2020).

S. aureus possesses small α-helical peptides called phenol soluble modulins (PSM) which act as surfactants and disrupt cell–cell interactions within the biofilm better than proteases, unlike other bacteria that commonly use nucleases and proteases for biofilm detachment (Otto, 2008). The detachment and dispersal of planktonic cells are mediated by changes conferred in pH, nutrition, waste accumulation, oxygen depletion, etc. (Otto, 2008).

Once colonization is established, the bacteria adhere and start multiplying on wounded tissues, resulting in the upregulation of virulence genes and the production of toxins that further aid in disease progression. The initially expressed adhesion genes are now downregulated (Novick, 2003; Foster et al., 2014).

During the onset of infection, 95% of iron is within host cells in the form of serum iron bound to host proteins. In such a situation of iron starvation, S. aureus secretes high-affinity iron-binding proteins such as staphyloferrin (Drechsel et al., 1993) and aureochelin (Courcol et al., 1997). S. aureus can also initiate transcription of Isd (iron surface determinants)-mediated heme-iron transport that facilitates the release of heme from hemoglobin, haptoglobin, and hemopexin. This free heme transports across the plasma membrane via the iron complex and free iron is released in the cytoplasm of bacterial cell as a result of oxidative degradation (Maresso and Schneewind, 2006). This helps the bacterium in survival along with evasion from host defense (Liu, 2009).

Gram-positive bacteria including S. aureus secrete a diverse array of chemo-attractants such as peptidoglycan (Dziarski and Gupta, 2005), N-terminal lipoylated structure of lipoproteins (Nguyen and Götz, 2016), formylated peptides (Krepel and Wang, 2019), and unmethylated CpG sequences in DNA (Hemmi et al., 2000). This gradient of chemo-attractants induces proinflammatory signaling and activation of local immune response by recruitment of neutrophils and macrophages to the site of infection via a process called chemotaxis (Kolaczkowska and Kubes, 2013). Moreover, PSMs secreted by S. aureus sheds lipoproteins that also act as neutrophil chemo-attractants (Hashimoto et al., 2006; Hanzelmann et al., 2016). These structures collectively termed as “pathogen-associated molecular patterns” (PAMPs) are specific to bacteria and hence are recognized by the host immune system which activates Toll-like receptors (TLRs; Kumar et al., 2011).

A fibrin pseudo-capsule termed as abscess is formed within 2–6 days of infection (Kobayashi et al., 2015) with the help of coagulase proteins secreted by S. aureus which surround bacterial cells and recruited polymorphonuclear leukocytes (PMNs). After recruitment, S. aureus further inhibits neutrophil extravasation, activation, and chemotaxis in various ways. The binding of the members of the staphylococcal superantigen-like protein (SSL) family viz. SSL2 and SSL4 to TLR2, SSL10 to CXCR4, and SSL5 to GPCRs, such as P-selectin glycoprotein ligand- 1 (PSGL-1; which normally binds to the P-selectin anchor on endothelial cells) on the leukocyte surface, subsequently blocking neutrophil adhesion and extravasation to the site of infection. SelX protein has also been reported to have a similar function as SSL5 (Cheung et al., 2021). CHIP (chemotaxis inhibitory protein) blocks neutrophil recognition of chemotactic factors while Eap (extracellular adhesion protein) prevents neutrophil binding to endothelial adhesion molecule (ICAM-1; Chavakis et al., 2002; de Haas et al., 2004). S. aureus protease staphopain degrades CXCR2 (which recognizes cytokines), leading to the inhibition of neutrophil migration toward cytokines (Laarman et al., 2012). Another Geh lipase has been reported that removes the pro-inflammatory lipoylated N- terminus of the bacterial lipoproteins, thereby disguising these PAMPs from neutrophils (Chen and Alonzo, 2019).

