The role of human extracellular matrix proteins in defining Staphylococcus aureus biofilm infections

Abstract Twenty to forty one percent of the world’s population is either transiently or permanently colonized by the Gram-positive bacterium, Staphylococcus aureus. In 2017, the CDC designated methicillin-resistant S. aureus (MRSA) as a serious threat, reporting ∼300 000 cases of MRSA-associated hospitalizations annually, resulting in over 19 000 deaths, surpassing that of HIV in the USA. S. aureus is a proficient biofilm-forming organism that rapidly acquires resistance to antibiotics, most commonly methicillin (MRSA). This review focuses on a large group of (>30) S. aureus adhesins, either surface-associated or secreted that are designed to specifically bind to 15 or more of the proteins that form key components of the human extracellular matrix (hECM). Importantly, this includes hECM proteins that are pivotal to the homeostasis of almost every tissue environment [collagen (skin), proteoglycans (lung), hemoglobin (blood), elastin, laminin, fibrinogen, fibronectin, and fibrin (multiple organs)]. These adhesins offer S. aureus the potential to establish an infection in every sterile tissue niche. These infections often endure repeated immune onslaught, developing into chronic, biofilm-associated conditions that are tolerant to ∼1000 times the clinically prescribed dose of antibiotics. Depending on the infection and the immune response, this allows S. aureus to seamlessly transition from colonizer to pathogen by subtly manipulating the host against itself while providing the time and stealth that it requires to establish and persist as a biofilm. This is a comprehensive discussion of the interaction between S. aureus biofilms and the hECM. We provide particular focus on the role of these interactions in pathogenesis and, consequently, the clinical implications for the prevention and treatment of S. aureus biofilm infections.


Epidemiology and significance of Staphylococcus aureus biofilms to human health
According to an announcement made by the National Institutes of Health, upw ar ds of 60% of all microbial infections are associated with biofilms (Lewis 2001 ).It is important to note that biofilms are not an exception but rather the predominant lifestyle in the microbial world (Costerton et al. 1987, Busscher and Van Der Mei 1995, Hall-Stoodle y and Stoodle y 2005, Lindsay and von Holy 2006, Ouidir et al. 2022 ).Biofilms are easily formed on both biotic and abiotic surfaces.Fifty to se v enty percent of all hospital-acquired infections are estimated to originate from the introduction of medical devices .T his includes the introduction of catheters, pacemakers , implants , and contact lenses , all of which can act as substrates for the development of Staphylococcus aureus biofilms (Dar ouic he 2004 , Stoodley et al. 2008, Kathju et al. 2014, Jamal et al. 2018 ).Regar dless of the point of entry, the biofilm lifec ycle involves the dispersion of bacteria to secondary locations, resulting in se v er e infections .For example , drinking water contaminated with Pseudomonas aeruginosa can de v elop into pneumonia and cause se v er e lung damage or death (Berrouane et al. 2000, Maharaj et al. 2017, Fujiki et al. 2022 ).Similarly, the introduction of pacemakers, potentially contaminated by transiently colonizing bacteria or through transport and handling by colonized hospital personnel, allows S. aureus biofilms to form and disperse, resulting in difficult-to-treat cases of sepsis and endocarditis (Rali et al. 2019, Gr a psa et al. 2022 ).
Although biofilm-forming bacteria are often isolated from hospitals, there is an underappreciation for this lifestyle, especially in the clinical management of disease (Lindsay and von Holy 2006 ).Biofilm infections are known to be up to 1000 times more recalcitrant to antimicrobial interventions, including treatment with antibiotics that are used successfully against planktonic bacteria, at the same or lo w er dosage (Ceri et al. 1999, Howlin et al. 2015 ).The failure of antimicrobials against biofilm infections is attributed to multiple biofilm-associated properties, including genetic alterations in the bacterium (Lewis 2001(Lewis , 2010 ) ).This can cause the de v elopment of additional antibiotic-toler ant and r esistant populations that confound the treatment of secondary infections.
The identification and treatment of biofilms is further complicated by the difficulty in isolating and culturing biofilm comm unities fr om patient samples.While biofilm biomass can often be isolated from the primary infection site (such as a catheter), tr aditional culturing tec hniques do not accur atel y ca ptur e bacterial burden, resulting in the incorrect estimation of antibiotic susceptibility and ultimately unsuccessful treatment of the infection (Høiby et al. 2015 ).Lastl y, m ultiple studies confirm that once a biofilm is formed, the ability of the host immune system to eradicate the infection decreases exponentially, conversely increasing the possibility of persistent infection (Brinkmann et al. 2004, Berends et al. 2010, Bhattacharya et al. 2020 ).

Staphylococcus aureus biofilm structure and general properties
The lifecycle of S. aureus biofilms can be br oadl y classified into four phases: attac hment, pr olifer ation, matur ation, and dispersion.Biofilms are formed when bacteria attach to a surface, prolifer ate, and ac hie v e sufficient population density to communicate with each other via chemical signals, in a process known as quorum sensing (Donlan andCosterton 2002 , Stoodley et al. 2002 ).In S. aureus, this function is performed by the activities of the a ccessory g ene r egulator (Agr) system.Quorum sensing by the Agr system r equir es the expr ession of four pr oteins.While AgrC and AgrA ar e histidine kinase-r esponse r egulator partners, AgrB is a tr ansmembr ane pr otein that facilitates the tr ansport of the autoinducing peptide encoded by agrD (No vick 2003 ).T his cir cuit activates do wnstream genes that allow for the expression of proteins and p olysaccharide i ntercellular a dhesin (PIA), which, together with extracellular DNA, comprise the bacterial-derived components of the e xtracellular p olymeric s ubstance (EPS) , allowing for maturation (Arciola et al. 2001, Boles et al. 2010, Speziale et al. 2014, Arciola et al. 2015 ) .
During the transition between each of the four phases of the biofilm lifecycle, the Agr regulatory network influences the expression of a majority of virulence factors made by the pathogen.This includes adhesins such as c lumping f actors A and B (ClfA, ClfB), fibrinogen-binding proteins A and B (Fnbp A, B), and others, which assist in the attachment to surfaces as well as the development of the biofilm EPS in vivo (Fig. 1 A) (Novick 1991(Novick , 2003 ) ). Agr positiv el y r egulates the expr ession of virulence factors suc h as alpha hemolysin, gamma hemolysin, and Panton Valentine leucocidin, whic h hav e specific functions in allowing biofilm bacteria to e v ade macr opha ge and neutr ophil-mediated killing (Sc herr et al. 2015, Bhattacharya et al. 2018 ).During the dispersion of bacteria from the biofilm, Agr positively regulates the expression of phenol-soluble modulins (PSMs), a group of peptides that act as surfactants, as well as DNases and proteases that can cleave matrix components, further supporting the transition to planktonic life (Peschel and Otto 2013, Dastgheyb et al. 2015, Bhattacharya et al. 2018 ).In addition to the Agr network, there are a handful of global regulators and two-component systems that can play significant roles during biofilm development and pathogenesis .T his includes at least 16 two-component systems that sense and respond to various environmental cues .T he reader is referred to these compr ehensiv e r e vie ws for a better understanding of the regulatory networks that interconnect with the Agr quorum sensing circuit (Jenul and Horswill 2019, Crosby et al. 2020, Bleul et al. 2022 ).
Staphylococcus aureus has e volv ed to be uniquely suited for survival in a human host.Unlike most bacterial pathogens, S. aureus can bind or utilize e v ery major hECM pr otein, ther eby causing a wide variety of infections with varied and specific pathogeneses (Howden et al. 2023 ).With a tight contr ol ov er the transcription of virulence factors, S. aureus is able to quic kl y sense its environment and tempor all y expr ess a signatur e of adhesins and secr eted pr oteins r equir ed to surviv e in a particular tissue nic he (Beenken et al. 2004 ).This r e vie w pr ovides a compr ehensiv e discussion of both partners in this interaction, i.e. the bacterium and the host.We ther efor e begin by elaborating on our current knowledge of factors that are expressed by S. aureus and allow it to bind and form biofilms with the hECM (Fig. 1 A).Following this, we provide a detailed description of the major hECM components that potentiate S. aureus biofilm infections in the human host (Fig. 1 B).We follow this with a discussion of some of the major biofilm-associated infections caused by S. aureus, with emphasis on the role of hECM components in each niche (Fig. 2 ).Lastly, this review briefly describes current and future avenues for anti-biofilm therapeutics that interfer e specificall y with these hECM-pathogen inter actions.

