➢ Tissue injury activates the acute-phase response mediated by the liver, which promotes coagulation, immunity, and tissue regeneration. To survive and disseminate, musculoskeletal pathogens express virulence factors that modulate and hijack this response. As the acute-phase reactants required by these pathogens are most abundant in damaged tissue, these infections are predisposed to occur in tissues following traumatic or surgical injury.
➢ Staphylococcus aureus expresses the virulence factors coagulase and von Willebrand binding protein to stimulate coagulation and to form a fibrin abscess that protects it from host immune-cell phagocytosis. After the staphylococcal abscess community reaches quorum, which is the colony density that enables cell-to-cell communication and coordinated gene expression, subsequent expression of staphylokinase stimulates activation of fibrinolysis, which ruptures the abscess wall and results in bacterial dissemination.
➢ Unlike Staphylococcus aureus, Streptococcus pyogenes expresses streptokinase and other virulence factors to activate fibrinolysis and to rapidly disseminate throughout the body, causing diseases such as necrotizing fasciitis.
➢ Understanding the virulence strategies of musculoskeletal pathogens will help to guide clinical diagnosis and decision-making through monitoring of acute-phase markers such as C-reactive protein, erythrocyte sedimentation rate, and fibrinogen.
Pathogenic bacteria possess an arsenal of virulence factors that allow them to invade, persist, and disseminate within the human body. Coevolution between pathogens and the human immune system has led to bacterial affinity for specific sites1,2. Pathogen tropism has been well documented, as nearly all bacteria exhibit selectivity for certain cells, tissues, or hosts3-6. In the context of musculoskeletal infection, bacterial pathogens have developed a tropism for sites of tissue damage. These bacteria take advantage of the transient immunocompromised state of injured tissue and express factors that hijack the host’s response to injury. This review focuses on the pathophysiology of musculoskeletal infection, in particular the virulence factors that enable pathogens to thrive in the context of tissue damage and exploit the body’s response to infection.
Tissue Injury and Repair: Coagulation-Controlled Compartmentalization
The essential elements of the musculoskeletal system are physically segregated from each other by matrix tissue such as fascia, paratenon, and joint capsule. Injury disrupts these well-defined anatomic compartments, predisposing to dysfunction, pathogen invasion, and bleeding (Fig. 1-A). The first response to hypoxic, injured tissue is activation of the acute-phase response. This dynamic biological response comprises coagulant, inflammatory, and regenerative processes7,8. First, the procoagulant arm of the coagulation system produces a fibrin or platelet web that seals off damaged musculoskeletal tissue both intravascularly and extravascularly9,10. The inflammatory system then responds to secreted signals from the injured tissue and the fibrin or platelet web to invade the zone of injury, to clear debris, and to kill invading pathogens (Fig. 1-B11-14)15,16. Lastly, the additional release of growth factors stimulates regenerative cells to reestablish tissue function and compartment segregation (Fig. 1-C)17. These processes are included in the acute-phase response (Fig. 218-22).
Following the sealing of the injured compartment by coagulant processes, the fibrinolytic arm of the coagulation system is required to break down fibrin or platelet clots and to promote efficient tissue regeneration. The protease plasmin is the principal mediator of this process, which occurs in a controlled manner to prevent unregulated loss of compartmentalization during tissue remodeling23-25. The fibrinolytic arm of wound-healing is emphasized by O’Keefe in his recent review, which highlights the importance of fibrinolysis in fracture-healing26.
Thus, during a musculoskeletal tissue injury, coagulation functions in two organized phases. Initially, the coagulant arm rapidly produces a fibrin or platelet web to seal off ruptured compartments. Later, the fibrinolytic arm removes this initial meshwork to allow the remodeling of injured tissue and the reestablishment of native compartments. Failure of the coagulant arm leads to the inability to sequester injury, and failure of the fibrinolytic arm leads to delayed tissue regeneration.