Phagocytosis is also inhibited by biofilm formation via PIA/PNAG production (refer Section 2.1.1), protective surface structures such as capsules (refer Section 2.1.1), and aggregation. The combined non-redundant activity of coagulase and von Willebrand factor-binding protein (vWbp) of S. aureus produces a protein called thrombi which binds to prothrombin (factor II of the coagulation process) and forms a complex called staphylothrombin (Friedrich et al., 2003; Cheng et al., 2010). In the absence of a vascular damage signal, staphylothrombin cleaves fibrinogen from the host cells and forms fibrin clots. Fibrinogen-binding proteins of S. aureus such as clumping factor A (ClfA) bind to these clots resulting in the formation of fibrin-containing bacterial aggregates (McAdow et al., 2011). FnBPA and FnBPB can also activate the aggregation of platelets (Fitzgerald et al., 2006). S. aureus cells that are already phagocytosed by PMNs fight for their survival by releasing cytolytic toxins which cause pore formation and eventually cell lysis (Figure 1; Peschel and Sahl, 2006; Chambers and DeLeo, 2009; Liu, 2009; Löffler et al., 2010; Spaan et al., 2013; Stapels et al., 2014).

The expression of the capsule, clumping factor A, Protein A, and various other complement inhibitors on the cell surface of the bacterium may help to overcome opsonophagocytosis, i.e., the marking of the bacterial pathogen by antibodies (immunoglobulins, Igs) or complement factors for efficient phagocytosis. The Igs bind to phagocytes by the F_c region and to the pathogen by the F_ab region. The presence of Igs on the bacterial surface not only marks it for opsonization but also activates the classical pathway of complement fixation (Cheung et al., 2021). S. aureus produces three proteins to overcome opsonization by antibodies. (1) Surface protein A (SpA) non-specifically binds to the F_c region of IgG (Forsgren and Sjöquist, 1966) and F_ab region of IgM, which acts as a B cell superantigen and causes B cell apoptosis. It also initiates the production of plasma B cells that specifically recognizes only protein A, thereby diverting the immune response away from other virulence factors (Goodyear and Silverman, 2003; Pauli et al., 2014). (2) Sbi (S. aureus binder of IgG) binds to the complement factor H and C3 apart from the F_c region of IgG (Zhang et al., 1998; Atkins et al., 2008). (3) SSL10 also binds to the F_c region of IgG and prevents receptor-mediated phagocytosis (Itoh et al., 2010).

All three pathways (lectin, classical, and alternative) of the complement system possess the C3 convertase which cleaves C3 into C3a and C3b. The deposition of C3b on the bacteria marks it for opsonization. Other complement factors such as C5a (formed via the interaction of C3-C3b) act as a chemoattractant for the recruitment of more immune cells to the site of infection (Rooijakkers et al., 2005). Staphylococcal complement inhibitor (SCIN) inhibits C3 convertase, thus reducing the C3b deposition and C5a chemoattractant formation leading to the blockage of all three pathways of the complement system. Extracellular fibrinogen binding protein (Efb) of S. aureus binds to the C3 component via the C-terminus and fibrinogen via its N-terminus, covering the bacteria in a fibrinogen layer that inhibits the activation of the complement system as it fails to sense the surface-bound C3b (Ko et al., 2013). A homologous protein of Efb i.e., extracellular complement binding protein (Ecb), although lacking the fibrinogen binding activity, can inhibit the C3 convertase of the alternative pathway and all C5 convertases (Jongerius et al., 2010). Some S. aureus proteins such as collagen adhesion protein (Cna) inhibit the classical pathway (Kang et al., 2013), SdrE protein inhibits the alternative pathway (Sharp et al., 2012; Zhang et al., 2017) and the extracellular adherence protein (Eap) inhibits both lectin and classical pathways (Woehl et al., 2014). Finally, the SSL7 protein binds to the C5 component of the complement system as well as to the F_c region of the IgA antibody, inhibiting its recognition (Langley et al., 2005).

Proteolytic activity of various S. aureus proteins such as staphylococcal serine protease (V8 protease; SspA), cysteine protease (SspB), metalloprotease (aureolysin; Aur), and staphopain (Scp) have also been reported to inhibit opsonization (Dubin, 2002). Though the primary function of these proteases is nutrient acquisition, they may also destroy various immune defense proteins such as aureolysin cleaves C3 (Laarman et al., 2011) and V8 protease cleaves Igs (Rousseaux et al., 1983).