Surface adhesins
Staphylococcus aureus strains can express at least 24 surfaceanc hor ed pr oteins that bind multiple known ligands from a single domain or multiple ligands from multiple domains of a single pr otein (Sc hnee wind and Missiakas 2019 ).A lar ge gr oup of these surface pr oteins ar e cr osslinked to the cell wall peptidoglycan via the activity of one or more of a group of sortase enzymes that can recognize the canonical LPXTG motif at the C-terminus of the protein (Fig. 1 A).Ligand binding commonly occurs at the Nterminus of surface-anc hor ed pr oteins; ho w e v er, binding of host ligands to additional domains on the C-terminal end has also been reported (FnbpA, FnbpB) (Bingham et al. 2008 ).While the overall structure of these surface proteins is similar, they can be grouped into subclasses depending upon their functional domains (Sdr domain, NEAT domain proteins etc.).For the purposes of this r e vie w, a brief discussion of these proteins, categorized based on known function and potential r ele v ance to biofilm formation, is provided here.For a more detailed analysis of the biochemistry and structure of these proteins, the reader is referred to the following expert r e vie ws (Foster et al. 2013 , Sc hnee wind andMissiakas 2019 ).
Fibronectin -binding proteins .FnbpA and B are sortase-anchored proteins that consist of an N-terminal A domain that is curr entl y c har acterized as binding to elastin, histones, plasminogen, and fibrinogen, follo w ed b y 10 (FnbpB) or 11 (FnbpA) fibronectin binding repeats at the C-terminus, each binding to the N-terminal domain of fibronectin with varying affinities using a β-zipper mechanism (Bingham et al. 2008, Pietrocola et al. 2016, Giampiero et al. 2019 ).While the A-domain is thought to promote interbacterial attachment and platelet aggregation, the C-terminus binds to fibronectin and often uses it as a bridge to interact with α 5 β 1 integrin, a common cell surface receptor (Maurer et al. 2016 ).
One study performed with isolates from device-related infections detected FnbpA in 99% of S. aureus samples (Arciola et al. 2005 ).Indeed, FnbpA has been found to have particular importance in periprosthetic implant-associated infections caused by S. aureus .Studies with human serum and joint synovial fluid demonstrate that FnbpA is required for optimum biofilm formation and that it protects bacteria against phagocytic attack (Gries et al. 2020, Pestrak et al. 2020 ).While deletion of genes encoding for both proteins has been shown to significantly affect biofilm formation, eliminating the expression of either FnbpA or FnbpB individually has not resulted in substantial effects on the capacity of S. aureus to form biofilms, indicating an ad diti v e r ole for the two proteins during infection (O'Neill et al. 2008 ).
Serine-aspartate-repeat (Sdr) domain proteins.ClfA and ClfB are canonical members of a subgroup of cell wall-anchored proteins known as the Sdr family of adhesins, characterized by a string of ∼150 serine aspartate repeats at the C-terminus .T his group The Sdr repeats characteristic of this group are glycosylated with N-acetyl glucosamine at e v ery serine residue .T he addition of this gl ycosyl gr oup is attributed to the combined enzymatic activity of two glycosyl transferases, namely, AggB/SdgA and AggC/SdgB.While a role for this repeated glycosylation is yet to be appreciated, the potential contribution of this modification to interbacterial attachment and therefore aggregate biofilm formation has been postulated (Hazenbos et al. 2013, Thomer et al. 2014 ).While ClfA and ClfB bind to soluble fibrinogen with high affinities, ClfA can additionally bind insoluble fibrin (polymerized fibrinogen).ClfA is essential for the survival of S. aureus during bloodstream-associated infections, attributed to its ability to cause bacterial-fibrinogen interactions that result in agglutination of blood plasma, a form of a ggr egate biofilm formation that is c har acteristic to S. aureus (McAdow et al. 2011 ).Additionally, ClfA can indir ectl y enga ge with v on W illebrand f actor (vWf), annexin 2a, and the platelet receptor glycoprotein IIIb, allowing numerous interactions to occur both in blood and tissue, leading to bacterial accumulation and biofilm formation (Mcdevitt et al. 1997, Ashraf et al. 2017, Claes et al. 2017, Herman-Bausier et al. 2018 ).The utility of ClfA as a ther a peutic tar get for S. aureus infections has resulted in multiple attempts at monoclonal vaccines targeting the adhesin (Ganesh et al. 2008, Tkaczyk et al. 2016 ).Indeed, recent studies demonstrate the efficacy of anti-ClfA antibodies in reducing the bacterial burdens of murine hematogenous implantassociated biofilms (Wang et al. 2017a ,b ).
ClfB plays a significant role in augmenting biofilm formation under conditions of calcium starvation in numerous strains of S. aureus (Abraham and Jefferson 2012 ).The role of ClfB as an important adhesion factor during the earl y attac hment phase of biofilm de v elopment is e vident fr om numer ous studies that demonstr ate the r equir ement of ClfB for the adhesion of bacteria to the nasal epithelium using murine models of colonization.This activity is attributed to the cytokeratin-10 binding capacity of the A-domain of ClfB (Schaffer et al. 2006, Wertheim et al. 2008, Sun et al. 2018 ).Another Sdr protein with noteworthy contributions to biofilm formation is SdrC.In addition to forming low-affinity homophilic bonds between neighboring cells, thereby facilitating bacterial accumulation, SdrC has also been shown to form hydrophobic bonds with abiotic surfaces, promoting the attachment of a ggr egates to a substr ate, suc h as would be found during implant-associated infections (Barbu et al. 2014, Feuillie et al. 2017 ).
Staphylococcus aureus surface (Sas) protein family.In addition to the Sdr group of proteins, S. aureus strains can also express up to eight surface-associated (Sas) pr oteins, initiall y named due to their discovery in association with the cell wall.SasG is the best-c har acterized of these pr oteins, particularl y for its ability to augment the formation of a ggr egate biofilms.With structur al and functional homology to the a ccumulation-a ssociated p rotein (Aap) of S. epidermidis as well as the plasmin-sensitive protein of S. aureus (Pls), SasG has a C-terminus comprised of multiple "B" domains containing 5-8 G5 subdomains ( ∼78 amino acids) separated by "E" subdomain linkers ∼50 amino acids long (Fig. 1 A).The B-domain of SasG is heavily processed by proteases, resulting in the formation of both secreted B-domain peptides and surfaceassociated B-domains formed following the cleav a ge of the Adomain.These B-domain variants are known to interact with each other, promoting the formation of biofilms, especially in the presence of physiological concentrations of zinc (Geoghegan et al. 2010, Gruszka et al. 2012 ).While the A-domain is not thought to contribute to interbacterial attachment, it has been shown to bind to desquamated epithelium, inv ariabl y assisting in the process of biofilm formation (Roche et al. 2003, Geoghegan et al. 2010 ).From studies with the comparatively better-characterized homolog Aap, it is clear that additional roles for SasG in S. aureus biofilm formation ar e likel y.Studies by Sc haeffer et al. ( 2015 ) demonstrate a significant reduction of biofilm burden in adult male Spr a gue-Dawley r ats when jugular catheters wer e infected with a mutant unable to express Aap, in comparison to an isogenic wild-type contr ol.Inter estingl y, this defect in virulence was onl y slightl y incr eased when aap bacteria were unable to produce PIA, indicating a lar ger r ole for proteins, as compared to PIA, in assisting interbacterial attachment during biofilm formation.Indeed, SasG-expr essing str ains of S. aureus wer e found ca pable of forming PIA-independent biofilms.Lastly, in addition to potentiating interbacterial attachment, the fibrillar nature of SasG has been shown to mask the activity of other surface-associated proteins, including FnbpA and B. The role of SasG in facilitating biofilm dev elopment may ther efor e depend on the tissue niche as well as the presence of specific host proteins.Additional Sas family proteins include SasA (SraP), SasB, SasD, SasF, SasJ, SasK, SasL, and SasH (AdsA).Although the ligands for most of these pr oteins ar e curr entl y poorl y c har acterized, SasX and SasC have both been shown to be important for cell a ggr egation, likel y pr omoting biofilm formation (Sc hr oeder et al. 2009, Li et al. 2012 ).
Immune evasion proteins.While providing multiple avenues of adherence to the human tissue substratum, the surface-anchored proteins of S. aureus also contribute to pathogenicity and immune e v asion mec hanisms .T he most versatile of these functions is performed by s urface p rotein A ( S pa), a cell surface-associated and secr eted super antigen that is best known for binding to the Fc domains of IgG molecules, often masking their pr otectiv e imm une functions (Becker et al. 2014 ).Spa plays a particularly significant role in assisting the survival of S. aureus during bloodstream infections .T his activity has specifically been correlated to an increased ability for r ecalcitr ance in subsequent invasion of joint tissue, resulting in S. aureus -induced septic arthritis (Palmqvist et al. 2002 ).Furthermor e, the imm unoglobulin binding activity of Spa was utilized to demonstrate that Spa forms an essential component of pol ysacc haride-independent S. aureus biofilm matrices, since the addition of either serum, immunoglobulin, or anti-Spa antibodies sho w ed a dose-dependent reduction of biofilm formation in a pol ysacc haride-independent manner (Merino et al. 2009 ).
As a prominent nasal colonizer, S. aureus expresses multiple surface proteins that facilitate bacterial binding to the nasal epithelium, some of which are described abo ve .Additionally, the expression of a c ollage n -b inding a dhesin (Cna) provides the pathogen with specific access to the most abundant protein on the skin (Ricard-Blum 2011 ).Cna has been shown to additionally bind laminin, a commonly found basement membrane protein.It is ther efor e not surprising that Cna contributes to S. aureus colonization and the establishment of skin and implantassociated infections (Rhem et al. 2000, Arciola et al. 2005 ).In a study that genetically characterizes biofilm-forming S. aureus isolates from implant-associated infections, although MRSA strains wer e lar gel y found to be Cna-negativ e, the adhesin was found to be expressed in ∼25% of implant-associated infections, indicating a specific role for the adhesin during biofilm formation in colla gen-ric h tissue envir onments (Arciola et al. 2005 ).As a consequence of its ability to bind collagen via its N-terminal domain, Cna has also been demonstrated to bind the colla gen-ric h triple helix stem domain of the serum complement activ ating pr otein, C1q (Kang et al. 2013 ).The stem of C1q is r equir ed for its activity.The Cna-C1q interaction therefore blocks complement activation, likely contributing to the evasion of innate immune defenses by S. aureus (Kang et al. 2013 ).Lastl y, colla gen has been reported to bind dir ectl y to fibrinogen and fibrin, a process that is crucial to wound healing (Hayuningtyas et al. 2021 ).The ability of S. aureus to bind colla gen may ther efor e act as a bridge to accumulate fibrinogen around forming aggregates and mask the pathogen from immune defenses.
The iron surface determinant (Isd) proteins .As the only limiting nutrient for bacterial growth during infection, iron is required for bacterial survival in the human host.Conditions of iron starvation can alter the surface hydrophobicity of S. aureus (Clarke et al. 2004, Skaar et al. 2004 ).The iron sequestering, surface-anc hor ed proteins of the Isd system, expressed via the isdA, isdB, isdCDEF-srtBisdG, isdH , and isdI transcriptional units, include IsdA, IsdB, IsdC, and IsdH.The promoters of these transcriptional units are r epr essed by the ferric uptake r epr essor pr otein (Fur) under ir onreplete conditions.Studies by Johnson et al. ( 2005 ) describe an increase in biofilm formation under conditions of iron starvation.The authors note that this effect was independent of PIA production, suggesting a greater contribution for ir on-scav enging pr oteins in the formation of PIA-independent matrix biofilms.Furthermor e, the pr esence of hemoglobin in planktonic cultur es has been associated with a downregulation of Agr, a process that occurs during the transition of planktonic populations to biofilm communities (Pynnonen et al. 2011 ).
In addition to their primary function, ir on-scav enging pr oteins hav e demonstr ated imm une e v asion pr operties that pr ovide further evidence of their role during biofilm-associated infections (Clark e et al. 2007, Clark e and Foster 2008, Visai et al. 2009, Zapotoczna et al. 2013). Torres et al. ( 2006 ) clearly demonstrate that IsdB is essential for the binding of hemoglobin to the bacterial surface and that in the absence of this protein, S. aureus is severely impaired in its ability to cause kidney abscesses in murine models of infection (Kim et al. 2010 ).IsdA, while a structur all y similar protein to IsdB, binds primarily to heme but has been shown to interact with additional ligands, including fibrinogen (Clarke et al. 2004 ).As a protein with a demonstr abl y significant contribution to nasal colonization, the expression of IsdA has been reported to reduce the hydrophobicity of the bacterial surface, consequentially making S. aureus tolerant to host fatty acids and immune cells as well as allowing for survival on human skin (Clarke et al. 2007 ).

