Structure‐function of CWA proteins.
Staphylococcus aureus is a commensal bacterium that causes infections such as sepsis, endocarditis, and pneumonia. S. aureus can express a variety of virulence factors, including surface proteins. Surface proteins are characterized by presence of a Sec‐dependent signal sequence at the amino terminal, and the sorting signal domain. Surface proteins are covalently attached to peptidoglycan and they are commonly known as cell wall–anchored (CWA) proteins. CWA proteins have many functions and participate in the pathogenesis of S. aureus. Furthermore, these proteins have been proposed as therapeutic targets for the generation of vaccines. In this chapter, different topics related to CWA proteins of S. aureus are addressed. The molecular structure of CWA proteins and their role as virulence factors of S. aureus are described. Furthermore, the involvement of CWA proteins in the processes of adhesion, invasion of host cells and tissues, evasion of the immune response, and the formation of biofilm is discussed. In addition, the role of CWA proteins in skin infection and the proposal to use them as potential vaccine antigens are described. The information contained in this chapter will help the readers to understand the biology of CWA proteins and to recognize the importance of surface molecules of S. aureus.
- Staphylococcus aureus
- CWA proteins
- surface proteins
2. Structure of CWA proteins
All CWA proteins contain a Sec‐dependent signal sequence at the amino terminal and at the carboxyl terminal a sorting signal and a hydrophobic domain (wall‐spanning W). The sorting signal domain contains the characteristic motif for breaking by the sortase LPXTG (Leu‐Pro‐X‐Thr‐Gly; wherein X represents any amino acid). The hydrophobic domain retains the protein in the bacterial membrane during secretion, so that the sortase can join and carry out its transpeptidase function. Between the amino and carboxyl terminal domains, there are different regions or functional domains. Based on its molecular structure and arrangement, the CWA proteins of
|Protein family||Structural motifs and domains*||Proteins||Function during infection|
|(1) MSCRAMMClf‐Sdr||N‐terminal A region (comprises subdomains N1, N2, N3); BSDR repeats(in SdrC, SdrD and SdrE); R region (known as SD region, contains serine‐aspartate repeats)||ClfAa||Adhesion to fibrinogen; degradation of C3b. Immune evasion|
|ClfB||Fibrinogen, keratin and loricrin binding. Nasal colonization by adhesion to desquamated epithelial cells|
|SdrC||β‐Neurexin binding. Adhesion to desquamated nasal epithelial cells|
|SrdD||Adhesion to desquamated nasal epithelial cells|
|FnBp||A region (subdomains N1, N2, N3); R region (contains fibronectin‐binding repeats)||FnBpA||Fibrinogen, fibronectin and elastin binding. Adhesion to extracellular matrix; cell host invasion.|
|FnBpB||Fibronectin‐binding. Adhesion to extracellular matrix; cell host invasion|
|Cna||A region (subdomains N1, N2, N3);BCNA repeats||Cna||Collagen binding. Adhesion to extracellular matrix|
|(2) NEAT||Near iron transporter motif; C‐terminal hydrophilic stretch (in IsdA)||IsdA||Heme, fibrinogen, fibronectin, cytokeratin and loricrin binding. Heme capture and iron uptake; nasal colonization|
|IsdBa||Heme, hemoglobin and 3β integrins binding. Heme capture and iron uptake; invasion of non‐phagocytic cells|
|IsdH||Heme, hemoglobin binding. Heme capture and iron acquisition; immune evasion by C3b degradation|
|(3) Three helical bundle||Tandemly linked triple‐helical bundle domains (known as EABCD);repeat‐containing Xr region; nonrepetitive Xc region||Protein A||IgG, IgM and TNRF1 binding. Evasion of immunity; increased inflammation during pneumonia and skin infection|
|(4) G5‐E||A region; alternating repeats of G5and E domains||SasG||Adhesion to desquamated epithelial cells; formation of biofilm|
2.1. The MSCRAMM family
The main feature of this family of proteins is its structural similarity and its mechanism for binding the ligand. The general structure of these proteins is a domain A at the amino terminal and a region R. The A domain is divided into subdomains: NI, N2, and N3, which integrate the ligand‐binding domain. The N2 and N3 subdomains form folding structures IgG‐like  that are important to form the ligand‐binding site. With respect to ligand‐binding mechanism of these proteins, they bind to fibrinogen through the mechanism “dock, lock, and latch” (DLLs) by N2 and N3 subdomains. The DLL mechanism occurs when the ligand dock to the open apo form and conformation changes create a closed form, in which the ligands are locked into a place . Clumping factor A (ClfA) and ClfB proteins of
