Review

Staphopains in Staphylococcus aureus bacteremia: Virulence activitiesrelated to the onset of septic shock, coagulation disorders,and infectious endocarditis

Takahisa Imamura n

Department of Molecular Pathology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan

a r t i c l e i n f o

Article history:Received 10 March 2014Received in revised form27 March 2014Accepted 1 April 2014Available online 2 May 2014

Keywords:Staphylococcus aureusStaphopainKininFibrinogenCollagen

a b s t r a c t

Background: Staphylococcus aureus is an important oral bacterium that enters the bloodstream followingdental procedures, causing bacteremia. Infectious endocarditis occurs due to the invasion of blood-bornepathogens into the endocardium. S. aureus is the most frequently isolated bacterium in Gram-positive sepsis,and shock and coagulation disorders are common and potentially fatal consequences of sepsis. Staphopainsare the most abundant proteases among extracellular proteolytic enzymes produced by staphylococci, andtheir virulence activities related to the pathophysiology of S. aureus bacteremia have been elucidated.Highlight: Staphopain A (ScpA)—not staphopain B (SspB)—releases bradykinin, and the two staphopainssynergistically release a novel kinin, Leu-Met-Lys-bradykinin, directly from human plasma kininogens. Thesekinins cause vascular leakage. ScpA, similar to the two kinins, lowers blood pressure in guinea pigs in abradykinin B2-receptor-dependent manner when administered intra-arterially, and produces septic shocksymptoms in them. Kinin generation from human plasma by ScpA is enhanced in the presence of SspB,strongly suggesting a shock induction by the bacterial proteases. Staphopains, SspB being threefold morepotent than ScpA, truncate fibrinogen by preferentially cleaving this coagulation factor at the C-terminalregion of the Aα chain, which results in loss of fibrinogen clottability, thereby causing bleeding tendency.Staphopains showed a degradation activity for type I collagen similar to that observed in ScpA-mediated skindestruction, indicating that they participate in S. aureus-induced endocardium destruction.Conclusion: Staphopains are potent virulence factors and are potentially involved in the onset of septic shock,coagulation disorders, and infectious endocarditis that occurs in S. aureus bacteremia.

& 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 822. Properties of staphopains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823. Virulence activities of staphopains in bacteremia/sepsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

3.1. Induction of vascular leakage and hypovolemic hypotension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 823.2. Induction of coagulation disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833.3. Collagen degradation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84Ethical approval. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84Conflict of interest. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85

Contents lists available at ScienceDirect

journal homepage: www.elsevier.com/locate/job

Journal of Oral Biosciences

http://dx.doi.org/10.1016/j.job.2014.04.0011349-0079/& 2014 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

Abbreviations: APTT, activated partial thromboplastin time; BK, bradykinin; DIC, disseminated intravascular coagulation; HK, high molecular weight kininogen; ScpA,staphopain A; SspB, staphopain B; VL, vascular leakage

n Tel.: þ81 96 373 5306; fax: þ81 96 373 5308.E-mail address: taka@kumamoto-u.ac.jp

Journal of Oral Biosciences 56 (2014) 81–85

1. Introduction

Staphylococcus aureus is an important oral bacterium [1] that isisolated in approximately 20% of specimens obtained from the oralcavity and facial area [2]. S. aureus was isolated from 12%of supragingival plaque samples in healthy dentate adults [3].Approximately 28% of tongue swabs obtained from children, whohad no diseases other than dental disease, were S. aureus-positive[4]. The isolation rate from oral cavity increases to more than 30%in periodontal disease patients [5]. Some oral infections are caused, atleast in part, by S. aureus, such as angular cheilitis [6], parotitis [7],and staphylococcal mucositis [8]. Bacteria in the oral cavity enter thebloodstream in high numbers after dental procedures, causingbacteremia [9,10]. The bacteremia rates were 60% (range: 18–85)following tooth extraction, 88% (range: 60–90) after periodontalsurgery, and 40% (range: 7–50) subsequent to brushing teeth orirrigation [11]. Periodontal probing can cause bacteremia in patientswith periodontitis [12]. The risk of bacteremia increases in patientswith periodontal disease due to the proximity of the organisms tothe bloodstream via ulcerations within gingival pockets or thegingival crevice epithelium [13]. S. aureus bacteremia is associatedwith various general diseases, including infective endocarditis andsepsis [10,14,15]. S. aureus is the most frequently isolated pathogen inGram-positive sepsis, which is as common as Gram-negative sepsis[16–18]. Shock and coagulation disorders are the common andpotentially fatal consequences of sepsis.