Secr eted pr oteins
In addition to a cache of secreted toxins and cytolytic enzymes used to lyse human cells, S. aureus expresses extracellular proteins that bind multiple host ligands, thereby facilitating a ggr egation and biofilm formation.The following section discusses these proteins and their roles in colonization and pathogenesis.
Coagulases .Survival in the bloodstream allows S. aureus to disseminate and cause life-threatening tissue-associated biofilm infections , including endocarditis , septic arthritis , and abscess formation.To interfere with blood homeostasis, S. aureus requires two coa gulases, namel y, sta phylocoa gulase (Coa) and vWf binding protein (VWbp), in conjunction with the fibrin binding capacity of ClfA.Indeed, the absence of any one of these secr eted pr oteins r esults in a significant loss of virulence in bloodstream-associated infections by S. aureus (McAdow et al. 2011 ).Coa and vWbp are both mosaic proteins with N-terminal fibrinogen-binding domains .T he N-terminus of both proteins also activates prothrombin.While the C-terminus of Coa acts as an additional fibrinogenbinding domain consisting of ∼27 tandem peptide repeats, the Cterminus of vWbp binds to fibronectin (Fig. 1 A).The C-terminus of vWbp encodes a binding site for vWf.Since this host protein is present on endothelial tissue and vWbp is secreted, the significance of this inter action r emains unclear.Recentl y, ho w e v er, ther e has been m uc h a ppr eciation for the r ole of vWbp as a protein that bridges the bacterial surface to the hECM (Claes et al. 2014, Viela et al. 2019 ).
The activation of the tissue factor pathway at the site of injury is r equir ed for r estoring homeostasis .T he activity of multiple plasma proteases leads to the accumulation of Factor Va and Xa, also known as the pr othr ombinase complex, whic h activ ates pr othr ombin to form thrombin.The two secreted coagulases, Coa and vWbp can bypass these steps to catalyze the formation of enzymaticall y activ e sta phylothr ombin complexes.Similar to the activity of thr ombin, sta phylothr ombin will cleave the fibrinopeptides A and B from fibrinogen to form fibrin cables (Fig. 1 B).ClfA and FnbpA subsequently attach fibrin cables to the bacterial surface, ther eby pr e v enting the proper fibrin crosslinking that is required for clot formation under normal physiological conditions.This results in dense aggregate biofilms of bacteria shielded by a matrix of fibrin and bacterial pr oteins, pr otected fr om imm une onslaught (T hamma vongsa et al. 2013 ).Since these bacterial proteins circumvent the numerous enzymatic steps that are involved in physiological clot formation, this likely fails to induce the appropriate inflammatory response required to restore homeostasis (Thomer et al. 2016 ).
DN A -binding proteins .T he c har acterization of pol ysacc harideor PIA-independent biofilm formation by S. aureus has led to a gr eater a ppr eciation for DNA and the pr oteins that comprise the EPS (Boles et al. 2010 ).The autolysin, Atl causes the enzymatic lysis of S. aureus gr owing as biofilms, r esulting in a r elease of DNA that gets incor por ated into the matrix (Bose et al. 2012 ).Additionall y, cytoplasmic pr oteins "moonlight" as DNA-binding components when released into the matrix upon cell lysis .T hese proteins likely bind to DNA and protect it from the activity of nucleases released by the biofilm (Foulston et al. 2014, Moormeier et al. 2014 ) .It is ther efor e e vident that DNA is an important component of S. aureus biofilms.Additionally, S. aureus secretes a group of proteins with non-specific DNA-binding activity.Primarily characterized for their function in planktonic cultur es, we r ecentl y identified these proteins from the matrix of PIA-independent biofilms using southwestern blotting and mass spectrometry (Kavanaugh et al. 2019 ).The most abundant of the proteins identified was the extr acellular adher ence pr otein, Ea p. IsaB, a second, wellc har acterized cell wall-associated DNA-binding protein, was also identified in this screen.These two proteins play ad diti ve roles in retaining DNA onto the biofilm matrix (MacKey-Lawrence et al. 2009 ).Eap is additionally implicated as being able to bind and aggr egate neutr ophil DNA.Eisenbeis et al. ( 2018 ) utilize c hemicall y induced neutrophil extracellular traps incubated with increasing concentrations of purified Eap to demonstrate a reduction in the induction of NETs in vitro .Ho w e v er, since NETosis is induced by S. aureus biofilms both in vitro and in vivo , the implications of this function r equir e further r esearc h (Bhattac harya et al. 2018 ).Lastly, Yonemoto et al. ( 2019 ) find that SasG can compensate for the absence of Ea p expr ession in an Ea p-ov er expr essing str ain of S. aureus .Their studies describe a role for Eap in maintaining the "ruggedness" of a biofilm, defined by the authors as a biofilm with decreased smoothness compared to a mutant unable to express SasG.Using gel shift assays with purified SasG, the authors conclude that SasG is also a DNA-binding protein and that Eap and SasG work to pr e v ent the degradation of eDNA incor por ated in the matrix.Whether this function contributes to the biofilmassociated phenotypes observed is unclear.
Metabolic proteins .Foulston et al. ( 2014 ) propose that S. aureus biofilm matrices are composed of cytoplasmic proteins released when bacteria enter into stationary phase, rather than the ex-pression of specific matrix proteins when bacteria transition from planktonic to biofilm communities .T he authors demonstrate that these intr acellular pr oteins r elocate to the cell surface in response to decreased pH and that this localization can be reversed with an increase in pH.Whether these proteins are actively translocated (secretion system) or released via autolysis, ho w ever, w arrants further investigation.Biofilm metabolism is often found to have a complex but specific relationship with host responses.As an interesting example of this concept, Tomlinson et al. describe the process by which S. aureus glycolysis induces mitochondrial stress in the airway, resulting in the synthesis of itaconate, which r e-dir ects S. aureus to w ar ds biofilm formation and aw ay from glycol ysis.In a gr eement with these observ ations, non-synon ymous mutations in pro-glycolytic genes were discovered in isolates from c hr onic airway infections with S. aureus .These included mutations in pyruvate kinase ( pyk ), lactate dehydrogenase ( ldh ), and aconitase ( acnA ) (Heim et al. 2020, Tomlinson et al. 2021 ).