The R region of the Sdr‐ and Clf proteins is composed by repeated Ser‐As, known as the SD region. However in the fibronectin‐binding protein A (FnBPA) or (FnBPB), the R region contains repeated fibronectin‐binding, which have the function of mediating ligand binding. The SdrC, SdrD, SdrE, and bone sialo‐binding protein (BBP) proteins, which are MSCRAMM of
An atypical MSCRAMM protein is collagen adhesin (Cna). This protein binds to collagen, also has a domain A in its N‐terminal and it is divided into three subdomains N1, N2, and N3. The Cna differs from the other members MSCRAMMs because its ligand‐binding domain (IgG‐folded) is composed of the N1 and N2 subdomains, and not composed of the N2 and N3 subdomains as other MSCRAMMs typical. Furthermore, the space between domain A and the cell wall‐spanning W domain consists of a variable number of repeated BCNA domains, which are different from BSDR subdomains. Another difference is that the Cna has a different ligand‐binding mechanism named as collagen hug.
2.2. NEAT motif family
The main feature of this family is the presence of near iron transporter (NEAT) motifs, which recognize and bind to heme or hemoglobin. Proteins iron‐regulated surface (Isd) A, B, and H contain NEAT motif (one NEAT motif for IsdA, two NEAT motif for IsdB, and three NEAT motif for IsdH) and these proteins are involved in the capture of heme from the hemoglobin. Isd is important for the survival of the bacterium into the host, where the iron is limited. Besides, Isd is involved actively in the metabolism of heme. Heme binds to Isd, and then heme binds to a membrane transporter protein, which transfers heme into the cytoplasm. In the cytoplasm, the iron is released from heme by hemoxygenases [4, 11]. The structure of the NEAT domain has been elucidated and the molecular mechanism of ligand‐binding was determined . Other Isd proteins can bind different ligands to the heme group, as the case IsdA that binds to fibrinogen, fibronectin, cytokeratin 10, and loricrin; and IsdB that binds to 3
2.3. Three‐helical bundle family
The main feature of this family is the presence of several single separately‐folded three‐helical bundles. Protein A of
Other proteins of
2.4. The G5‐E repeat family
The basic structure of this family is G5‐E repeat domain. Each domain G5 has five conserved glycine residues, which adopt a folding of
2.5. Other CWA protein families
There are other CWA proteins with different functional domains such as the legume lectin and the nucleotidase. These two groups of CWA proteins are classified outside the four families mentioned above, because they are not exclusive of
3. Posttranslational modifications of CWA proteins
The MSCRAMMs proteins achieve proteolytic posttranslational modification in the domain A. Proteases that remove subdomain N1 of MSCRAMMs are located on the bacterial cell surface. Proteolytic processing is conducted by a staphylococcal protease, called aureolysin, which cleaves between the subdomains N1 and N2 of ClfB and ClfA. For FnBPA, there is not a staphylococcal protease, the responsible of this processing is the thrombin of the host. Removal of N1 of ClfB can decrease the length of the protein and cause lack of binding fibrinogen . It is thought that the elimination of N1 subdomain reduces the ability of
Another posttranslational modification is the glycosylation of proteins Clf‐Sdr. It has been shown that the glycosyltransferases SdgB and SdgA of
4. CWA proteins as virulence factors
The generation of mutants is a useful tool to know the function of a gene; however, the study of CWA proteins has been complicated because the generation of defective mutants of CWA protein had generated, in some cases, unexpected results due to functional redundancy. For example,
Despite the difficulties mentioned above, the role of CWA proteins in virulence has been studied. Human population (20%) is permanently colonized by
4.1. CWA proteins in the invasion of epithelial and endothelial cells
In the case of FnBPA and FnBPB proteins, the binding of these proteins to fibronectin facilitates
4.2. Immune system and inflammation
The CWA proteins are involved in immune evasion. Protein A binds to the Fc region of IgG, this binding leads to an incorrect orientation of IgG antibody, preventing the recognition of the bacterium by neutrophils and the activation of the classical complement pathway . Furthermore, it has been demonstrated that in pulmonary epithelial cells, protein A is capable of interacting with tumor necrosis factor receptor 1 (TNFR1), triggering the production of interleukin‐8 (IL‐8) and the neutrophil recruitment, promoting inflammation and tissue damage . Also it has been reported the involvement of protein A in the production of interferon β (IFNβ) and IL‐6 in a mouse pneumonia model .