S. aureus infection-mediated diseases are caused by the viru-lence factors produced by this pathogen. In addition to enterotox-ins and haemolysins, S. aureus secretes several extracellularproteases, including the serine protease V8 protease, the metallo-protease aureolysin, and the cysteine proteases staphopainA (ScpA) and staphopain B (SspB). These enzymes are consideredas putative virulence factors [19,20]. Staphopains are the mostabundant proteases among extracellular proteolytic enzymes ofstaphylococci [19]. This review focuses on the pathophysiologyof S. aureus bacteremia and staphopain virulence activities asso-ciated with septic shock, coagulation disorders, and infectiousendocarditis.

2. Properties of staphopains

ScpA is encoded by scpA, and the mature form has a molecularmass of 20 kDa and is composed of 174 amino acid residues [21].SspB is encoded by sspB, and the mature form encompasses 174 or168 amino acid residues sharing 46% identity with ScpA [21]. Inspite of this similarity, the pI of ScpA is alkaline (pI 9.0), whereasthe pI of SspB is acidic (pI 4.64). Both ScpA and SspB are processedfrom large proenzymes containing 363 and 357 residues, respec-tively [19]. The tertiary structure of ScpA resembles the overall foldof papain with two interacting domains. As in the papain-likeproteases, the Cys24 and His120 (His121 in SspB) residues form thecatalytic dyad of ScpA, with Gln18 being involved in forming the“oxyanion hole,” and Asn141 interacting with the imidazolium ringof the catalytic His. SspB has a very similar fold and a topologyidentical to that of the ScpA active-site cleft.

ScpA has very broad substrate specificity, with no preferencefor a particular amino acid at any of the several positions on eitherside of the scissile peptide bond [22]. Despite the broad specificity,this protease shows a limited ability to cleave synthetic peptidesubstrates, which include ABZ(amino benzoic acid)-Phe-Gly-Ala-Lys-ANB-NH2(amide of 5-amino-2nitrobenzoic acid) [23], Z(ben-zyloxycarbonyl)-Leu-Leu-Glu-MCA, and Z-Leu-Leu-Arg-MCA. Thiolcompounds are necessary for the protease activity. In addition toheavy metals and oxidizing agents, ScpA is irreversibly inhibitedby E-64 and human α2-macroglobulin [24]. SspB has far more

restricted substrate specificity in comparison with ScpA, and itcleaves ABZ-Ile-Ala-Ala-Gly-ANB-NH2 [23] and Z-Phe-Arg-MCA.E-64 inhibits SspB, but no effective inhibitors for this protease inhuman plasma have been identified thus far.

3. Virulence activities of staphopains in bacteremia/sepsis

In bacteremia/sepsis, staphopains, released by S. aureus in thebloodstream, cleave plasma proteins, thereby exerting their viru-lence activities.