The Trap: A Secondary Role of the Fibrin or Platelet Web
In addition to isolating damaged tissue, the coagulation system also serves as the initial defense against bacterial invasion. Coagulation provides antimicrobial activity by immobilizing bacteria within clots and recruiting leukocytes to the site of infection through integrin expression on fibrin27-29. In addition, activated platelets promote the formation of neutrophil extracellular traps, an important bactericidal mechanism utilized by neutrophils in innate immunity (Fig. 330,31). Neutrophil extracellular traps are extracellular DNA fibers with associated proteins that function like fibrin to immobilize and destroy pathogens32-34. In a positive feedback mechanism, the histone components of neutrophil extracellular traps promote platelet activation and thrombosis as well35. Platelet activation provides additional immune activity through the release of antimicrobial proteins from alpha granules (thrombocidins) following stimulation with thrombin. Previous studies have demonstrated that alpha granules possess activity against isolates of Staphylococcus aureus and other pathogens36-38.
Remarkably, selective pressure exerted by bacterial pathogens has also resulted in genetic polymorphisms within the coagulation system. The common prothrombotic genetic variant Factor V Leiden, which is resistant to cleavage inactivation by activated protein C (APC), has been shown to improve survival in patients with sepsis and mice exposed to bacterial lipopolysaccharide (LPS) endotoxin39. The survival benefit of this mutation, which is present in up to 5% of Caucasians, likely contributes to the stability of this polymorphism in the population40. Other independent polymorphisms of Factor V have also been described (Factor V Hong Kong and Factor V Cambridge)41,42, suggesting that this protein may have a crucial role at the intersection of coagulation and immunity.
Hijacking the Acute-Phase Response: Bacterial Targeting of Injured Tissue
Although the acute-phase response is essential for wound-healing and pathogen clearance, aspects of the response can be dysregulated and can be manipulated by pathogens (Video 1). The coagulation response is a primary target for musculoskeletal pathogen virulence factors43,44. This review will focus on the mechanisms of invasion, proliferation, and dissemination of Staphylococcus aureus and Streptococcus pyogenes, the most common causes of musculoskeletal infections in the United States.
Staphylococcus aureus: Manipulate the Web and Then Break It
Staphylococcus aureus has a variety of virulence factors that manipulate the acute-phase response. Staphylococcus aureus initially uses procoagulant factors to form a fibrin and platelet abscess where it can proliferate without interference from host immune responses. Later, the staphylococcal abscess community produces proteins that activate the fibrinolytic system to break apart the abscess and disseminate.
The most well-known Staphylococcus aureus virulence factor is coagulase, which was discovered by Loeb using goose and rabbit plasma in 190345. Staphylococcus aureus secretes coagulase into the extracellular environment, where it associates with and activates prothrombin to thrombin46,47. The coagulase-thrombin complex catalyzes the cleavage of fibrinogen to fibrin to promote clot and abscess formation48,49. Although this complex catalyzes clot formation at a slower rate than thrombin, the enzymes are bound together irreversibly and neither antithrombin III nor exogenous thrombin inhibitors affect its activity50-52. The von Willebrand factor binding protein (vWBP), discovered about 100 years later, plays a similar role in Staphylococcus aureus virulence, complexing with prothrombin to further promote antithrombin-resistant fibrin formation53-55. Additionally, vWBP activates platelets and promotes bacterial adhesion to blood vessel walls, enabling Staphylococcus aureus to resist the shear stress exerted by blood flow. Adherence to vessel endothelium promotes Staphylococcus aureus tissue invasion, a key step in the hematogenous spread of disease56.
Coagulase and vWBP activation of prothrombin promotes abscess formation, thereby allowing Staphylococcus aureus to resist phagocytic clearance57-59. Within an abscess, two concentric structures are formed: an inner pseudocapsule formed predominantly by coagulase and a thicker outer meshwork dependent on vWBP (Fig. 4, Video 2). Together, these redundant mechanisms generate a double-layered, fibrin-dependent protective barrier to prevent neutrophil access and clearance60. This process allows Staphylococcus aureus to form its own compartment within the body by hijacking the normal coagulation response to the initial site of tissue injury.
Several other Staphylococcus aureus factors have been described that promote platelet activation and aggregation. Clumping factors A and B (ClfA/B) and fibronectin binding proteins A and B (FnBPA/B) activate platelets via the glycoprotein IIb/IIIA receptor in a fibrinogen-dependent manner. In addition, by binding fibrin, ClfA tethers Staphylococcus aureus to the fibrin meshwork generated by coagulase and vWBP, further protecting the bacterium from phagocytosis61.