ClfA and Can are involved in evading the immune system by recruiting regulators of complement pathway . Furthermore, ClfA is involved in bacterial survival by binding to fibrinogen, because in a sepsis model this interaction reduces the probability of
One of the major virulence factors of
5. Involvement of CWA proteins in skin infections
The study of the participation of CWA proteins in skin infections and abscess formation has been achieved mainly in animal models with CWA protein mutant strains of
|Infection model||Mutant CWA in ||Result|
|Murine kidney abscess||Sortase||No abscess formation in the kidneys|
|Murine skin infection||ClfA||Decreased CFU in the skin abscess|
|Murine skin infection||FnBPA and FnBPB||Decreased CFU in the skin abscess|
|Murine skin infection||SasX||Smaller abscesses in the skin|
|Mice inoculated subcutaneously||Protein A||Decreased CFU in the skin abscesses|
|Rabbit skin infection||Wild‐type||High transcription level of the |
In a murine skin abscess model, infected with
6. CWA proteins as vaccines
Currently, there is a proposal to use recombinant CWA proteins as potential vaccine antigens. In animal models, the use of CWA proteins has induced immunological protection against
6.1. CWA proteins such as T‐cell antigen
Up to date, the mechanism of immune system activation by CWA proteins is unknown except for the protein A that binds to TNFR1 and induces the production of interleukin‐8 (IL‐8) and the neutrophil recruitment . The anti‐
Currently, ClfA protein is used in multivalent vaccines. Thus, the vaccine designed by Pfizer, with the status of Phase II clinical trials, is made with ClfA antigens, capsular polysaccharide MNTC, and two proteins (CP5 and CP8) [76, 77]. This vaccine induces a high production of antibodies; however, there are no studies on its cellular immunity. NovaDigm I developed a vaccine with homologues of ClfA and Als3p ; in phase I clinical test, the vaccine showed an increase in the production of specific antibody titer and induced Th1 and Th17 cell response in humans . ClfA is emerging as a potent stimulator of T‐cells and it is a promising antigen vaccine development; however, there is little research on the potential of other CWA proteins to activate T‐cells. Therefore, studies to determine which CWA proteins cause a high T‐cell response should be performed to identify potential proteins for future vaccines.
Although it has recognized the role and ligands for some CWA proteins of
On the other hand, the immune response of the CWA protein also requires more studies, since the mechanism by which CWA proteins interfere with the host innate immune response is unknown, in particular regulation of complement activation. In addition, determining CWA proteins causing a cellular immune response is crucial for the generation of new vaccines.
Most studies of CWA proteins have been conducted with laboratory strains. These studies should be extended in clinical isolates, where the variation of ligand binding of the CWA proteins is considerable. Additionally, the regulatory system of the expression of CWA proteins is still insufficient, because the expression of CWA proteins depends on the strain understudy.
Surface proteins have a wide range of functions that are essential for colonization and survival of
This work was supported by the Grant SIP20160325, SIP20161111, and SIP20160135 from Instituto Politécnico Nacional (IPN). JCCD, JJR, SRM, and MECD appreciate the COFAA and EDI, IPN fellowships, and the support provided by SNI-CONACYT. EGG thanks the CONACYT Postdoctoral fellowship and the support provided by SNI‐CONACYT.