Host extracellular matrix proteins that are exploited for biofilm formation
The study of microbial biofilms often focuses on the properties of the micr obe.Similarl y, r esearc h with host-biofilm relationships is often limited to the properties of immune cells.While this has gr eatl y adv anced the field of infectious disease contr ol, ther e can be no effective treatment against S. aureus biofilm infections without a gr eater a ppr eciation for the host-derived molecules that interact with and often assist the formation of S. aureus biofilms in vivo.In this section, we ther efor e focus on the general properties and known functions of some of the hECM molecules that are significant to S. aureus biofilm formation (Fig. 1 B).

Collagen
As the most abundant component of the extracellular matrix, eac h colla gen molecule is composed of 3 polypeptide alpha chains with a c har acteristic Gl y-X-X' motif, wher e X is most often a proline and X' is either a hydr oxypr oline or hydr oxyl ysine (Fig. 1 B).These α-chains assemble as a right-handed helical structure and this motif or collagen-like domain can be additionally found in numer ous human pr oteins, including complement C1q, pulmonary surfactant proteins, and ficolins (Ricard-Blum 2011 ).Vertebrates express up to 46 distinct chains that can form 28 types of collagen and assemble as fibrils or networks, ubiquitous to the hECM in most tissues.While multiple hECM proteins contain collagenous subdomains , many hECM components , including fibrinogen and fibronectin can bind directly to collagen.This is of particular significance when thinking of S. aureus since the pathogen expr esses pr oteins to bind colla gen, fibrinogen as well as a fibronectin (Fig. 1 A,B) (Bingham et al. 2008, Ganesh et al. 2008, O'Neill et al. 2008, Valotteau et al. 2017 ).
Vascular injury exposes underl ying colla gen, whic h binds to platelets with high affinity under shear conditions present in blood vessels ( ∼800 s −1 ) (Westerbacka et al. 2002 ).This interaction between collagen and platelets is additionally significant during the inflammatory stage of wound healing.Cytokines released in part due to the formation of fibrin clots will result in the influx of fibroblasts, epithelial, and endothelial cells.Fibroblasts can additionall y deposit colla gen at the wound site, contributing to wound closure .T he breakdown of collagen, likely by matrix metalloproteases, results in the synthesis of growth factors and fibroblast pr olifer ation, whic h further assist the processes of angiogenesis and re-epithelialization (Mathew-Steiner et al. 2021 ).Some of the common manifestations of collagen overproduction include fibrosis , cirrhosis , and scar tissue.For example, patients born with mutations in the c ystic f ibr osis (CF) tr ansmembr ane conductance r egulator ar e pr edisposed to the de v elopment of lung fibrosis.Studies with 3D-reconstructed CF airway stromal connectiv e tissue demonstr ate an incr eased pr oduction of collagen from CF-fibroblasts, in comparison to a non-CF control (Mazio et al. 2020 ).Similarl y, alv eolar tissue fr om CF patients was found to contain significantly higher le v els of colla gen as compar ed to a ge-matc hed healthy individuals, further underlining the central role of this hECM protein during disease (Ulrich et al. 2010 ).

Fibronectin
Fibronectin is a ubiquitously distributed, multidomain, 440 kDa gl ycopr otein that plays a vital role in the cellular de v elopment of v ertebr ates and is subsequentl y involv ed in numer ous homeostatic functions, including wound healing and blood clotting.Early r esearc h with this protein demonstrated that mouse embryonic de v elopment was se v er el y impair ed in the absence of fibronectin (Boucaut et al. 1984, George et al. 1993, Speziale et al. 2019 ).Fibronectin is found either as a soluble dimer in plasma (pFN1) or in its insoluble fibrillar form in tissue hECM (cFN1), where it plays various tissue-specific roles (Fig. 1 B).Both types of fibronectin can self-associate, bind to cellular integrin receptors (primarily α5 β1), and additionally to other hECM proteins, including but not limited to heparin, fibrin, collagen, tenascin, and fibrillin (Henderson et al. 2011, Spada et al. 2021 ).Cellular fibronectin is particularly well researched for its ability to carry out fibrillogenesis, a process that ultimately expands and provides architectural properties to the hECM.This is essential for the ability of cytokine, c hemokine, and gr owth factor diffusion into tissue en vironments , ther eby indir ectl y playing a major role in the host response to infection and disease (Spada et al. 2021 ).While adhesins of S. aureus are the best-characterized of the bacterial proteins that bind to fibronectin, both Gram-positive ( Streptococcus , Enterococcus , Clostridia , and Listeria species) and Gr am-negativ e ( Esc heric hia , Campylobacter , Salmonella and Borrelia ) bacterial pathogens have been shown to express proteins that can bind fibronectin (Speziale et al. 2019 ).

Laminin
Laminin composition dictates the structure of basement membranes, a specialized hECM that is required for physically connecting all intra-and intercellular tissue en vironments .While laminins are a large group of glycoproteins that can vary in size and structure, they are primarily composed of three disulfidelink ed polype ptides, namely the α, β, and γ chains (Fig. 1 B).While the N-terminus of the gl ycopr otein is thought to be responsible for binding to other hECM components, including heparin and collagen, the C-terminus ar guabl y performs the most important general function of binding to plasma membr ane-anc hor ed pr oteins of neighboring cells, thereby allowing communication between the intra-and extracellular compartments (Singh et al. 2012, Mckee et al. 2019 ).
Of note, the C-terminus of laminin is also where bacterial adhesins can bind, thereby potentially blocking essential intercellular biochemical signals from being transmitted, while utilizing laminin as a component of the biofilm matrix (Carneiro et al. 2004 ).Studies with mouse models of meningitis indicate that three major blood-brain barrier pathogens-Neisseria meningitidis , Haemophilus influenzae , and Streptococcus pneumoniae -can all manipulate the laminin binding receptor sites on brain endothelial cells, utilizing them as binding sites (Orihuela et al. 2009 ).These studies, as well as additional and similar investigations with P .aeruginosa , Streptococcus agalacticae , Bacillus anthracis , and S. aureus indicate a correlation between the ability to bind laminin and the potential for inv asiv eness of the pathogen (Carneiro et al. 2004, Tenenbaum et al. 2007, Wang et al. 2016, Paulsson et al. 2019 ).

Elastin
Soluble tropoelastin monomers of ∼60 kDa multimerize to form elastin fibers .T he monomer itself can extend up to eight times its own length (Uitto 1979, Swee et al. 1995, Baldock et al. 2011 ).As fibers, elastin is a highly hydrophobic molecule with a low elastic modulus that can resist acid and alkali attacks.As the name suggests, elastin is responsible for imparting elasticity to various cells, the most significant of these being the vascular tissue.Major blood vessels and aortas are composed of 25%-32% dry weight elastin (Uitto 1979, Ozsvar et al. 2021, Wang et al. 2021 ).As a consequence of its ubiquity in blood vessels, perturbations in elastin composition are associated with numerous cardiovascular conditions, including ather oscler osis, my ocar dial isc hemia, r eperfusion injury, and atrial fibrillation (Wang et al. 2021 ).The property to confer tissue elasticity is also of particular significance to the cells of the lung and female r epr oductiv e tr act with elastin being abundant in the uterus and placental tissue (Ozsvar et al. 2021 ).Among the pathogens with documented ability to bind elastin to its adv anta ge, P. aeruginosa is of particular note for expressing an elastase, pseudolysin, that can rapidly degrade the elastin present in lung tissue.Indeed, the significance of this singular interaction to the bacterium is evident in the fact that it is the most abundant extracellular virulence factor and protease that is expressed by clinicall y r ele v ant str ains of the pathogen (Hamdaoui et al. 1987 ).This elastase has also been demonstrated to play a significant role in degrading elastin fibers during bloodstream infections as well as corneal keratitis, further indicating its importance as a virulence factor, likely underlying some of the tissue pr efer ence observed with P. aeruginosa infections (Hamdaoui et al. 1987, Matsumoto 2004, Yang et al. 2015 ).
While binding to elastin has been observed with S. aureus , the consequences of this interaction to infection are currently undera ppr eciated (Downer et al. 2002 ).Inter estingl y, similar to colla gen, the concentrations and synthesis of elastin were also found to be higher in patients with cystic fibrosis and COPD, conditions that are often associated with biofilms of P. aeruginosa and S. aureus .Mycobacterium tuberculosis, the causative agent of tuberculosis, a de v astating pulmonary disease, expr esses thr ee major secreted antigens, namely Ag 85A, Ag 85B, and Ag 85C, collectiv el y known as the antigen 85 complex.In addition to playing significant roles during pathogenesis, these pr oteins wer e found to interact with tropoelastin and facilitate the binding of M. tuberculosis .Authors found that in the absence of tropoelastin binding ability, there was a significant reduction in the capacity of M. tuberculosis to invade cells (Kuo et al. 2013 ).These and similar studies provide further evidence of the central role that elastin can play during bacterial infection, warr anting further inv estigations of its r ole in S. aureus biofilm structure and pathogenesis.