Staphylococcal protein A (SpA) is another protein that binds von Willebrand factor and activates platelets to promote coagulation62. However, unlike other virulence factors, SpA also interferes with the immune system by interacting with antibodies. It binds to the conserved Fc (fragment crystallizable) region of antibodies and acts as a superantigen by cross-linking surface immunoglobulin G (IgG) on B cells, thereby hijacking the immune and inflammatory responses63-66.
Staphylococcus aureus further manipulates the immune system by interfering with neutrophil extracellular traps through expression of nuclease and adenosine synthase67. In a two-step process, nuclease cleaves neutrophil extracellular traps and then adenosine synthase converts the resulting degradation products to deoxyadenosine, a factor that triggers macrophage apoptosis. On histologic examination, staphylococcal colonies lacking nuclease and adenosine synthase have increased concentrations of macrophages at the abscess border, suggesting that these factors protect the staphylococcal abscess community from phagocytosis68,69.
Extracellular adherence protein also upregulates the acute-phase response by inducing interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and other cytokines through interaction with immune cells70. However, extracellular adherence protein simultaneously inhibits other aspects of the acute-phase response by preventing neutrophil adherence, leukocyte recruitment, and T-cell activation71-73. The complex actions of extracellular adherence protein exemplify the generalized dysregulatory influence that Staphylococcus aureus exerts on the human acute-phase response. Other proteins such as staphylococcal super antigen-like (SSL) protein have similarly broad effects on inflammation, coagulation, and innate immunity74,75.
Although most Staphylococcus aureus virulence factors promote coagulation to form abscesses and to evade immune clearance, staphylokinase promotes fibrinolysis through activation of plasminogen to plasmin. Although the resulting staphylokinase-plasmin complex is initially susceptible to alpha-2 antiplasmin inhibition through covalent binding, binding of the complex to fibrin or Staphylococcus aureus surface proteins renders alpha-2 antiplasmin inhibition ineffective25,76,77. By catalyzing the rapid cleavage of fibrin, the complex is able to lyse tenfold more clot than plasmin alone in 5 hours78. Staphylokinase expression is regulated by the accessory gene regulator system, which modulates gene expression as Staphylococcus aureus transitions from the rapid exponential growth phase to the stationary phase79. When the staphylococcal abscess community reaches quorum, which is the colony density that enables cell-to-cell communication and coordinated gene expression, Staphylococcus aureus expresses staphylokinase to promote fibrin degradation, abscess rupture, and dissemination (Fig. 5)60,77.
Streptococcus pyogenes: Bypass the Web Altogether
Streptococcus pyogenes uses a different approach for invasion and dissemination. Instead of initially producing procoagulant factors to form an abscess, it bypasses this step and immediately expresses fibrinolytic proteins (Fig. 6, Video 3).
The best studied of these fibrinolytic factors is streptokinase, which was serendipitously discovered in 1933 when Tillet failed to empty a set of test tubes and returned to find that previously coagulated blood had been liquefied80. Streptokinase was subsequently identified, was purified, and was adopted for therapeutic use in thrombotic disease by 194780. Like staphylokinase, streptokinase binds plasminogen and activates it via conformational change. The resulting alpha-2 antiplasmin-resistant streptokinase and plasminogen complex activates circulating plasminogen 5 times faster than tissue plasminogen activator (tPA) and 30 times faster than urokinase plasminogen activator (uPA)81,82, leading to rapid degradation of both fibrin and fibrinogen78.
There are several surface-bound plasminogen receptors that augment the fibrinolytic activity of streptokinase83,84. Three of these proteins are plasminogen binding group A streptococcal M protein (PAM), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and alpha-enolase. These plasminogen receptors mimic fibrinogen by using lysine residues to bind plasmin. Substitution of lysine in these receptors reduces the bacteria’s plasminogen binding affinity and virulence85-89. The coat of plasmin on the bacterial surface produced by these proteins promotes dissemination through human tissue via the activation of matrix metalloproteinases and cleavage of both fibrin clots and laminin basement membranes84,90. The inability of the host to sequester a Streptococcus pyogenes infection is demonstrated by the invasiveness of necrotizing fasciitis infection and its disregard for tissue planes, physiologic compartments, or extracellular matrix barriers91.