Despite the several mechanisms to evade phagocytosis, neutrophils can still manage to engulf S. aureus cells. Primary granules of neutrophil synthesize the enzyme Myeloperoxidase (MPO) which produces reactive oxygen species (ROS) and antimicrobial peptides (AMPs) such as defensins. Secondary granules of neutrophil secrete antimicrobial proteins such as lysozyme. S. aureus has evolved with various mechanisms to overcome both ROS and AMPs (Cheung et al., 2021). The orange pigment that gives S. aureus (aureus stands for golden) its name, Staphyloxanthin has been reported to scavenge the free radicals originating from ROS activity (Pelz et al., 2005; Clauditz et al., 2006). S. aureus synthesizes the enzyme superoxide dismutase which converts superoxide to less toxic H₂O₂ (Mandell, 1975; Clements et al., 1999). Furthermore, the catalase KatA and alkyl hydroperoxide reductase C AhpC detoxify the H₂O₂ by converting it into oxygen and water (Mandell, 1975; Cosgrove et al., 2007). MPO is directly inhibited by a staphylococcal peroxidase inhibitor (SPIN; De Jong et al., 2017).

Defensins and other AMPs are usually positively charged and hence are attracted to the negatively charged cell membrane and exhibit their bactericidal activity by forming pores in the bacterial membrane (Joo and Otto, 2015). The dlt operon of S. aureus esterifies hydroxyl groups in teichoic acids with alanyl residues which imparts an extra positive charge per alanine into the bacterial cell membrane, increasing the net charge and thereby inhibiting the binding of AMPs (Peschel et al., 1999). Moreover, a membrane-bound enzyme MrpF (multiple peptide resistance factor) adds Lys-PG on the outer layer of the cell membrane which also decreases the affinity of AMP binding (Peschel et al., 2001; Ernst et al., 2015). Finally, S. aureus secretes an enzyme named OatA, which acetylates the muramic acid residues of peptidoglycan reducing the efficacy of the neutrophil secreted antibacterial protein lysozyme which is otherwise very effective against other Gram-positive bacteria (Bera et al., 2005).

Neutrophil extracellular traps (NETs) are considered to be an integral component of the human immune system as they play a major role in host defenses during bacterial infections. NETs consist of activated neutrophils and DNA backbone along with proteins of various biological functions. NETs can ensnare but not kill S. aureus. The trapped bacteria from NETs can be released by degradation of the DNA backbone via S. aureus DNase leading to the persistence of the chronic infection. Eap, a protein secreted by S. aureus, can bind and aggregate linearized DNA, hindering the formation of NETs. Apart from this, the pathogen overcomes NET-mediated killing by expressing the surface protein FnBPB, which can neutralize the bactericidal activity of histones (Speziale and Pietrocola, 2021).

When encountered by neutrophils, S. aureus biofilms release the leukocidins such as LukAB and PVL (Panton-Valentine Leukocidin), which not only promotes S. aureus survival during phagocytosis but also induces the formation of NETs. Neutrophils and NETs can penetrate S. aureus biofilm but are unable to disrupt it (Malachowa et al., 2013). NETs can accumulate in organs and can cause tissue damage, as the infection progresses (Saffarzadeh et al., 2012; Weber, 2015). Therefore, the induction in the formation of NETs rather than blocking its antibacterial activity benefits the bacteria by favoring the colonization of deeper tissues, and thus providing better access to metabolic resources. This ensures the safer and optimal survival of bacteria in the host (Speziale and Pietrocola, 2021).

The abscess initially formed and matured within 6–14 days of infection accompanied by fibroblastic proliferation and tissue repair at the abscess margin and the formation of a fibrous capsule at the periphery (Kobayashi et al., 2015). The disruption of abscesses leads to the spread of S. aureus beneath the skin surface as well as in the bloodstream causing bacteremia. The bacterium can now adhere to endothelial surfaces and platelets causing endocarditis, metastatic abscesses, and bacterial uptake into endothelial cells where antibiotics and host defense molecules struggle to reach (Chavakis et al., 2005; Weidenmaier et al., 2005; Löffler et al., 2014). If endovascular spread cannot be controlled then systemic blood coagulation, massive production of microorganism-associated molecular pattern molecules (MAMP) and superantigen toxin-mediated cytokine storms can result in systemic inflammation, sepsis, multiple organ failure, and eventually death (Thomer et al., 2016; Lee et al., 2018).