Fibrinogen
Fibrinogen is a 340-kDa gl ycopr otein that polymerizes into fibrin and is essential for clotting and homeostasis.Fibrin was discovered as early as 1666 b y Mar celo Malpighi in his seminal report titled "De polypo cordis" (Forrester 1995 ).The soluble fibrinogen monomer is primarily synthesized in hepatocytes and is a ho-modimer consisting of 2A α, 2B β, and 2 γ polypeptides linked by 29 disulfide bonds to form hexameric complexes (Fig. 1 B).In healthy human subjects, soluble fibrinogen is present at 2-5 mg/ml but this concentration can go up to ∼7 mg/ml under acute inflammatory conditions .T he pol ymerization of fibrinogen to fibrin r equir es the plasma-associated pr otease, thr ombin to cleav e fibrinopeptides from the N-terminus of A α and B β polypeptide chains.Subsequently, the exposed "knobs" of these subunits are then inserted into "holes" in the β and γ chains of adjacent monomers .T his results in the formation of a "protofibril."These protofibrils will then a ggr egate and cr osslink to form fibrin cables that form the network of a blood clot.As described in pr e vious sections, S. aureus coagulases (Coa, vWbp) and ClfA can hijack this homeostatic process to form "thrombi" containing bacteria shielded by fibrin, a form of biofilm formation that is c har acteristic to S. aureus (Thomer et al. 2016 ).
The structure and stability of fibrin clots can depend on integral blood properties, including but not limited to the concentration of anticoa gulants, pH, temper atur e, blood flow, platelet, and red blood cell compositions.Lastly, perturbations in the structure and concentration of fibrinogen itself can alter the physical properties of fibrin and its subsequent effectiveness at restoring hemostasis .T hese changes can occur due to m ultiple pr e-disposing conditions, including but not limited to ischemic strok e, m y ocar dial infarction, aortic aneurysms, and thromboembolisms (Kattula et al. 2017 , Pieters andWolberg 2019 ).

Skin infections
Human skin forms the outermost barrier that protects the body against pathogens.Some of the innate properties of skin that prevent bacterial proliferation include low pH, sebaceous gland secr etions, antimicr obial peptides, and lipids.Normal flora can colonize e v ery major la yer of the skin, including the epidermis , dermis , subcutaneous , adipose , and muscle fascia.These populations often provide an additional barrier against pathogens by outcompeting foreign species.Staphylococcus epidermidis is a commensal on healthy skin and is the most pr ominent anta gonist for the development of S. aureus biofilms (Lunjani et al. 2019 ).
Specificall y, S. epidermidis secr etes a pr otease (Esp) that can reduce the ability of S. aureus biofilm formation.Indeed, nasal swabs obtained from patients with Esp-secreting S. epidermidis were associated with the absence of S. aureus, indicating that this commensal can activ el y pr e v ent S. aureus nasal colonization (Dubin et al. 2001, Vandecandelaere et al. 2014 ).Upon perturbation of the normal flora or when allo w ed to gain entry via abrasions or injections , S. aureus in v ariabl y causes de v astating skin infections that can lead to c hr onic biofilm-associated conditions, the most common of which are discussed below.

Atopic dermatitis
Atopic dermatitis is a c hr onic inflammatory skin disease commonly associated with S. aureus (39%-70%) and prominent in childr en (15%-20%) (P ark et al. 2013(P ark et al. , Totté et al. 2016 ).AD patients with large numbers of S. aureus often correlate with lo w er densities of the commensal, S. epidermidis .This pattern is also associated with se v er e cases of AD.Also of particular interest is the association of S. aureus biofilm-forming propensity with severe AD cases.Gonzalez et al. ( 2021 ) used 400 AD isolates of S. aureus to show that 62% had high to moderate biofilm-forming capacity.Additionally, although 68% of those that were co-colonized with S. epidermidis and S. aureus formed mixed species biofilms, se v er e AD was only associated with strong biofilm-forming isolates of S. aureus and not S. epidermidis .In another study performed with S. aureus isolates from patients with AD, Cho et al. ( 2001 ) demonstrate that strains expressing collagen-binding adhesin as well as fibr onectin-binding pr oteins wer e better able to adher e to str atum corneum isolated from AD patients as compared to healthy skin as well as bacteria that could not express adhesins required to bind to either collagen (Cna) or fibronectin (Fnbps).Two additional cell wall-associated proteins, ClfB and FnbpB, have specifically been implicated in this increased binding to AD stratum corneum.Additionally, Towell et al. ( 2021 ) elegantl y demonstr ate the direct binding of ClfB and FnbpB to corneodesmosin, the major protein found in corneocytes on the skin.Of note, the stratum corneum of AD patients contains markedly lo w er le v els of natur al moisturizing factor, which leads to an abnormal le v el of exposed corneodesmosin, likely allowing for S. aureus proteins to bind and potentiate biofilm formation.These studies indicate the significance of hECM-binding proteins as well as biofilm formation, to the pathogenesis of S. aureus AD infections.
Chronic Wounds .A wound that is physiologically impaired due to a disruption in the wound healing cycle, often resulting in a prolonged inflammatory phase, is defined as a c hr onic wound.These include diabetic ulcers, venous ulcers, and pr essur e ulcers (Bjarnsholt et al. 2008(Bjarnsholt et al. , P er cival et al. 2012 ) ). Ther e ar e four main stages of wound healing namely hemostasis, inflammation, prolifer ation, and r emodeling.Clotting and platelet plug formation are necessary to achieve hemostasis.Specifically, platelets bind to collagen exposed in the endothelial wall as well as fibrinogen and fibrin, resulting in the development of a plug (Wilkinson and Hardman 2020 ).This process can be interrupted by multiple S. aureus proteins, as described in previous sections .T he inflammatory phase of wound healing begins with the recruitment of neutrophils as a response to platelet signals.Neutrophils use phagocytosis, degranulation, and NETosis to eliminate the infection, but often fail (Schierle et al. 2009, Roy et al. 2014, Jeffery Marano et al. 2015, Wilkinson and Hardman 2020 ).This is in part due to a variety of exotoxins and immune evasion factors expressed by the pathogen.Staphylococcus aureus biofilms in particular are recalcitrant to neutrophil defense mechanisms, expressing leucocidins that induce the release of NETs, which fail to clear the biofilm (Bhattacharya et al. 2018(Bhattacharya et al. , 2020 ) ). Simultaneous to the neutrophil r esponse, monocytes ar e r ecruited to the site of infection by hECM pr oteins, including colla gen, fibr onectin, serum complement factors, and elastin.Monocytes will differentiate into macr opha ges and assist the r emov al of neutrophil and bacterial debris generated in the process .T he last cells to be recruited are lymphocytes that respond to similar cues, including complement, degr aded imm unoglobulin, and cytokines.Resolution of the inflammatory phase involves the replenishment of some hECM components such as collagen, by resident fibroblasts (Wilkinson and Hardman 2020 ).Pr olifer ation, also known as gr anulation tissue formation, includes the de v elopment of ne w connectiv e tissue with r a pid and extensiv e r eplenishment of hECM pr oteins c har acterized by the replacement of the fibronectin matrix by a stronger, collagen I and collagen III-rich scaffold (Wilkinson andHardman 2020 , Mathew-Steiner et al. 2021 ).
Multiple studies demonstrate that S. aureus is one of the most commonly isolated pathogens in both acute ( ∼60%) and chronic wound infections ( ∼65%) (Gjødsbøl et al. 2006, Kirketerp-Møller et al. 2008, Fazli et al. 2009, P er cival et al. 2010 ).Other pathogens fr equentl y isolated include P. aeruginosa and Enterococcus species, both prominent biofilm-forming bacteria.Indeed, a growing body of liter atur e demonstr ating the pr esence of biofilms in these wounds indicates a need to consider biofilm de v elopment as one of the hallmarks of c hr onic wound infection by bacterial pathogens (Gjødsbøl et al. 2006, Bjarnsholt et al. 2008, Kirketerp-Møller et al. 2008, Fazli et al. 2009 ).Studies with both acute and c hr onic models of wound infections indicate that S. aureus biofilms delay wound healing by causing inflammation and delayed r e-epithelialization (Sc hierle et al. 2009, Chaney et al. 2017 ).A number of infection-related phenotypes are associated with r esponses gener ated thr ough the host extr acellular matrix.Of note, c hr onic wounds infected with S. aureus cause the activation of tissue-and neutr ophil-deriv ed metallopr oteases, whic h br eakdown collagen.Seminal studies in the field of biofilm-associated wound infections describe a role for matrix metalloproteinase-2 in reducing the ratio of collagen1/collagen 3 present at the wound edge, resulting in a loss of skin tensile strength (Roy et al. 2020 ).Results from this study support the recurrence of S. aureus biofilms in these poorly re-epithelialized wounds.Other gr oups hav e also shown a corr elation between the pr esence of Staphylococci in wounds and an increase in epithelial gap size (Xu et al. 2021 ).

