Multiple mechanisms for the activation of human platelet aggregation by Staphylococcus aureus: roles for the clumping factors ClfA and ClfB,the serine-aspartate repeat protein SdrE and protein A

The ability of Staphylococcus aureus cells to induce platelet aggregation has long been recognized. However, despite several attempts to identify the mechanisms involved in this interaction, the nature of the bacterial receptors required remains poorly understood. Using genetic manipulation, this study for the first time provides clear evidence that several S. aureus surface proteins participate in the inter-action with platelets. Mutants of S. aureus strain Newman lacking one or more surface proteins were tested for their ability to stimulate platelet aggregation. This approach was complemented by the expression of a number of candidate proteins in the non-aggregating Gram-positive bacterium Lacto-coccus lactis. S. aureus-induced aggregation was monophasic and was dependent on the platelet receptor GPIIb/IIIa. The fibrinogen-binding proteins, clumping factors A and B and the serine-aspartate repeat protein SdrE could each induce aggregation when expressed in L. lactis. Although protein A expressed in L. lactis was not capable of inducing aggregation independently, it enhanced the aggregation response when expressed on the surface of S. aureus. Thus, S. aureus has multiple mechanisms for stimulating platelet aggregation. Such functional redundancy suggests that this phenomenon may be important in the pathogenesis of invasive diseases such as infective endocarditis.     

Crystal Structures Reveal the Multi-Ligand Binding Mechanism of Staphylococcus aureus ClfB

Staphylococcus aureus (S. aureus) pathogenesis is a complex process involving a diverse array of extracellular and cell wall components. ClfB, an MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules) family surface protein, described as a fibrinogen-binding clumping factor, is a key determinant of S. aureus nasal colonization, but the molecular basis for ClfB-ligand recognition remains unknown. In this study, we solved the crystal structures of apo-ClfB and its complexes with fibrinogen α (Fg α) and cytokeratin 10 (CK10) peptides. Structural comparison revealed a conserved glycine-serine-rich (GSR) ClfB binding motif (GSSGXGXXG) within the ligands, which was also found in other human proteins such as Engrailed protein, TCF20 and Dermokine proteins. Interaction between Dermokine and ClfB was confirmed by subsequent binding assays. The crystal structure of ClfB complexed with a 15-residue peptide derived from Dermokine revealed the same peptide binding mode of ClfB as identified in the crystal structures of ClfB-Fg α and ClfB-CK10. The results presented here highlight the multi-ligand binding property of ClfB, which is very distinct from other characterized MSCRAMMs to-date. The adherence of multiple peptides carrying the GSR motif into the same pocket in ClfB is reminiscent of MHC molecules. Our results provide a template for the identification of other molecules targeted by S. aureus during its colonization and infection. We propose that other MSCRAMMs like ClfA and SdrG also possess multi-ligand binding properties.     

Identification of novel inhibitors of bacterial surface enzyme Staphylococcus aureus Sortase A

In-silico virtual screening of bacterial surface enzyme Staphylococcus aureus Sortase A against commercial compound libraries using FlexX software package has led to the identification of novel inhibitors. Inhibition of enzyme catalytic activity was determined by monitoring the steady state cleavage of a model peptide substrate. Preliminary structure activity relationship studies on the lead compound resulted in the identification of compounds with improved activity. The most active compound has an IC50 value of 58 microM against the enzyme.     

Discovery and development of substituted thiadiazoles as inhibitors of Staphylococcus aureus Sortase A

High-throughput screening of small-molecule libraries has led to the identification of thiadiazoles as a new class of inhibitors against Staphylococcus aureus sortase A (SrtA). N-(5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide (IC₅₀ = 3.8 µM) was identified as a potent inhibitor of SrtA after synthetic modification of hit compounds. Additional ligands developed in this study displayed affinities in the low micromolar range without affecting bacterial growth in vitro. The study also suggest a new mode of action through covalent binding to the active site cysteine.     