Streptococci have further interaction with the coagulation system through the expression of streptolysin O, which activates platelets to complex with lymphocytes and cause microvascular occlusions. The resulting vascular dysfunction may lead to shock and organ failure, further contributing to the virulence of Streptococcus pyogenes92.
To evade sequestration by extracellular traps produced by neutrophils, streptococci also express DNAse to cleave DNA and to prevent immobilization by this antimicrobial trap93. Studies have demonstrated a dose-dependent clearance of neutrophil extracellular traps in the presence of Streptococcus pyogenes. Furthermore, inhibition of DNAse improves neutrophil clearance of bacteria93.
Overall, Streptococcus pyogenes virulence depends heavily on dysregulation of the host fibrinolytic system to rapidly invade and to disseminate throughout the body.
Additional Pathogens Targeting Plasminogen
Virulence factors designed to hijack the host acute-phase response and coagulation system are not limited to Staphylococcus aureus and Streptococcus pyogenes. Numerous other bacteria have independently evolved virulence factors targeting these systems, which emphasizes the importance of this virulence strategy in bacterial infection. Plasminogen activation, in particular, has been targeted by bacteria including Escherichia coli, Salmonella typhi, Neisseria meningitidis, and Haemophilus influenzae94-99. The surface protease plasminogen activator (pla) of Yersinia pestis is another well-characterized plasminogen activator that plays a crucial role in infection dissemination and virulence. One study has demonstrated that the lethal dose of Yersinia pestis is approximately 1 million-fold higher in plasminogen-deficient mice compared with wild-type mice, revealing the importance of host plasminogen for virulence100. Borrelia burgdorferi, the bacteria responsible for Lyme disease, activates plasminogen contained in a tick’s blood meal to mediate transmission from the tick digestive system to the human bloodstream101,102. Pseudomonas aeruginosa binds plasminogen to its extracellular surface to degrade fibrin and extracellular barriers99,103. These bacterial mechanisms of dissemination mirror mechanisms of metastasis in tumor pathogenesis104.
The importance of plasmin-mediated pathogen dissemination is clear on the basis of its ubiquitous utilization. Interestingly, the widespread utilization of this virulence mechanism has resulted in selective pressure in humans for the production of plasminogen variants that may confer resistance to infection. In some Asian countries, an A601T mutation in plasminogen is present in approximately 2% of the population105. Although this mutation leads to a moderate prothrombotic state, it may confer a compensatory selective survival advantage against certain pathogens that have coevolved to exploit plasmin protease activity.
Clinical Correlations for Musculoskeletal Infection
Correlation between pathogenic virulence mechanisms and clinical findings allows clinicians to accurately predict disease severity and clinical outcomes. Musculoskeletal infection may be viewed as a form of continuous tissue injury that intensely activates the acute-phase response through persistent production of IL-6. The tissue damage caused by infection generates measurable increases of acute-phase reactants in serum, and numerous studies have demonstrated that inflammatory and coagulation markers (i.e., C-reactive protein, erythrocyte sedimentation rate, fibrinogen, platelet count) are sensitive indicators of musculoskeletal infection severity106-110. In addition, unpublished data from the authors have demonstrated significant differences in acute-phase, inflammatory markers for disseminated infection compared with those for compartmentalized infections. However, it must be noted that acute-phase reactants are not specific indicators for infectious disease, as many aseptic conditions are associated with elevated acute-phase reactants as well.
Severe cases of musculoskeletal infection lead to clinically relevant dysregulation of the coagulation system, resulting in thromboembolism, sepsis, or disseminated intravascular coagulopathy. Venous thrombosis is a well-documented complication of musculoskeletal infection associated with osteomyelitis and Staphylococcus aureus infection111,112. Epidemiologic studies have detected venous thromboembolism in up to 7 (10%) of 70 pediatric patients with hematogenous osteomyelitis113,114. Additional studies have demonstrated that systemic dissemination of musculoskeletal infection predisposes patients to developing sepsis and coagulopathy115,116. Understanding how pathogens dysregulate and exploit the coagulation system will allow physicians to diagnose severe cases at earlier stages by following relevant indices (i.e., fibrinogen, d-dimer, international normalized ratio). Improved anticipation and diagnosis of systemic complications will lead to more timely and effective interventions and improved patient outcomes.