The structural proteins, namely IcaA, IcaD, IcaB, and IcaC encoded by the ica operon synthesize PIA/PNAG. IcaA and IcaD are transmembrane proteins that simultaneously work as N-acetylglucosaminyltransferase-converting NAG monomers to PNAG oligomers of < 20 residues in length. The membrane-bound IcaC protein transports the PNAG oligomers across the cell membrane. Bacterial cell surface-associated IcaB protein deacetylates the PIA/PNAG oligomers that impart a positive charge to them and thus facilitates binding with the negatively charged bacterial cell surface (Cue et al., 2012).

cidABC and lrgAB operons of S. aureus encodes bacteriophage such as holins and antiholins, respectively (Brunskill and Bayles, 1996b). These operons have also been reported to have a crucial role in staphylococcal murein hydrolase activity (Groicher et al., 2000; Rice et al., 2003). In bacteriophages, holins are responsible for the transport of murein hydrolases across the cytoplasmic membrane for cell lysis and antiholins have an inhibitory activity on the holins (Young, 1992). Similarly, in S. aureus the cell lysis and eDNA release are controlled by the antagonistic activity of cid and lrg gene products. Mutations in either cid or lrg operons lead to variation in biofilm formation which suggests that fine-tuning between survival and death is essential for a robust biofilm formation (Rice et al., 2007; Mann et al., 2009).

CP5 and CP8 capsules (prevalent among S. aureus isolates) consist of repeating units of N-acetyl mannosaminuronic acid, N-acetyl L-fucosamine, and N-acetyl D-fucosamine. S. aureus have different serotypes due to the difference in glycosidic linkages between the sugars and the sites of O-acetylation of the mannosaminuronic acid residues of the capsule (O'Riordan and Lee, 2004; Kuipers et al., 2016). The pathway for capsule (CP) biosynthesis occurs in the cytoplasm via three distinct reactions, in which the universal cell envelope substrate UDP-D-N-acetylglucosamine (UDP-D-GlcNAc) is converted into the three different nucleotide-coupled sugars: UDP-N-acetyl-D-fucosamine (UDP-D-FucNAc), UDP-N-acetyl-L-fucosamine (UDP-L-FucNAc), and UDP-N-acetyl-D-mannosaminuronic acid (UDP-D-ManNAcA; Figure 6; Rausch et al., 2019).

The first reaction is catalyzed by the enzymes CapD and CapN that converts UDP-D-GlcNAc into the first soluble precursor UDP-D-FucNAc. The phosphosugar moiety of UDP-D-FucNAc is transferred to the membrane-anchored lipid carrier undecaprenyl-phosphate (C₅₅P) by CapM, yielding lipid I_cap (Li W. et al., 2014). The second reaction is catalyzed by the enzymes CapE, CapF, and CapG which convert UDP-D-GlcNAc into a second soluble precursor UDP-L-FucNAc. The transferase CapL further attaches L-FucNAc to lipid I_cap, resulting in the formation of second CP lipid intermediate, lipid II_cap (Kneidinger et al., 2003). The third reaction is catalyzed by the epimerase CapP and the dehydrogenase CapO, which converts UDP-D-GlcNAc into a third soluble precursor UDP-D-ManNAcA (Kiser et al., 1999; Portols et al., 2001). The transmembrane protein CapI transfers the UDP-D-ManNAcA moiety to lipid II_cap, generating the final capsule precursor lipid III_cap (Rausch et al., 2019). The modification of C₅₅P coupled trisaccharide is carried out by acetyltransferase CapH, which catalyzes the O- acetylation of L-FucNAc residues at the C3 position in CP5 strains (Bhasin et al., 1998). The putative flippase CapK and the polymerase CapJ translocate the modified precursor to the outer surface of the cell membrane where polymerization occurs (Sau et al., 1997; O'Riordan and Lee, 2004). The attachment of CP precursors to the MurNAc (N-acetylmuramic acid) moiety of peptidoglycan occurs via an unknown mechanism that possibly involves a member of the LCP (LytR-CpsA-Psr) family of proteins (Kawai et al., 2011; Chan et al., 2014). This process has been assumed to release the lipid carrier C₅₅P, which enters a new synthesis cycle (Rausch et al., 2019).