牛源金黄色葡萄球菌粘附因子Efb与ClfA的免疫生物学特性比较

为评价与比较金黄色葡萄球菌的粘附因子Efb(Extracellular fibrinogen-binding protein)和Clf A(Clumping factor A)的抗原性、粘附特性及其抗血清对金黄色葡萄球菌菌体的识别能力、抗粘附能力及免疫保护力等免疫生物学特性,分别构建Efb和Clf A的重组表达载体,并用纯化的重组蛋白免疫实验动物,分别检测家兔抗血清对菌体的识别能力、抗粘附能力以及小鼠抗血清的抗体效价和免疫保护作用。结果显示,Efb和Clf A均有与纤维蛋白原(Fibrinogen,Fg)结合的能力,且Efb蛋白对纤连蛋白(Fibronectin,Fn)的结合能力优于Clf A(P0.01),Clf A免疫组抗血清对全细菌的识别能力优于Efb免疫组(P0.01)。与对照组相比Efb和Clf A免疫组的抗血清均能显著抑制金黄色葡萄球菌对Fg和Fn的粘附(P0.01),Efb免疫组对金黄色葡萄球菌的粘附抑制优于Clf A,两种粘附因子免疫小鼠后产生的血清抗体效价与对照组比较有显著的升高,抗体滴度可达1∶40 500,攻毒结果显示Efb与Clf A免疫组均对小鼠具有良好的保护效果。该研究结果对Efb在金黄色葡萄球菌的亚单位疫苗的应用奠定了基础。     

The binding mechanism,multiple binding modes,and allosteric regulation of Staphylococcus aureus Sortase A probed by molecular dynamics simulations

Sortase enzymes are vitally important for the virulence of gram‐positive bacteria as they play a key role in the attachment of surface proteins to the cell wall. These enzymes recognize a specific sorting sequence in proteins destined to be displayed on the surface of the bacteria and catalyze the transpeptidation reaction that links it to a cell wall precursor molecule. Because of their role in establishing pathogenicity, and in light of the recent rise of antibiotic‐resistant bacterial strains, sortase enzymes are novel drug targets. Here, we present a study of the prototypical sortase protein Staphylococcus aureus Sortase A (SrtA). Both conventional and accelerated molecular dynamics simulations of S. aureus SrtA in its apo state and when bound to an LPATG sorting signal (SS) were performed. Results support a binding mechanism that may be characterized as conformational selection followed by induced fit. Additionally, the SS was found to adopt multiple metastable states, thus resolving discrepancies between binding conformations in previously reported experimental structures. Finally, correlation analysis reveals that the SS actively affects allosteric pathways throughout the protein that connect the first and the second substrate binding sites, which are proposed to be located on opposing faces of the protein. Overall, these calculations shed new light on the role of dynamics in the binding mechanism and function of sortase enzymes.     

Single mutation on the surface of Staphylococcus aureus Sortase A can disrupt its dimerization

Staphylococcus aureus Sortase A (SrtA) is an important Gram-positive membrane enzyme which catalyzes the anchoring of many cell surface proteins conserved with the LPXTG sequence. Recently SrtA has been demonstrated to be a dimer with a Kd of 55 microM in vitro. Herein, we show that a single point mutation of amino acid residue on the surface of SrtA can completely disrupt the dimerization. Native polyacrylamide gel electrophoresis and analytical gel filtration chromatography were used to detect the dimer-monomer equilibrium of SrtA mutants. Circular dichroism spectrum experiments were performed to study the conformational change of each SrtA mutant. An enzyme activity assay confirmed that all the SrtA mutants were active in vitro. Our results not only are important for understanding the SrtA protein self-associating mechanism but also provided the necessary starting materials for the study of sortase A pathway in vivo, which may have significant implications for discovering microbial physiology and give a potential target for novel Gram-positive antibiotics.     