Lung infections
While the cellular composition of the human lung is incr edibl y complex and yet to be fully understood, it can lar gel y be divided into the par enc hyma and the interstitial space.A c har acteristic feature of the lung epithelial barrier is the presence of a ciliated pseudostratified columnar epithelium coated with mucus that is produced by goblet cells found throughout the airwa y.T his is arguably the most important defense against those pathogens that can gain entry to the r espir atory system through the nose and mouth (Burgstaller et al. 2017 , Hall-Stoodley andMcCoy 2022 ).Pr oteomic studies r e v eal that the pulmonary interstitial space can contain at least 150 hECM components, with perturbations in the le v els of these constituents often resulting in deleterious effects such as heightened disease progression (Shimbori et al. 2013, Eurlings et al. 2014 ).For example, r esearc hers hav e found that the r egener ation of lung matrix components by resident fibroblasts plays an important role in maintaining homeostasis after injury or infection (Sivakumar et al. 2012, Shimbori et al. 2013 ).
Regardless of location, collagen is the most abundant protein in the lung.Since the lung is a diverse microenvironment, the composition of hECM as well as collagen subtypes observed differs depending upon whether tensile strength or elasticity take functional pr efer ence (Bur gstaller et al. 2017 , Hall-Stoodley andMcCoy 2022 ).For example, the alveoli, responsible for gas exchange, are provided with an elastic hECM matrix composed mainly of collagen I and III as well as elastin, which allows this environment to maintain non-linear str ess-str ain behaviors that ar e c har acteristic of connective tissue.Another important hECM component in the alveolus is the negatively charged proteoglycan heparin sulfate, whic h inter acts with numerous bioactive compounds and is often shed into the alveolar space for up to 3 weeks post-insult.This can result in detrimental effects to the host, including inflammation and decreased barrier function (Haeger et al. 2018, LaRivière et al. 2020 ).T he o v er all mec hanical integrity of the lung is lar gel y pr ovided by the fibrillar, less elastic colla gen types I, II, III, V, and IX.In addition to collagen and elastin, lung hECM also consists of gl ycosaminogl ycans, fibr onectin, laminin, heparin sulfate, and pr oteogl ycans suc h as decorin and bigl ycan among others.
Although the structural complexity of the lung impedes the ease of demonstrating biofilm-associated infections in this envir onment, r ecent adv ances in confocal microscopy and 16s RNA sequencing have provided clear evidence of the presence of these communities .For example , Kolpen et al. ( 2022 ) collected sputum samples from 43 patients (16 pneumonia, 13 COPD, and 14 CF) and sho w ed that biofilm communities of > 100 um 3 could be observed in 40 of these samples .T hese studies pro vide evidence for the presence of biofilms in both acute and c hr onic airway infections.Some k e y examples of these biofilm-associated infections ar e pr ovided her e.
Pneumonia.Bacteria and viruses are common causes of infection in the lung.This pneumonia is often c har acterized by inflammation of the lung tissue and accumulation of fluid or pus in alveoli.Two of the most common types of pneumonia associated with S. aureus infections include hospital-a cquired p neumonia and ventilator-associated pneumonia (V AP).V AP is caused in patients that r equir e mec hanical v entilation following tr ac heal intubation, often utilized as a method to alleviate breathing distr ess fr om pr e vious conditions suc h as emphysema, heart failur e, or se v er e tr auma (Bec ker and K err 1960 , P arker and Prince 2012 , Pickens and Wunderink 2022 ).These infections are most common in patients with compromised immunity such as infants ( < 5 yrs) and elderly patients ( > 65 yrs) as well as patients undergoing prolonged treatment ( Risk Factors for Pneumonia | CDC 2022 ; Parker and Prince 2012 ).Colonization of the nasal epithelium is a pr er equisite to the de v elopment of lung infection.Two surface adhesins, namely clumping factor B (ClfB) and collagen-binding adhesin (Cna) have been found to be associated with efficient colonization.While ClfB is r equir ed for attac hment to cytoker atin in the nasal epithelium, Cna was found to be important in bacterial attac hment to colla gen I and IV as well as laminin, prominent in the basement membrane tissue .T he expression of Cna is known to be a predisposing factor for the ability of S. aureus to cause necrotizing pneumonia (De Bentzmann et al. 2004 ).It is likely that since S. aureus colonizes the anterior nares of ∼30% of the population, pneumonia can arise as a secondary infection, caused by multiple primary insults.Staphylococcus aureus is commonly acquired as a secondary, se v er e infection following influenza.Recent studies by Langouët -Astrié et al. ( 2022 ) demonstrate that the damage caused to lung tissue, specifically the alveolar lining, following an influenza infection, results in excessi ve shed ding of he parin sulfate, a prominent pulmonary glucosaminoglycan.This causes a significant increase in the expression of cytotoxins by S. aureus, often resulting in fatality due to secondary bacterial infection (Teng et al. 2019 ).Inter estingl y, r esearc h with mixed biofilms of S. aureus and S. pneumoniae , another prolific cause of community-acquired pneumonia, demonstrates that when mice colonized with both bacteria are challenged with a secondary influenza virus infection, S. pneumonia biofilms can disperse, while pr e v enting the dispersal and ther efor e the tr ansition to inv asiv e disease of S. aureus (Reddinger et al. 2018 ).
Although m uc h r emains to be understood about the pathogenicity of biofilms in the lung, the de v elopment of tools r equir ed to demonstr ate the pr esence of these comm unities during pneumonia infection is r a pidl y gr owing.Baid ya et al. ( 2021 ) anal yzed br onc hoalv eolar lav a ge and deep aspir ate fr om 70 patients with VAP to show that biofilms could be observed in 56.3% of these samples.Additionally, studies by Hook et al. make dir ect confocal observ ations of USA300 S. aureus labeled with gr een fluor escent pr otein and sho w that follo wing inhalation of these bacteria by mice, S. aureus tr av els to the lungs, where they form micr oa ggr egates in > 50% of the alveoli observed.These comm unities wer e found to be stable and could not be flushed out with buffers .Furthermore , these microaggregates express cytotoxins, caused inflammation and localized lung injury while being impermeable to antibiotic-sized solutes (Hook et al. 2018 ).Together, these results emphasize a need for the consideration of biofilms as a common phenotype in bacterial pneumonia.
Cystic Fibrosis.A monogenic r ecessiv e m utation in the c ystic f ibrosis t ransmembrane r eceptor (CFTR) causes impaired mucociliary clearance and increased susceptibility to numerous pathogens, including but not limited to P. aeruginosa, S. aureus, Haemophilus influenzae, Burkholderia cepacia , and Mycobacterium abscesses (Harrison 2007 , Hall-Stoodley andMcCoy 2022 ).The CFTR is responsible for the transport of chloride and bicarbonate across the epithelial lining.This m utation, ther efor e, has widespr ead effects, including a disruption of pancreatic and gastrointestinal function, in addition to its most prominent manifestations in the lung (Wallis 1997 , Cohen andPrince 2012 ).The most common consequence of CFTR mutations is the excessive uptake of water by epithelial cells, which leads to a concomitant depletion of fluidity on the airway surface fluid and thickening of mucus.Mucociliary clear ance is hamper ed, allowing for pathogens to be retained and infections to de v elop with m uc h gr eater ease than they would in a healthy lung (Gilligan 1991, Hauser 2009, Zemanick and Hoffman 2016 ).The lung of a CF patient is a uniquely harsh environment for the growth of microbes.Increased acidity, o xidati ve stress, hyperinflammation, and competition between multiple microbial species result in the selection of microbial sub-populations that adapt to withstand these conditions .T his includes the development of mucoidy, small colony variants, and biofilms (Pietruczuk-Padzik et al. 2010, Høiby et al. 2017 ).
As a result of numerous insults to the lung, tissue scarring or fibrosis causes difficulty in breathing.The process of fibrosis in and of itself is defined by the ov er pr oduction of hECM (Henderson et al. 2020 ).Staphylococcus aureus is one of the first bacteria detected in infants and c hildr en with CF (Harrison 2007 ).Recent patient registry information from Europe and the USA shows that 60%-80% of CF patients under the age of 20 are colonized with S. aureus (ECFS Patient Registry Annual Data Report 2020 , Patient Registry | Cystic Fibrosis Foundation 2021 ).As a common resident of the anterior nares, S. aureus finds easy access to the respiratory tract of this imm unocompr omised population.Additionall y, with fr equent hospital sta ys , the introduction of S. aureus biofilms via biotic surfaces such as syringes , catheters , and endotr ac heal tubes as well as through surgical procedures and contact with healthcare workers is common (Saiman et al. 2014, Savant et al. 2014, Bell et al. 2018 ).
Patients with CF develop hyperinflammation months after birth.In combination with repeated microbial infections, the cycle of inflammation and tissue destruction per petuates ov er their lifetimes (Cantin et al. 2015 ).Neutrophil-induced inflammation is a pathophysiological hallmark of CF disease and results in the release of numer ous pr oteol ytic factors, including matrix metallopr oteases, Cathepsin G, and neutr ophil elastase that destr oy the connective barrier of the lung, reducing the capacity of tissue to expand and contract while breathing.Interestingly, Cathepsin G can effectiv el y disrupt S. aureus biofilm i n vitro.Whether this activity can clear biofilms in the lung environment, ho w ever, remains to be investigated (Kavanaugh et al. 2021 ).
Cr oss-sectional bioc hemical anal yses performed on br onc hioalv eolar lav a ge fluid fr om c hildr en with CF describe an extensiv e r emodeling of the lung hECM with a dr amatic incr ease in the le v els of elastin, colla gen, and gl ycosaminogl ycans.Earl y work in the field describes an increased concentration of collagen-deri ved pe ptides and elastin degradation products (desmosins) measured in urine collected from CF patients, when compared to healthy individuals (Stone et al. 1995 ).Since then, studies with CF sputum samples show significantl y ele v ated le v els of collagenase, specificall y deriv ed fr om neutr ophils (Po w er et al. 1994). Recentl y, Pinezic h et al. ( 2022 ) conducted an elegant mass spectrometric analysis of explanted lungs from end-stage CF patients in comparison to non-CF transplant donors to c har acterize the major changes that occur in the matrix of these tissues .T heir results sho w ed that there is a significant degree of degradation in the extr acellular matrix structur e and composition in CF patients, when compared to non-injured donors.Similar pathology is observed in a less common form of fibr osis, namel y idiopathic pulmonary fibr osis.Incr eased deposition of collagen is often reported, with decline in neutrophil function and a concomitant prevalence of S. aureus infections (Warheit-Niemi et al. 2020 ).Together, these studies indicate that hECM disbalance occurs and significantly contributes to the pathology during CF.As a majority of the research in the field of CF focuses on P. aeruginosa , the most common biofilm-forming pathogen infecting adults, m uc h r emains to be understood about the consequence of these hECM perturbations to the de v elopment of S. aureus biofilm infections, pr e v alent in c hildr en with CF.