WTA biosynthesis was first characterized in Bacillus subtilis 168, which makes poly(glycerol) phosphate, and hence the genes involved in its synthesis were known as tag genes (for teichoic acid glycerol). Bacillus subtilis W23 and S. aureus make poly(ribitol) phosphate and hence the genes involved in its synthesis were known as tar genes (for teichoic acid ribitol; Ward, 1981; Figure 7).

The first three steps of the biosynthetic pathway are catalyzed by the enzymes TagO (TarO), TagA (TarA), and TagB (TarB), respectively. In the cytoplasm, N-acetylglucosamine phosphate (GlcNAc-1 phosphate) is transferred to an undecaprenyl phosphate (bactoprenyl phosphate) by a reversible phosphosugar transferase enzyme, TagO (TarO; Soldo et al., 2002; Price and Tsvetanova, 2007). Furthermore, TagA (TarA), an N-acetylmannosaminyl transferase, catalyzes the transfer of ManNAc from UDP-ManNAc to the C4 hydroxyl of GlcNAc residue to form ManNAc-β1,4-GlcNAc disaccharide (Ginsberg et al., 2006; Zhang et al., 2006). Finally, TagB (TarB), a glycerophosphotransferase, catalyzes the transfer of a single phosphoglycerol unit from CDP- glycerol (synthesized by TagD or TarD) to the C4 hydroxyl of ManNAc to complete the synthesis of linkage unit (Ginsberg et al., 2006; Bhavsar et al., 2007). The WTA linkage unit is highly conserved in all Gram-positive bacterial strains characterized so far. After these first three initial steps, the WTA pathways diverge (Brown et al., 2013).

In S. aureus, TarF (homolog of TagF that acts as a polymerase), which acts as a primase, adds one additional glycerol phosphate unit to the linkage unit (Swoboda et al., 2010). The assembly of the poly(ribitol) phosphate main chain is carried out by one of the two bifunctional poly(ribitol) phosphate primases/polymerases known as TarK/TarL. S. aureus TarL is a bifunctional enzyme having both primase and polymerase activities (Meredith et al., 2008). A cytidylyl transferase TarI and an alcohol dehydrogenase TarJ together synthesize the CDP-ribitol substrate that is utilized by TarL. TarL attaches more than 40 ribitol phosphates to complete the polymer synthesis (Brown et al., 2013). tarK gene is highly homologous to the tarL gene, suggesting that it might have the same enzymatic function. The reason for the presence of two homologous sets of tarIJL genes (tarI'J'K genes) with the same function (Qian et al., 2006) in S. aureus is still not clear; however, Meredith et al. (2008) have reported that tarK can nullify the loss of tarL and have an enzymatic function similar to tarL the gene product. However, strains that produced only TarL or TarK produced two electrophoretically distinct poly (ribitol) phosphate WTAs, known as L-WTA or K-WTA, respectively. K-WTA was found to be significantly shorter than L-WTA based on PAGE analysis. tarK gene expression is negatively regulated by agr quorum sensing system. As the expression of tarK can shorten K-WTA length by 50%, it is presumed that WTA chain length is dynamically regulated by S. aureus between pro-adhesion and low adhesion state to promote adhesion and dispersal of biofilm during different stages of infection. Once the polymerization is completed in the cytoplasm, glycosylation occurs and the polymer is flipped out by an ABC-dependent transporter complex TarGH followed by which ligation to the cell wall occurs (Swoboda et al., 2010).

The agr locus (first reported by Peng et al., 1988) is 3.5 kb in size and consists of two divergent promoters P2 and P3 that generates the transcripts of RNAII and RNAIII, respectively. RNAII locus comprises of four genes namely, agrB, agrD, agrC, and agrA. agrD transcript encodes a 7–9 amino acid long autoinducing peptide (AIP), which also plays a role in extracellular quorum sensing signal in S. aureus (Ji et al., 1995). AgrB is a multifunctional endopeptidase that is responsible for maturation (thiolactone modification and C terminal cleavage) and the export of AIP across the cell membrane. AgrC and AgrA constitute the two-component signal transduction system, of which AgrC is a membrane-bound histidine kinase, which is auto-phosphorylated upon the binding of AIP. It then trans-phosphorylates the response regulator AgrA. Activated AgrA can bind to the P2 and P3 promoters of agr locus and can regulate the expression of downstream genes (Novick et al., 1995; Queck et al., 2008; Le and Otto, 2015; Figure 8).

The majority of the virulence factors that are under the control of agr system are regulated by RNAIII. It is a messenger RNA that contains the hld gene for delta toxin or delta hemolysin (Janzon et al., 1989). It also activates the transcription of the hla gene for an alpha toxin or alpha hemolysin. RNAIII controls the expression of surface proteins, such as Protein A, coagulase, and fibrinogen binding protein, and repressor of toxin (Rot) protein by antisense base pairing with 5′ untranslated regions (5′ UTR) and forming RNA duplexes (Boisset et al., 2007). Rot protein binds to the promoter of many exoproteins (α, β, and γ hemolysin), enterotoxins (Toxic shock syndrome toxin), and leukocidins, inhibiting their transcription. The inhibition of Rot and surface proteins by RNA III leads to an upregulation of virulence factors along with the dispersal of biofilm. AgrA can also upregulate the transcription of phenol-soluble modulins psmα and psmβ operons in an RNAIII-independent manner (Queck et al., 2008; Le and Otto, 2015).

agr system is a global regulatory system of S. aureus for the upregulation of virulence factors, which aids S. aureus to cause several types of infections (Li S. et al., 2014; Tuchscherr and Löffler, 2016) and downregulation of surface proteins for disruption of biofilm. During the initial course of bacterial infection when the cell density is low, agr system is downregulated, resulting in an increased production of adhesins and surface proteins. Once colonization is established and nutrients become limited, the upregulation of degradative exoenzymes and toxins mediated by agr occurs, which helps not only to acquire nutrition for the cell but also to evade the host immune system (Fowler et al., 2004; Cheung et al., 2014). Yarwood and Schlievert (2003) have reported thicker and smoother biofilms on medical devices formed by agr mutant strains, leading to chronic and persisting infections in the host due to their inability to disseminate from the patient's body (Shopsin et al., 2010). The ability of Staphylococcus aureus to form biofilm in chronic relapsing infections associated with medical devices such as urinary catheters, intravenous catheters, and orthopedic prosthetics (Singh and Ray, 2014) shields the bacteria from the host immune system as well as antibiotics (Waters et al., 2016).

S. aureus strains can be classified into four agr groups (agrI, agrII, agrIII, and agrIV) based on the agrD gene (which encodes the synthesis of AIPs) and agrC gene (which encodes the receptor of agr TCS). The four AIP molecules are similar enough to bind with the AgrC receptor of the different groups but cannot activate the AgrA protein unless it is activated by the AIP of a similar group (Jabbari et al., 2012). Ikonomidis et al. (2009) and Khoramrooz et al. (2016) have reported that strains of agr group II and agr group III are more potent biofilm producers. Nichol et al. (2011) have also established a relationship between antibiotic resistance and agr groups in S. aureus. agr group I is widely associated with CA-MRSA genotypes, whereas agr group II is associated with HA-MRSA genotypes in humans.

The sae (staphylococcal accessory element) system was first identified by Giraudo et al. (1994) while studying a Tn551 insertional mutant in which the production of exoproteins such as nuclease, coagulase, α hemolysin, and β hemolysin was found to be altered. The sae operon consists of four genes, i.e., saeP, saeQ, saeR, and saeS, which are under the regulation of P1 and P3 promoters. The sae locus primarily comprises of a two-component system in which SaeR is the response regulator and SaeS is the histidine kinase (Giraudo et al., 1999). This system is activated by external stimuli such as H₂O₂ and α defensins and repressed under low pH and high NaCl concentrations, both resulting in membrane perturbation (Geiger et al., 2012; Haag and Bagnoli, 2015). The signal is sensed by SaeP which interacts with the extracellular linker peptide of SaeS leading to its autophosphorylation at His131 which then transphosphorylates SaeR at Asp51. SaeR can now bind to SBS (SaeR binding sequence), leading to the upregulation of saeR-mediated virulence genes (Figure 9).

Very little was known about the role of auxiliary proteins SaeP and SaeQ in Sae TCS activity and disease progression. Collins et al. (2020) generated mutants of saeP, saeQ, and saeP-saeQ double mutant of the USA300 strain of S. aureus to analyze their function. The survival of S. aureus USA300ΔsaeP increased compared to the wild type USA300 strain post phagocytosis by neutrophils probably due to increased expression of bicomponent leukocidins by the former. The USA300ΔsaeQ also yielded a similar phenotype but the neutrophil interaction results were comparatively less significant. However, the saeP-saeQ double mutant strain (USA300ΔsaePsaeQ) exhibited a drastic increase in neutrophil survival and virulence during murine bacteremia as compared to the USA300ΔsaeP single mutant strain. These observations suggested the role of SaeP in the survival of bacterium after phagocytosis by neutrophils and the combined effect of SaeP and SaeQ in in vivo pathogenesis.

Flack et al. (2014) have reported that SaeS more specifically detects α-defensin 1/Human neutrophil peptide 1 (HNP-1) and human polymorphonuclear leucocytes (PMNs). SaeR and SaeS are transcribed via the P3 promoter located within the saeQ coding sequence. The P1 promoter is located upstream of the saeP gene and can transcribe all four genes of the locus. The P1 promoter has been reported to exhibit 2–30 times higher activity compared to the P3 promoter. Jeong et al. (2011) have reported that only basal level expression of saeRS genes from the P3 promoter is sufficient for activation of sae target genes and exoprotein production. P1 promoter has two saeR binding sites and thus it can be auto-induced by saeR transcripts produced by basal level expression of saeRS TCS via P3 promoter (Figure 7). The target genes of the sae system have been bifurcated into two classes: Class I target genes require high levels of phosphorylated SaeR for its activation, viz., fnbA, coa, P1 promoter, sae, and eap, while Class II target genes require basal/lower levels of phosphorylated SaeR for its activation, viz., hla and hlb genes (Mainiero et al., 2010; Haag and Bagnoli, 2015). SaeP and SaeQ have been reported to form a protein complex with SaeS and induce the sensory kinase phosphatase activity resulting in dephosphorylation of activated SaeR which downregulates the virulence genes regulated by saeR (Jeong et al., 2012; Haag and Bagnoli, 2015).

Depending on growth conditions, sae system can both positively (upregulation of biofilm forming genes, i.e., hla, hlb, coa, emp, eap, fnBPA, and fnBPB) and negatively (upregulation of biofilm dispersal factors such as nucleases and proteases) affect biofilm formation (Caiazza and O'Toole, 2003; Johnson et al., 2008; O'Neill et al., 2008; Huseby et al., 2010; McCourt et al., 2014; Zapotoczna et al., 2015; Liu et al., 2016). Moormeier et al. (2014) have reported that nuc transcription is positively regulated by the SaeRS system, as both sae and nuc mutant were unable to cause the dispersal of biofilms, which likely explains the degradation of eDNA during the dispersal stage of biofilm formation. Moreover, SaeS has been reported to exhibit polymorphism across S. aureus strains as a point mutation can lead to hyperactivation of SaeRS TCS leading to the inability of such strains to form robust biofilms (Mainiero et al., 2010; Cue et al., 2015). saeRS locus also acts synergistically with sarA to inhibit the production of extracellular proteases, resulting in an improved ability for biofilm formation (Arya and Princy, 2013).

SaeRS system positively regulates NETs (Berends et al., 2010; McDonald et al., 2012), SCIN, CHIPS (Rooijakkers et al., 2006), leukocidins (Münzenmayer et al., 2016), α hemolysin (Hla; Nygaard et al., 2013), pro-inflammatory cytokines, proteases, and toxins production (Watkins et al., 2011; Zurek et al., 2014; Cho et al., 2015). However, S. epidermis, which also possesses a SaeRS two-component system and is closely related to S. aureus, does not possess these virulence genes (Handke et al., 2008; Ravcheev et al., 2011). This indicates that S. aureus has acquired these virulence genes during evolution, which are being maintained by it under the control of the SaeRS two-component system (Liu et al., 2016).