The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a bifunctional protein that also binds to fibrinogen

Staphylococcus aureus is an important pathogen capable of causing a wide spectrum of diseases in humans and animals. This bacterium expresses a variety of virulence factors that participate in the process of infection. These include MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that mediate the adherence of the bacteria to host extracellular matrix components, such as collagen, fibronectin (Fn), and fibrinogen (Fg). Two Fn-binding MSCRAMMs, FnbpA and FnbpB, have been previously identified. The Fn binding activity has been localized to the approximately 40-amino acid residue D repeats in the C-terminal part of these proteins. However, no biological activity has yet been attributed to the N-terminal A regions of these proteins. These regions exhibit substantial amino acid sequence identity to the A regions of other staphylococcal MSCRAMMs, including ClfA, ClfB, and SdrG (Fbe), all of which bind Fg. This raises the question of whether the Fn-binding MSCRAMMs can also bind specifically to Fg. In this report, we show that a recombinant form of the A region of FnbpA does specifically recognize Fg. We localize the binding site in Fg for recombinant FnbpA to the gamma-chain, in particular to the C-terminal residues of this polypeptide, the site also recognized by ClfA. In addition, we demonstrate that recombinant FnbpA can compete with ClfA for binding to both immobilized and soluble Fg. By the use of surface plasmon resonance spectroscopy and fluorescence polarization, we determine the dissociation equilibrium constant for the interaction of recombinant FnbpA with intact immobilized Fg and with a synthetic C-terminal gamma-chain peptide, respectively. Finally, by overexpressing FnbpA in a mutant strain of S. aureus that lacks the expression of both ClfA and ClfB, we show that native FnbpA can mediate the interaction of S. aureus with soluble Fg.     

Clumping factor B (ClfB), a new surface-located fibrinogen-binding adhesin of Staphylococcus aureus

The surface-located fibrinogen-binding protein (clumping factor; ClfA) of Staphylococcus aureus has an unusual dipeptide repeat linking the ligand binding domain to the wall-anchored region. Southern blotting experiments revealed several other loci in the S. aureus Newman genome that hybridized to a probe comprising DNA encoding the dipeptide repeat. One of these loci is analysed here. It also encodes a fibrinogen-binding protein, which we have called ClfB. The overall organization of ClfB is very similar to that of ClfA, and the proteins have considerable sequence identity in the signal sequence and wall attachment domains. However, the A regions are only 26% identical. Recombinant biotinylated ClfB protein bound to fibrinogen in Western ligand blots. ClfB reacted with the α- and β-chains of fibrinogen in the ligand blots in contrast to ClfA, which binds exclusively to the γ-chain. Analysis of proteins released from the cell wall of S. aureus Newman by Western immunoblotting using antibody raised against the recombinant A region of ClfB identified a 124 kDa protein as the clfB gene product. This protein was detectable only on cells that were grown to the early exponential phase. It was absent from cells from late exponential phase or stationary phase cultures. Using a clfB mutant isolated by allelic replacement alone and in combination with a clfA mutation, the ClfB protein was shown to promote (i) clumping of exponential-phase cells in a solution of fibrinogen, (ii) adherence of exponential-phase bacteria to immobilized fibrinogen in vitro, and (iii) bacterial adherence to ex vivo human haemodialysis tubing, suggesting that it could contribute to the pathogenicity of biomaterial-related infections. However, in wild-type exponential-phase S. aureus Newman cultures, ClfB activity was masked by the ClfA protein, and it did not contribute at all to interactions of cells from stationary-phase cultures with fibrinogen. ClfB-dependent bacterial adherence to immobilized fibrinogen was inhibited by millimolar concentrations of Ca²⁺ and Mn²⁺, which indicates that, like ClfA, ligand binding by ClfB is regulated by a low-affinity inhibitory cation binding site.     

An exhaustive yet simple virtual screening campaign against Sortase A from multiple drug resistant Staphylococcus aureus

Methicillin resistant Staphylococcus aureus (MRSA) is one of the challenging bacterial pathogen due to its acquired resistance to the β lactam antibiotics. The Sortase A is an enzyme of Gram-positive bacteria including S. aureus to anchor surface proteins to the cell wall. Sortase A is well studied enzyme and considered as the drug target against MRSA. Sortase A plays active role in anchoring the virulence proteins on the cell wall of the Gram-positive bacteria. The inhibition of Sortase A activity results in the separation of S. aureus from the host cells and ultimately alleviation of the infection. Here, we adapted a structure-based virtual screening protocol which helped in identification of novel potential inhibitors of Sortase A. The protocol involved the docking of a chemical library of druglike compounds with the Sortase A binding site represented by multiple crystal structures. The compounds were ranked by multiple scoring functions and shortlisted for future experimental screening. The method resulted in shortlisting of three compounds as potential novel inhibitors of Sortase A out of a large chemical library. The high rankings of shortlisted compounds estimated by multiple scoring functions showed their binding potential with Sortase A. The results are proved to be a simple yet efficient choice of structure-based virtual screening. The identified compounds are druglike and show high rankings among all set protocols of the virtual screening. We hope that the study would eventually help to expedite the discovery of novel drug candidates against MRSA.     