3.1. Induction of vascular leakage and hypovolemic hypotension

The pathogenesis of Gram-positive sepsis has not been fullyelucidated. However, the plasma levels of the plasma kallikrein/kinin system components, factor XII, prekallikrein, and high mole-cular weight kininogen (HK), are low in sepsis patients, indicat-ing activation and subsequent consumption of these components[25–28]. The activation of the plasma kallikrein/kinin system in ananimal bacteremia model causes lethal hypotension [29,30];hence, plasma kallikrein/kinin system activation appears to con-tribute to septic shock. In human plasma, S. aureus induces therelease of bradykinin (BK) [31], the final product of the plasmakallikrein/kinin system activation, which causes vascular leakage (VL)[32] leading to hypotension. This bacterium has a high negative netsurface charge because of the presence of cell wall teichoic acid andlipoteichoic acid [33], and can activate the plasma kallikrein/kininsystem as efficiently as lipopolyscaccharide and lipid A from Gram-negative bacteria do in vitro [34]. Thus, these cell wall moleculeshave the potential to activate the plasma kallikrein/kinin system inS. aureus bacteremia. However, heat-labile extracellular products ofS. aureus were found to be far more potent as lethal factors than itscell wall components when evaluated in a mouse sepsis model [35].Among the secreted proteases, V8 proteinase was shown to releasekinin from HK; however, this activity was not affected by thepresence of serine proteinase inhibitors [36], suggesting that con-taminating proteolytic enzymes have been responsible for kiningeneration. Therefore, heat-labile staphopains were examined fortheir role in kinin release.

Intradermal injection of ScpA into guinea pigs induced VL in aconcentration-dependent manner starting at an enzyme concen-tration of 20 nM (2 pmole/100 ml) [37]. Inhibition of this activityby E-64 and treatment of the animals with the BK B2-receptorantagonist HOE 140 suggested that BK was generated by ScpAproteolytic activity. Indeed, this enzyme induced VL via releaseof BK directly from both human HK and low molecular weightkininogen (Fig. 1) but not through the activation of factor XII or

bradykinin

---G M I S L M K R P P G F S P F R S S R I G E--- human kininogen

ScpA ScpASspBcleavage site

ScpAScpA ScpA + SspB

R P P G F S P F R R P P G F S P F R +L M K R P P G F S P F R

bradykinin B2 receptor-dependentinduction of vascular leakage/shock

Fig. 1. Human kininogen cleavage by staphopains. Arrows indicate staphopaincleavage sites at kininogen neighboring the bradykinin sequence.

T. Imamura / Journal of Oral Biosciences 56 (2014) 81–8582

prekallikrein. Interestingly, although SspB enhanced VL activityinduced by ScpA in a concentration-dependent manner, it showedno VL activity alone; this is attributed to the synergistic cleavage ofkininogen. SspB cleaves HK at -Ile-Ser-↓-Leu-Met-, and togetherwith cleavage at the C-terminus of the BK sequence by ScpA, itreleases Leu-Met-Lys-BK (Fig. 1), which exhibits VL activity almostequal to that of BK. Thus, coexistence of the two staphopainsincreases VL activity through the additional release of Leu-Met-Lys-BK. An important pathophysiological mechanism of septicshock is hypovolemic hypotension, caused by plasma leakage intothe extravascular space. ScpA, similar to the two kinins, lowersblood pressure in a BK B2-receptor-dependent manner whenadministered as an intra-arterial injection to guinea pigs andproduces septic shock symptoms in them. Kinin generation fromhuman plasma by ScpA is enhanced in the presence of SspB,strongly suggesting that these proteases can cause shock inpatients with S. aureus sepsis.

3.2. Induction of coagulation disorders

Disseminated intravascular coagulation (DIC) is a common andpotentially fatal consequence of sepsis. In as many as 40% of septicpatients, DIC leads to multiple organ failure [38], and is directlylinked to a high mortality rate. Clotting induction and thesubsequent bleeding tendency due to the consumption of coagu-lation factors are the prominent clinical features of DIC. In S. aureussepsis, staphopains may participate in the onset of the clottingdisorder through the activation or inactivation of plasma coagula-tion factors by a proteolytic cleavage. However, the ability ofstaphopains to affect plasma clotting has not been studied.

Activated partial thromboplastin time (APTT) shows plasmaclottability through the intrinsic coagulation pathway. Both sta-phopains prolonged APTT and SspB was found to be approximatelythreefold more potent than ScpA [39]. Plasma thrombin timedemonstrates the clottability of fibrinogen and similar effects ofthe enzymes on this assay indicates that staphopains predomi-nately degrade fibrinogen to reduce its clottability. Staphopains,indeed, efficiently prolonged fibrinogen thrombin time in a con-centration- and protease activity-dependent manner. SDS-PAGEanalysis and N-terminal sequencing of fibrinogen fragmentshave revealed that staphopains preferentially cleave the Aα-chain

(Fig. 2). First, ScpA rapidly cuts out a 16-kDa fragment from the αC(C-terminal two/thirds region) of the Aα-chain (66 kDa) [40],resulting in the formation of a 50-kDa fragment. Subsequent slowcleavage within this fragment reduces its molecular mass to34 kDa. The αC is also targeted by SspB, which generates 41-,37-, and 35-kDa fragments (Fig. 2). It is thought that SspB initiallyreleases the 41-kDa fragment by proteolysis within the αC, at arate slower than that of the ScpA-mediated cleavage to release the16-kDa fragment. The 41-kDa fragment is further cut at the αC,sequentially generating 37- and 35-kDa fragments. In contrast tothe Aα-chain, the Bβ- and γ-chain comparatively resist cleavage bystaphopains. Plasmin degrades all three chains of fibrinogen, andcleaves the Aα-chain at various sites, including peptide bonds atthe C-terminal sites of Arg252, Arg424, Arg491, and Lys508 [41].Proteolysis at these sites generates fragments with molecularmasses different from those released by the staphopains-inducedcleavage of the Aα-chain. Therefore, it is clear that staphopains cleavethe fibrinogen Aα-chain at sites different from those at whichplasmin cleaves. Thrombin cuts off fibrinopeptide A from theAα-chain, and fibrinopeptide B from the Bβ-chain [42], causing thepolymerization of generated DesAA-fibrinogen into protofibrils.The protofibrils subsequently laterally associate with one anotherthrough the binding of the αC regions, (Fig. 3) [43], thus convertingfibrinogen to a fibrin clot. Charged amino acid residues necessary forlateral association are located in the αC [44]. The very efficientcleavage of the αC by SspB truncates majority of the αC protein todisrupt the necessary interaction for lateral association of protofibrils(Fig. 3). ScpA also cleaves fibrinogen at the αC, more rapidly thanSspB, generating the 50-kDa fragment first, but this cleavage seemsunlikely to cause a reduction in clotting factor function. The nextcleavage, by which a 34-kDa fragment is generated, is slow butrenders fibrinogen unclottable (Figs. 2 and 3). The difference incleavage sites explains why SspB impairs fibrin clot formation moreefficiently than ScpA. Thus, fibrinogen cleavage by staphopains inS. aureus sepsis results in the loss of plasma clottability, leading to agreater tendency to bleed, a fatal consequence of sepsis.

3.3. Collagen degradation

Invasion of S. aureus into the endocardium, leading to infectiousendocarditis, requires the degradation of connective tissue extracellular

35k37k41k

34k

50k

ScpA

A B γγ

αC

CHO

CHO

SspB

Fibrinogen (AαBβγ)2

αC

βC γC

αC

βCγC

D domain D domainE domain

βC γC

βCγC

αC αC staphopain

staphopain

FPAFPA

FPBFPB

35k37k41k

34k

50k

ScpA

Aα Bβ γ

αC

CHO

CHO

SspB

αC

βC γC

αC

βCγC

D domain D domainE domain

βC γC

βCγC

αC αC staphopain

FPAFPA

FPBFPB

Fig. 2. Fibrinogen structure, staphopain cleavage sites at the αC domain, and changes in fibrinogen after staphopain cleavage. The arrow and molecular weight indicate thecleavage site and the molecular weight of the cleaved fragment, respectively. Aα, Bβ, and γ indicate fibrinogen chains. αC: C-terminal domain of the Aα-chain, βC: C-terminaldomain of the Bβ-chain, γC: C-terminal domain of the γ-chain, FPA: fibrinopeptide A, FPB: fibrinopeptide B, and CHO: N-bound carbohydrate.

T. Imamura / Journal of Oral Biosciences 56 (2014) 81–85 83

matrix proteins and is facilitated by proteases. In this context,staphylococcal proteases may contribute to this invasion, althoughlittle is known about the ability of S. aureus-derived enzymes todegrade matrix proteins. The extracellular matrix degradation essentialfor the pathogenicity of S. aureus is inferred from the ability of ScpA toinduce widespread plasma leakage via BK release following itsintradermal injection into guinea pigs[37]. This is in stark contrast tothe localized plasma leakage induced by the injection of BK alone. Thewidespread leakage of plasma is thought to be due to degradation ofextracellular matrix proteins, including elastin, which can be cleavedby ScpA [24]. The activity of SspB on matrix proteins has not beeninvestigated.

Both staphopains degraded type I collagen in a concentration-dependent manner at concentrations as low as 10 nM and inactiva-tion of these proteases inhibited their collagenolytic activity [39].Although the two staphopains differ in their fibrinogenolytic activity,they exert the same collagenolytic activity. The ability to degradecollagen strongly suggests that staphopains can participate in endo-cardium destruction caused by S. aureus. Adherence of S. aureus toendothelial cells is the initial step in the development of endocarditis.In this process, fibrinogen acts as a bridging molecule betweenS. aureus and the endothelium [45], and the ability of this pathogento bind to fibrinogen is recognized as an important factor for valveinfection and invasion in experimental endocarditis [46]. Thus, theinteraction of S. aureus with fibrinogen plays a crucial role in thepathogenicity of endocarditis caused by this bacterium. Moreover, a135-kDa cell surface protein of S. aureus [47] binds specifically tocollagen [48–51], constituting another important virulence trait inexperimental endocarditis [52].

4. Conclusions

S. aureus in the oral cavity invades the bloodstream in highnumbers following dental procedures and releases staphopains

into the plasma. By cleaving kininogens and fibrinogen in plasma,staphopains lower blood pressure through kinin release andimpair plasma clottability. Staphopains degrade type I collagen,which possibly contributes to endocardium destruction byS. aureus. These pathogenic activities of staphopains suggest thatthe S. aureus-produced proteases are potent virulence factors andare potentially involved in the induction of septic shock andcoagulation disorders, and in the onset of infectious endocarditisin S. aureus bacteremia. In addition, such staphopain activities areassociated with local S. aureus infections. Edema occurs throughkinin release, unclottable fibrinogen generation impedes inductionof the host-defense coagulation against pathogens, and collagendegradation destroys tissues, thereby facilitating S. aureus invasioninto the circulation and its dissemination. Therefore, staphopainsmay be novel targets for drug development for therapy ofinfectious diseases caused by S. aureus.

Ethical approval

Ethical approval is not required.

Conflict of interest

There is no conflict of interest in this review

Acknowledgments

The author is grateful to Prof. Ryutaro Kamijo, Department ofBiochemistry, Showa University School of Dentistry, and Prof.Hayato Ohshima, Division of Anatomy and Cell Biology of the HardTissue, Department of Tissue Regeneration and Reconstruction,Niigata University Graduate School of Medical and Dental Sciences,

Fibrinogen

Protofibril formation

Lateral associationof protofibrils

Bundling andbranching of fibers

Fibrin networks

Fibrin formation process

Thrombin

FPA

Thrombin

FPB

Thrombin

FPA

Thrombin

FPB

staphopain

X

Protofibrils are unableto associate laterally

Fig. 3. Fibrin formation process and loss of the protofibril lateral association in fibrinogen cleaved by staphopains. FPA: fibrinopeptide A and FPB: fibrinopeptide B.

T. Imamura / Journal of Oral Biosciences 56 (2014) 81–8584

for their encouragement while writing this review. The author'sstudies included in this review were supported in part by a Grant-in-Aid for Scientific Research (B) from the Japanese Ministry ofEducation and Science (Grant No: 18390125).

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