Future Directions for Therapy
The introduction of antibiotics drastically improved treatment of musculoskeletal infection, but few ground-breaking pharmacologic advances have been made since then to improve outcomes. Extensive research has been directed toward vaccine development against both Staphylococcus aureus and Streptococcus pyogenes. Although vaccines targeting Staphylococcus aureus antigens including coagulase, vWBP, and M protein have been effective in mouse studies57,117,118, active vaccine trials in humans have not shown the same promise119. Given the wide range of virulence factors expressed by Staphylococcus aureus, vaccines targeting single antigens are unlikely to be protective. Current approaches targeting multiple Staphylococcus aureus antigens or secreted toxins may prove more successful.
Numerous Streptococcus pyogenes antigens have also been targeted for vaccination, with M protein being the most popular target. However, a roadblock in the development of a Streptococcus pyogenes vaccine occurred after a clinical trial in 1968 when administration of an M protein vaccine resulted in cross-reactivity that led to an increased development of acute rheumatic fever120,121. This trial led to a prohibition on human administration of Streptococcus pyogenes derivatives122 despite additional studies demonstrating the protective effects of novel vaccines without the induction of acute rheumatic fever123,124. Since the ban was lifted in 2006, several polyvalent vaccines targeting the N-terminus of M protein have been under development118,125,126.
Monoclonal antibodies targeting bacterial antigens and virulence factors are another therapeutic avenue currently being pursued. Recent studies have demonstrated improved survival in mouse models using monoclonal antibodies against a number of targets including Staphylococcus aureus coagulase, staphylococcal protein A, and enterotoxin B57,127,128. However, there are no therapies that have demonstrated efficacy in humans129-132. A possible barrier to monoclonal antibody treatment is binding and cleavage of immunoglobulins by Staphylococcus aureus and Streptococcus pyogenes virulence factors such as staphylococcal protein A, staphylokinase, and streptokinase65,66,133,134. Antibodies targeted against the surface of either pathogen must avoid interference from these proteases to be effective.
In addition, therapeutics that alter inflammatory and coagulation cascades are being developed. APC is a well-studied anticoagulant that inactivates factors V and VIII to inhibit coagulation. In early studies, administration of APC (Xigris) during sepsis reduced the absolute risk of death by 6.1% (210 of 850 compared with 259 of 840), possibly by inhibiting the production of TNF and other proinflammatory cytokines. However, this therapy was withdrawn from the market after larger studies showed no benefit and increased risk of bleeding135-139. Further research into therapeutic targets that modulate coagulation and inflammation (i.e., corticosteroids and vitamin K) may yield more promising results.
Tissue injury ruptures anatomic compartment boundaries, leading to contaminated microenvironments that require complex physiologic processes for proper temporal repair. Staphylococcus aureus and Streptococcus pyogenes have evolved sophisticated mechanisms for evading and hijacking the hemostatic, tissue regenerative, and antimicrobial properties of the acute-phase response. Of particular interest, it was recently determined that the fibrinolytic arm of the coagulation system is essential in breaking down the initial fibrin or platelet web required for bone regeneration24,26. However, Staphylococcus aureus and Streptococcus pyogenes employ this same system to disseminate within host tissue, highlighting the paradoxical dilemma of targeting microbial mechanisms of invasion without impairing musculoskeletal regeneration. These bacteria also possess many other virulence factors including toxins and other modulators of the acute-phase response that are beyond the scope of this review. Further understanding of the virulence strategies utilized by such pathogens will inform infection diagnosis, will improve patient outcomes, and will facilitate the development of better therapeutics in the future.
Investigation performed at the Vanderbilt University School of Medicine, Nashville, Tennessee
Disclosure: There was no source of external funding for this study. The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article.
- Copyright © 2016 by The Journal of Bone and Joint Surgery, Incorporated