Staphylococcus aureus Sortase A-Mediated Incorporation of Peptides: Effect of Peptide Modification on Incorporation

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall.     

Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole

Sortase cysteine transpeptidases covalently attach proteins to the bacterial cell wall or assemble fiber-like pili that promote bacterial adhesion. Members of this enzyme superfamily are widely distributed in Gram-positive bacteria that frequently utilize multiple sortases to elaborate their peptidoglycan. Sortases catalyze transpeptidation using a conserved active site His-Cys-Arg triad that joins a sorting signal located at the C terminus of their protein substrate to an amino nucleophile located on the cell surface. However, despite extensive study, the catalytic mechanism and molecular basis of substrate recognition remains poorly understood. Here we report the crystal structure of the Staphylococcus aureus sortase B enzyme in a covalent complex with an analog of its NPQTN sorting signal substrate, revealing the structural basis through which it displays the IsdC protein involved in heme-iron scavenging from human hemoglobin. The results of computational modeling, molecular dynamics simulations, and targeted amino acid mutagenesis indicate that the backbone amide of Glu²²⁴ and the side chain of Arg²³³ form an oxyanion hole in sortase B that stabilizes high energy tetrahedral catalytic intermediates. Surprisingly, a highly conserved threonine residue within the bound sorting signal substrate facilitates construction of the oxyanion hole by stabilizing the position of the active site arginine residue via hydrogen bonding. Molecular dynamics simulations and primary sequence conservation suggest that the sorting signal-stabilized oxyanion hole is a universal feature of enzymes within the sortase superfamily.     

金黄色葡萄球菌ClfA蛋白Th表位鉴定及其免疫原性分析

【目的】ClfA蛋白是金黄色葡萄球菌粘附于宿主细胞的最主要粘附因子之一,筛选鉴定ClfA蛋白的Th表位,研制基于表位的联合疫苗将成为该菌疫苗新的发展方向。【方法】本研究采用生物信息学软件预测ClfA可能的H-2d限制性Th表位,并以CD4+T淋巴细胞增殖实验及流式细胞分析进行筛选和鉴定。【结果】获得BALB/c小鼠H-2d限制性Th1表位(C335)和Th2表位(C214,C286和C436)。【结论】筛选出优势性Th细胞表位,并对表位的免疫学特性进行实验免疫研究,为最终研制保护作用强、安全性高的新型疫苗奠定基础。     

Sortase from Staphylococcus aureus does not contain a thiolate-imidazolium ion pair in its active site

Many surface proteins are anchored to the cell wall by the action of sortase enzymes, a recently discovered family of cysteine transpeptidases. As the surface proteins of human pathogens are frequently required for virulence, the sortase-mediated anchoring reaction represents a potential target for new anti-infective agents. It has been suggested that the sortase from Staphylococcus aureus (SrtA), may use a similar catalytic strategy as the papain cysteine proteases, holding its Cys184 side chain in an active configuration through a thiolate-imidazolium ion interaction with residue His120. To investigate the mechanism of transpeptidation, we have synthesized a peptidyl-vinyl sulfone substrate mimic that irreversibly inhibits SrtA. Through the study of the pH dependence of SrtA inhibition and NMR, we have estimated the pKas of the active site thiol (Cys184) and imidazole (His120) to be approximately 9.4 and 7.0, respectively. These measurements are inconsistent with the existence of a thiolate-imidazolium ion pair and suggest a general base catalysis mechanism during transpeptidation.     

A Novel Gene,fudoh, in the SCCmec Region Suppresses the Colony Spreading Ability and Virulence of Staphylococcus aureus

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis.