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Title: Clumping factor B is an important virulence factor during Staphylococcus aureus skin infection and a promising vaccine target}
Article:
Abstract:
Staphylococcus aureus expresses a number of cell wall-anchored proteins that mediate adhesion and invasion of host cells and tissues and promote immune evasion, consequently contributing to the virulence of this organism. The cell wall-anchored protein clumping factor B( ClfB) has previously been shown to facilitate S. aureus nasal colonization through high affinity interactions with the cornified envelope in the anterior nares. However, the role of ClfB during skin and soft tissue infection( SSTI) has never been investigated. This study reveals a novel role for ClfB during SSTIs. ClfB is crucial in determining the abscess structure and bacterial burden early in infection and this is dependent upon a specific interaction with the ligand loricrin which is expressed within the abscess tissue. Targeting ClfB using a model vaccine that induced both protective humoral and cellular responses, leads to protection during S. aureus skin infection. This study therefore identifies ClfB as an important antigen for future SSTI vaccines.
Introduction:
Staphylococcus aureus is the leading cause of skin and soft tissue infections( SSTIs) in humans[1, 2] with S. aureus SSTIs resulting in over 11 million outpatient visits and almost 500, 000 hospital admissions in the United States annually[3]. S. aureus SSTIs routinely manifest as cutaneous abscesses which limit the penetration and efficacy of antibiotics[4]. Community-acquired methicillin resistant S. aureus( CA-MRSA) skin infections are increasing in frequency in healthy individuals[5] and the treatment of these infections has become increasingly difficult due to the emergence of antibiotic resistance. The development of an anti-S. aureus vaccine offers a potential solution to prevent infection regardless of antibiotic resistance; however, although significant efforts have been made, an effective anti-S. aureus vaccine remains elusive. Given the wide spectrum of pathologies caused by this bacterium it is unlikely that a universal anti-S. aureus vaccine will ever be realised[6], and instead, a vaccine targeting specific clinical manifestations may need to be pursued. As skin is the most frequent site of S. aureus infection[7], a vaccine specifically targeted against SSTIs would be of great benefit. If prophylactic vaccines or other forms of immunotherapy to treat S. aureus SSTIs are to be developed as an alternative to antibiotics, a greater understanding of the specific role of individual virulence factors during infection at this site is needed to identify important targets for future therapy. S. aureus expresses up to 25 different cell wall-anchored( CWA) proteins, which are covalently linked to the peptidoglycan layer by the enzyme sortase A and are primarily involved in adhesion and invasion of host cells and tissues, biofilm formation and immune evasion[8]. Sortase A-deficient mutants, which lack the majority of cell surface bound CWA proteins, have reduced virulence in a murine kidney abscess model[9, 10], while in a skin abscess model, a sortase A-deficient mutant resulted in lower bacterial burden in the skin and a significantly reduced pathology compared to wild-type infected mice[11]. These studies indicate that CWA proteins are important during SSTIs and suggest that CWA proteins may be particularly important during the process of abscess formation. However, there is a paucity of information regarding the role of individual surface proteins during SSTIs. Clumping factor A has previously been shown to play a role in the pathogenesis of S. aureus SSTI, as mice inoculated with a ClfA-deficient mutant of S. aureus strain Newman demonstrated a lower bacterial burden in the skin compared to the wild type strain at Day 2 post-inoculation[11]. However, vaccination strategies targeting ClfA only provided modest protection in this model[12] suggesting that other surface proteins may also play an important role in the development of S. aureus SSTI. S. aureus attachment to the anterior nares during colonization is facilitated by the CWA protein clumping factor B( ClfB) through high affinity interactions with the cornified envelope. Through this interaction ClfB has been shown to promote nasal colonization in both rodents and humans[13–15]. ClfB is expressed in the early exponential phase of growth and is absent from cells in the late and stationary phase. The N-terminus of ClfB consists of a signal sequence followed by its binding domain, region A. This is a 540 amino-acid long segment containing 3 independently folded subdomains, N1, N2 and N3[16]. Similar to ClfA, the ligand-binding region of ClfB has been localised to the N2N3 domain of region A. The N1 domain has no known binding function[16]. The clfB gene is carried by most strains of S. aureus[17]. Sequence variation in the ClfB protein occurs between different clonal complexes of S. aureus, with the variant proteins sharing at least 94% amino-acid identity to each other[18]. ClfB binds to plasma fibrinogen[19], cytokeratin 10[20], which is the dominant component of the interior of squamous cells, and to loricrin, which is the most abundant protein of the cornified envelope of squamous cells[21]. Rates of S. aureus nasal colonization were significantly reduced in loricrin-knockout mice compared to wild-type mice, demonstrating that loricrin is a critical ligand for ClfB in vivo, at least in mice[13]. The contribution of ClfB to S. aureus skin infection however, has never previously been addressed. This study aims to elucidate its role during S. aureus skin infection and to assess its potential as a candidate vaccine antigen specifically targeting SSTIs.
Discussion:
Previous to this study, the main function attributed to ClfB was in facilitating S. aureus nasal colonization due to its ability to bind host ligands loricrin and cytokeratin 10 in the anterior nares[13–15]. This study identifies, for the first time, an important role for ClfB in the pathogenesis of SSTIs and provides proof-of-concept that ClfB represents a good candidate antigen for inclusion in next-generation vaccines targeting S. aureus SSTI. Using a clfB mutant in the clinically relevant CA-MRSA USA300 strain, LAC::lux, we demonstrate that ClfB is a virulence determinant during S. aureus skin infection. Crucially, ClfB appears to exert its effect early during the infection process during initial formation of the abscess. Immunohistochemistry analysis revealed that loricrin, an important ligand for ClfB, was present in the outermost layer of the skin, the corneal layer, as expected; however, loricrin also appeared to be present within the wall of the skin abscess structure of LAC::lux infected mice. Although loricrin is normally confined to the granular and corneal layers of the skin[22, 23], the expression and localisation of loricrin is disrupted during wound healing[24, 25]. This may account for the presence of loricrin in the subcutaneous tissue. We propose that expression of loricrin within the damaged tissue facilitates the binding of S. aureus via ClfB which in turn promotes the development of the abscess. To investigate this proposed ClfB-loricrin interaction in the skin, loricrin deficient mice were used. Lor-/- mice infected s. c. with LAC::lux formed significantly smaller abscess, with lower bacterial burden and reduced pathology compared to WT infected mice, suggesting that the interaction between S. aureus and loricrin is crucial during SSTIs. This effect appears to be ClfB-specific, and not due to ClfB binding to an alternative ligand in the skin, such as fibrinogen or keratin, as Lor-/- mice infected with LAC::lux ΔclfB did not display any further significant reduction in abscess formation or the overall severity of skin infection. Furthermore, the interaction between S. aureus and loricrin could be blocked in vivo using recombinant loricrin. Pre-incubation with recombinant loricrin L2v abolished the ability of ClfB to bind to native ligands in the skin. Throughout the infection period, mice infected with LAC::lux pre-incubated with L2v had reduced bacterial burden and abscess lesion size compared to the control group. These data reveal a novel, previously unidentified role for ClfB-loricrin binding in the pathogenesis of S. aureus SSTI; thus, targeting this pathway may represent a novel approach for the prevention of S. aureus SSTI. Interestingly, ClfB has also been shown to be a major player in mediating S. aureus adhesion to skin corneocytes in atopic dermatitis[26]. We therefore decided to investigate if vaccination with ClfB could protect against subsequent skin infection. Vaccination with ClfB has previously been shown to reduce murine nasal colonization[14], and vaccination with ClfB in combination with Freund’s adjuvant reduced bacterial burden in the kidneys of intravenously infected mice[27]. However, vaccination with ClfB has never previously been shown to protect against skin infection. In fact, only a handful of studies have attempted to vaccinate against S. aureus skin infection. These have included vaccination with ClfA[12], Als3p[28] and adenosine synthase A[29] in combination with alum and SasX combined with Freund’s adjuvant[30]. These vaccines have had variable efficacy, with only modest protection against SSTIs when ClfA[12] was used as vaccine antigen, supporting the notion that multiple CWA proteins may need to be targeted simultaneously, whereas Als3p[28] and adenosine synthase[29] offered significant protection. In the case of SasX[30], Freund’s adjuvant was used, limiting the potential for translation to humans. This study utilised the adjuvant, CpG, which due to its low reactogenicity and strong adjuvanticity is currently considered a prime adjuvant for future vaccines against infectious diseases[31]. In this current study, a model vaccine was formulated with ClfB in combination with CpG which has successfully driven Th1 and Th17 responses during immunization in mice[32–36]. Immunization with ClfB+CpG induced significant expansion of antigen-specific CD4+ and CD8+ T cells capable of producing IFNγ, IL-17 and IL-22. This correlates with our previous findings which demonstrates ClfB is capable of inducing antigen-specific T cell responses in human CD4+ T cells by inducing the activation of Th1 and Th17 cells[34]. IL-17 and IL-22 cytokine responses are known to be particularly important for protection during cutaneous infection with S. aureus through these cytokines ability to promote neutrophil recruitment to the infection site and also to drive local production of antimicrobial peptides, both important effector mechanisms for effective clearance of the bacterium[28, 37–40]. Vaccination with ClfB+CpG also activated humoral immune responses by inducing significant levels of ClfB-specific neutralizing antibodies compared to controls. This demonstrates that ClfB can activate both the humoral and cellular arms of the immune system which combined led to a substantial reduction in the severity of the subsequent LAC::lux skin infection, with reduced bacterial burden throughout the course of infection and a significant reduction in the abscess lesion area when compared to the adjuvant alone control group. Since ClfB is exposed on the bacterial cell surface and interacts directly with loricrin, we propose that antibodies produced in response to vaccination with ClfB may provide a protective effect by promoting opsonophagocytosis and by interfering with binding of ClfB to its ligand loricrin, which we have shown is present within the damaged skin and thus delays and prevents formation of a robust abscess structure. Importantly we demonstrated that direct blockade of the ClfB-loricrin interaction in vivo using recombinant L2v significantly inhibited pathogenesis in this model. Simultaneously the expansion of ClfB-specific T cells producing effector cytokines such as IFNγ, IL-17 and IL-22 and their downstream effects on phagocytes[41–43] and the induction of antimicrobial peptide expression[37, 44, 45] will directly contribute to the clearance of invading bacteria. Furthermore IL-22 has previously been shown to down regulate the expression of loricrin[46], which would reduce the levels available for ClfB to bind to thus further inhibiting abscess formation. Taken together, the results of this study highlight the importance of ClfB as a virulence determinant during S. aureus SSTIs which plays a role in determining the bacterial burden at the site of infection but most importantly identifies for the first time how ClfB also affects the structure and formation of the skin abscess. ClfB exerts its effect in the early stage of infection and its interaction with loricrin appears to play a role during pathogenesis. This interaction is likely specific to the skin as previous studies have demonstrated no difference in systemic S. aureus infection in WT and Lor-/- mice[13]. The data presented herein therefore support the targeting of ClfB in future vaccines specifically for the prevention of S. aureus SSTIs. These vaccines will likely be multivalent and will potentially include multiple cell wall-anchored proteins in addition to secreted proteins, and will be required to drive protective cellular in addition to humoral responses.
Staphylococcus aureus is the leading cause of skin and soft tissue infections ( SSTIs ) , the treatment of which is becoming increasingly difficult due to antibiotic resistance . An anti-S . aureus vaccine offers a potential solution , but a better understanding of how S . aureus causes pathology during SSTI is required to identify effective vaccine targets . Here , we identify an important virulence determinant during S . aureus SSTI . Clumping factor B ( ClfB ) , a surface protein expressed by S . aureus is shown to promote skin abscess formation by binding to the host protein loricrin . Targeting ClfB using a model vaccine conferred significant protection during S . aureus SSTI . In this study , we uncover an entirely novel mechanism by which S . aureus forms abscesses during skin infection , identifying an important therapeutic target for treating S . aureus SSTI .