De vice-rela ted infections
Hospital-acquired infections often arise from contaminated medical and prosthetic devices .T he most significant number of these cases are caused by S. aureus and S .epidermidis (Stoodley et al. 2013 ).Due to its ability to r a pidl y acquir e toler ance and r esistance to numerous antibiotics, S. aureus is a particularl y pr oblematic cause of foreign body-associated infections.Devices most commonly associated with S. aureus infections include catheters (centr al v enous and urinary), heart v alv es , pacemakers , endotr ac heal tubes , contact lenses , and periprosthetic devices (Moran et al. 2010, Peel et al. 2011 ).The most likely mechanism for entry of the pathogen and colonization on these devices is during surgical procedures or during the insertion of the device in patients that are colonized with S. aureus (McConoughey et al. 2014 ).Infection in the device can often spread to neighboring soft tissue and bone and may result in sepsis, especially when the device is inserted into the heart or circulatory system (Manne et al. 2012, Osmon et al. 2013 ).
Once an implant has been inserted into the body, it is immediatel y cov er ed in plasma pr oteins, including vWf, fibrinogen, fibr onectin, and vitr onectin.This pr ocess is crucial in allowing the implant to be accepted as "self" by the host immune system.Unfortunatel y, this coating pr ovides for a "conditioned" surface that is perfect for bacteria to attac h to, pr olifer ate on, and form biofilms .T his en vir onment is ther efor e particularl y favor able for biofilm formation by S. aureus , owing to its ability to bind each of these hECM components.Pestrak et al. ( 2020 ) demonstrate the nature of this double-edged sw or d in that while synovial fluid surr ounding peripr osthetic joints pr e v ents bacterial attac hment to the implant surface, fibrin and fibrinogen present in the fluid can promote the formation of bacterial a ggr egate biofilms that survive in synovial fluid to potentially cause secondary infections.As described by these studies, the fibr onectin-binding pr otein A (FnbpA) is of particular r ele v ance to implant-associated infections.FnbpA is a surface-associated protein that was found to be encoded in ∼99% of strains isolated from 191 implant-associated infections (Arciola et al. 2005 ).In a rodent model of implant infection, Gries et al. ( 2020 ) demonstrate that the expression of FnbpA allows for S. aureus to form structur ed comm unities on Tefloncoated intr av enous catheters and pr e v ents bacterial clear ance by macr opha ges.Additionall y, the authors describe a role for this protein in allowing for S. aureus to spread to neighboring soft and bone tissue.
The fibrinogen-binding ability of S. aureus can change the course of a bacterial infection to the detriment of the host.While this is discussed pr edominantl y in the context of the bloodstream, it is extremely relevant to device-related infections by the pathogen.Seminal studies by Vanassche et al. utilized dabigatran, a pharmacological inhibitor of staphylothrombin formation, to show that treatment with this drug could significantly reduce the capacity of S. aureus to form biofilms on jugular vein catheters in a mouse model of infection.This was accompanied with a reduction in fibrin deposition and increased efficacy of vancomycin tr eatment (Vanassc he et al. 2013 ).The conditioning of implant material is ther efor e a critical consider ation that is often absent from in vitro analyses aiming to understand the pathogenesis of S. aureus biofilms during de vice-r elated infections.

Endocarditis
Endocarditis is the inflammation of the endocardium, the inner squamous epithelial lining of the heart c hambers, whic h connects the heart to the circulatory system through blood vessels.An infection of this lining termed infective endocarditis (IE), most often pr ov es fatal.As earl y as 1973, Durac k et al. ( 1973 ) ) used a rabbit model of endocarditis to demonstrate that the endocardium is recalcitrant to bacterial colonization.The de v elopment of an infection in this tissue ther efor e r equir es perturbations in the valvular surface that create a favorable environment for such infections .T his often occurs as a result of injury, surgical intervention, or the introduction of foreign bodies, including pacemakers and heart v alv es.Other major factors that facilitate the de v elopment of endocardial biofilms or "vegetations" include the de v elopment of bacteremia caused by a primary infectious focal point such as an infected catheter or skin wound (Mylonakis andCalderwood 2001 , Werdan et al. 2014 ).As the causative agent of 15%-40% of IE in Europe and the USA, mortality rates for cases caused by S. aureus are 20%-30% (Asgeirsson et al. 2018 ).
An incr easingl y pr oblematic cause of IE is the intr av enous use of drugs that provide direct access for S. aureus colonizing the skin or unhygienic abiotic surfaces into the bloodstream (Wang et al. 2023 ).One such case reported by Rali et al. ( 2019 ) at the University of Kansas Medical Center provides a compr ehensiv e example for the transmission, manifestation, and treatment of IE.In this study, a 56-yr-old male was pr e viousl y hospitalized due to the de v elopment of c hr onic purulent bilater al cellulitis and osteomyelitis caused by S. aureus .These infections were traced back to repeated intr av enous injections in the lo w er extremities.As a testament to the difficulty in pr e v enting biofilm-associated infections, especially IE, upon admission, while a t rans t horacic e chocardiogram (TTE) was negative for IE, S. aureus was positiv el y cultur ed fr om the blood and urine of the patient.Soon after presentation, this patient was admitted to the intensive care unit, where a subsequent TTE demonstrated the presence of a ∼0.5 cm vegetation.This was accompanied by vegetations in the mitral, tricuspid, and aortal v alv es, whic h pr ogr essed to the lungs and br ain, pr oving fatal.This is one of many cases where the short time frame of biofilm de v elopment combined with the inability to make early detections in heart tissue, leads to de v astating consequences, further emphasizing the need to acknowledge biofilms as a serious, unique infectious disease hurdle (Guler et al. 2013, Wang et al. 2023 ).The modified Duke's criteria, widely used by clinicians to diagnose endocarditis are only useful once blood cultures turn positive for the presence of a particular pathogen, thus catching the infection long after biofilm formation would hav e occurr ed (Li et al. 2000 ).
When considering the extracellular matrix of the heart, it is important to note that the r egener ativ e ca pacity of this organ is poor.This means that injury to cardiac tissue and muscle results in very low levels of replacement with healthy cells, as is the case during my ocar dial infar ction (Bergmann andJ o vinge 2014 , Silva et al. 2020 ).The hECM in and around the endocardium is rich in collagens I, III, and IV, with types I and III being responsible for maintaining the mechanical strength of the compartments.Additionally, the major constituents of the hECM include fibronectin, elastin, laminin, proteoglycans, and glycosaminoglycans (Silva et al. 2020 ).In part due to the challenges of studying heart infections, especially in vitro , there is relatively little known about the pathogenesis of S. aureus -induced IE.In a well-designed attempt to impr ov e some of these techniques, Liesenborghs et al. ( 2019 ) describe the de v elopment of a murine model for the study of endocarditis.By comparing the effects of inflammation and v alvular dama ge, the authors ar e able to ima ge the infiltr ation of granulocytes and platelets to the site of S. aureus biofilm formation.Using these methods, the authors were able to provide in vivo evidence for some k e y events that occur during IE with S. aureus .They show that v alv e dama ge and inflammation can cause differ ential imm une r esponses to ensue.While both processes involved vWf, inflammation resulted in a predominantly platelet-driv en mec hanism of tr a pping the bacteria, independent of bacterial cell wall-anc hor ed pr oteins .Con v ersel y, v alvular damage was accompanied by S. aureus binding dir ectl y to vWF (via the vWbp coagulase) and fibrinogen/fibrin via ClfA, which deposited onto the damage valve .T his is one of very few examples of an in vivo exploration of the pathogenesis of S. aureus IE and brings up an important and underestimated aspect of S. aureus infection, namel y the r ole of platelets (Niemann et al. 2004, Ali et al. 2017 ).A significant impediment in the ability of a bacterium to cause bacter emia and ther efor e IE is the r equir ement to attac h to v alvular surfaces in the presence of extreme shear stress generated by the flow of blood.While most IE pathogens bind to platelets in order to gain entry , S. aureus uses vWbp to mimic the mechanisms used by platelets binding to vWf that are exposed on the endothelial lining of damaged valves (Claes et al. 2014 ).

Current therapeutics and future prospects
One mechanism for preventing the formation of an S. aureus biofilm in vivo is to block the activity of adhesins that bind hECM components.Curr entl y, ther e is no a ppr ov ed v accine av ailable to pr e v ent S. aureus infections, in part due to its ability to use multiple virulence strategies against the host.Numerous studies have attempted to de v elop v accines that target the hECM binding domains of surface proteins, to varying degrees of success.An additional hurdle is the selection of a vaccine target that is conserved among strains of S. aureus .
Mazmanian and Schneewind demonstrated that Sortase A is essential for the cell wall anchoring of 23 surface-associated adhesins (Mazmanian et al. 1999 ).Further studies show that srtA sortase mutants of S. aureus cannot colonize the nasal passages or cause abscess formation in a well-established murine model of bacterial sepsis (Mazmanian et al. 2000, Cheng et al. 2009 ).Owing to its importance during infection, multiple efforts have been made to de v elop a m ultiv alent v accine that tar gets m ultiple sortase-anc hor ed pr oteins.Stranger-J ones et al. ( 2006 ) performed one of the most compr ehensiv e studies to this effect by generating antibodies against 19 highly conserved sortase-anchored proteins.Protection studies with mice sho w ed that the most protective antibodies were generated against IsdA, IsdB, SdrD, and SdrE.The V710 vaccine was developed against IsdB and reached phase 3 clinical trials performed on patients undergoing cardiothoracic surgery.Administering V710 was not only ineffective at preventing infection but caused a higher degree of mortality in patients who de v eloped S. aureus infections.While the reasons for this safety failur e wer e not clear, it pr ovides an insightful example of the complexity in pathogenesis and the need for the de v elopment of tissue-specific interventions (Fowler et al. 2013 ).There is an ongoing effort to discover inhibitors of Sortase A activity.The disadv anta ge of these compounds to clinical use, ho w e v er, is that they are often not bactericidal and ar e ther efor e unlikel y to er adicate the infection (Sc hnee wind and Missiakas 2019 ).
Coating peripr osthetic de vices with anti-biofilm compounds is a burgeoning field of research for the prevention of the attachment phase of biofilm formation.Most of these efforts have focused on antimicrobial compounds and taken adv anta ge of the hydr ophobic natur e of bacteria by cr eating hydr ophilic and ther efor e r epellant implant surfaces (Bussc her and v an der Mei 2012, McConoughey et al. 2014 ).While these methods are useful in the pr e v ention of bacterial adhesion to surfaces, they do not alter the ability of bacteria to form a ggr egates with plasma-deriv ed matrices.Consequentl y, anti-fibrinol ytic a gents suc h as dabigatran, plasmin, and warfarin are clinically utilized and can break up S. aureus a ggr egates that form with fibrinogen, fibrin, and fibronectin.Hogan et al. ( 2018 ) used a combination of antimicrobial and anti-fibrinol ytic a gents to demonstr ate a significant r eduction of bacterial growth in a rat model of catheter-associated biofilms.Similarly, Kwiecinski et al. ( 2016 ) show that coating polyester-based coverslips with tissue plasminogen activ ator, r equir ed for fibrin cleav a ge, was effectiv e at r educing biofilm formation in vivo and improving the efficacy of clinically prescribed anti-S.aureus drugs.These studies demonstrate the need to target more than one biofilm properties in order to eradicate the infection.
A major issue with the use of agents that target bacterial-hECM inter actions, is the r equir ement for localized deliv ery.While these methods are effective for implant-associated biofilms, the administration of antithrombotic agents can cause disastrous effects if deliv er ed systemicall y.Additionall y, the localized delivery of antibiotics would be more effective than systemic delivery routes (Howlin et al. 2015 ).Lastly, the formation of IE vegetation biofilms occurs similarly to blood clots and r equir es the activation of platelets.While a combination of anti-platelet and anti-thr ombotic a gents has successfull y been utilized to show incr eased surviv al of r ats in an in vivo model of IE, results with clinical trials show mixed results, with one study showing increased potential for bleeding when aspirin was used as an anti-platelet compound (Chan et al. 2003, Anavekar et al. 2007, Veloso et al. 2015 ).T hese studies , while unsuccessful to v arying degr ees, pr ovide additional pieces to the puzzle of treating a biofilm infection and clearly define the essential role of ECM components to the survival of S. aureus biofilms in vivo.

Conclusions
The effectiv e tr eatment of S. aureus -associated infections r equir es a gr eater a ppr eciation of biofilms as the pr edominant bacterial lifestyle (Costerton et al. 1978, Costerton et al. 1999, Donlan and Costerton 2002, Hall-Stoodley et al. 2004, Høiby et al. 2011 ).Con-centrations of antibiotics used against planktonic S. aureus populations will ther efor e not be effective against biofilms and can lead to the de v elopment of extr emel y r ecalcitr ant subpopulations, per petuating the pr oblems of antibiotic toler ance and r esistance (Stewart andWilliam Costerton 2001 , Donlan andCosterton 2002 ).An additional hurdle that comes with the study and treatment of S. aureus biofilms, lies in the pathogenic versatility of the bacterium, expr essing a div erse combination of virulence factors, the permutations, and combinations of which are highly complex and under tight temporal control.Therapeutics to eradicate this pathogen will ther efor e r equir e a m ulti-tar get a ppr oac h (Bhattacharya et al. 2015 ).Unlike most pathogens, S. aureus is uniquely designed to thrive in the human body during both colonization and infection.This is in part due to the expression of one or more surface or secreted bacterial components (Fig. 1 A) with specific domains that form strong physical interactions with host extracellular ligands (Fig. 1 B) (Foster 1998 , Sc hnee wind andMissiakas 2019 ).The ability to eradicate S. aureus biofilms is ther efor e intricately linked to the framework of the host itself and requires a deeper understanding of the biochemistry and functionality of various host proteins , carbohydrates , and lipids that actively interact with S. aureus to potentiate the formation and/or survival of biofilms in vivo ( Deadly Staph Infections Still Threaten the USA | CDC Online Ne wsr oom | CDC 2019 ;Bhattac harya et al. 2015 ).This r e vie w pr ovides a compr ehensiv e discussion of human extracellular proteins that are particularly relevant to the pathogenesis of S. aureus.As e vident fr om the summary provided in Fig. 2 , the complexity of these interactions can be highly dynamic and dependent on the tissue-specific abundance of each host protein.Dispersion of biofilm communities and the transition to more inv asiv e bacterial virulence mec hanisms ar e additional considerations that need to be recognized in order for r esearc h on S. aureus pathogenesis to r eac h gr eater clinical r ele v ance.

Figure 1 .
Figure 1.Proteins associated with S. aureus biofilms in vivo.Gr a phical r epr esentation pr oviding a comparison of the functional motifs of surface and secr eted pr oteins with significant contributions to S. aureus biofilm formation, as discussed in this r e vie w article.Symbols r eflect pr otein size (pr ovided in a ppr o ximate kDa) (A).De piction of the structur es of 5 major hECM pr oteins that can be incor por ated into the S. aureus biofilm EPS, as discussed in this r e vie w.Staphylococcus aureus surface and secr eted pr oteins that ar e known to use eac h of these hECM pr oteins as ligands ar e shown in eac h panel.Pr oteins fr om A that ar e not depicted in B ar e known to hav e r oles in inter-bacterial a ggr egation (B).Ima ge was cr eated with BioRender.com.

Figure 2 .
Figure 2. Human extracellular matrix differentially contributes to S. aureus biofilm infections.Dia gr ammatic summary of some k e y examples of biofilm-associated S. aureus infections as discussed in this r e vie w.Colors r epr esent the r elativ e, known contributions of v arious extr acellular matrix-associated proteins (see legend) to S. aureus infections .T he distribution of these colors is based on the known contributions of each hECM component during S. aureus biofilm-associated infections, to the extent that has been discussed in this manuscript.Distribution of colors in each chart is not intended to reflect a quantitative measurement.Image was created with BioRender.com