Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA

The Staphylococcus aureus sortase transpeptidase SrtA isoform is responsible for the covalent attachment of virulence and colonization-associated proteins to the bacterial peptidoglycan. SrtA utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-D-isoGlu-Lys(epsilon-Gly(5))-D-Ala-D-Ala (branched Lipid II) and secreted proteins containing a highly conserved C-terminal LPXTG sequence. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly(5) portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we describe the development of a general in vitro method for elucidating the substrate specificity of sortase enzymes. In addition, using immunofluorescence, cell adhesion assays, and transmission electron microscopy, we establish links between in vitro substrate specificity and in vivo function of the S. aureus sortase isoforms. Results from these studies provide strong supporting evidence of a primary role of the SrtA isoform in S. aureus adhesion and host colonization, illustrate a lack of specificity cross talk between SrtA and SrtB isoforms, and highlight the potential of SrtA as a target for the development of antivirulence chemotherapeutics against Gram-positive bacterial pathogens.     

Kinetic mechanism of Staphylococcus aureus sortase SrtA

Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. We have also demonstrated in this study that the nucleophile binding site of S. aureus sortase SrtA is specific for diglycine. While S1' and S2' sites of the enzyme both prefer a glycine residue, the S1' site is exclusively selective for glycine. Lengthening of the polyglycine acceptor nucleophile beyond diglycine does not further enhance the binding and catalysis.     

Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).     

Development of a high-performance liquid chromatography assay and revision of kinetic parameters for the Staphylococcus aureus sortase transpeptidase SrtA

The SrtA isoform of the Staphylococcus aureus sortase transpeptidase is responsible for the covalent attachment of virulence- and colonization-associated proteins to the bacterial peptidoglycan. Sortase utilizes two substrates, undecaprenol-pyrophosphoryl-MurNAc(GlcNAc)-Ala-d-isoGlu-Lys(-Gly5)-d-Ala-d-Ala (branched Lipid II) and secreted proteins containing a highly conserved LPXTG sequence near their C termini. SrtA simultaneously cleaves the Thr-Gly bond of the LPXTG-containing protein and forms a new amide bond with the nucleophilic amino group of the Gly5 portion of branched Lipid II, anchoring the protein to this key intermediate that is subsequently polymerized into peptidoglycan. Here we show that reported fluorescence quenching activity assays for SrtA are subject to marked fluorescence inner filter effect quenching, resulting in prematurely hyperbolic velocity versus substrate profiles and underestimates of the true kinetic parameters kcat and Km. We therefore devised a discontinuous high-performance liquid chromatography (HPLC)-based assay to monitor the SrtA reaction employing the same substrates used in the fluorescence quenching assay: Gly5 and Abz-LPETG-Dap(Dnp)-NH2. Fluorescence or UV detection using these substrates facilitates separate analysis of both the acylation and the transpeptidation steps of the reaction. Because HPLC was performed using fast-flow analytical columns (<8min/run), high-throughput applications of this assay for analysis of SrtA substrate specificity, kinetic mechanism, and inhibition are now feasible. Kinetic analysis using the HPLC assay revealed that the kinetic parameters for SrtA with Abz-LPETG-Dap(Dnp)-NH2 are 5.5mM for Km and 0.27s-1 for kcat. The Km for Gly5 was determined to be 140microM. These values represent a 300-fold increase in Km for the LPXTG substrate and a 12,000-fold increase in kcat over literature-reported values, suggesting that SrtA is more a robust enzyme than previous analyses indicated.     

Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA

In Staphylococcus aureus, virulence and colonization-associated surface proteins are covalently anchored to the cell wall by the transpeptidase Sortase A (SrtA). In order to better understand the contribution of specific active site residues to substrate recognition and catalysis, we performed mutational analysis of several key residues in the SrtA active site. Analysis of protein stability, kinetic parameters, solvent isotope effects, and pH-rate profiles for key SrtA variants are consistent with a reverse protonated Cys184-His120 catalytic dyad, and implicate a role for Arg197 in formation of an oxyanion hole to stabilize the transition state. In contrast, mutation of Asp185 and Asp186 produced negligible effects on catalysis, and no evidence was found to support the existence of a functional catalytic triad. Mutation of Thr180, Leu181, and Ile182 to alanine produced modest decreases in SrtA activity and led to substrate inhibition. Thermodynamic stability measurements by SUPREX (stability of unpurified proteins from rates of H/D exchange) revealed decreases in conformational stability that correlate with the observed substrate inhibition for each variant, signifying a potential role for the conserved 180TLITC184 motif in defining the active-site architecture of SrtA. In contrast, mutation of Thr183 to alanine led to a significant 1200-fold decrease in kcat, which appears to be unrelated to conformational stability. Potential explanations for these results are discussed, and a revised model for SrtA catalysis is presented.     

Mutagenesis studies of substrate recognition and catalysis in the sortase A transpeptidase from Staphylococcus aureus

The Staphylococcus aureus transpeptidase sortase A (SrtA) is responsible for anchoring a range of virulence- and colonization-associated proteins to the cell wall. SrtA recognizes substrates that contain a C-terminal LPXTG motif. This sequence is cleaved following the threonine, and an amide bond is formed between the threonine and the pentaglycine cross-bridge of branched lipid II. Previous studies have implicated the beta6/beta7 loop region of SrtA in LPXTG recognition but have not systematically characterized this domain. To better understand the individual roles of the residues within this loop, we performed alanine-scanning mutagenesis. Val-168 and Leu-169 were found to be important for substrate recognition, and Glu-171 was also found to be important, consistent with its hypothesized role as a Ca(2+)-binding residue. Gly-167 and Asp-170 were dispensable for catalysis, as was Gln-172. The role of Arg-197 in SrtA has been the subject of much debate. To explore its role in catalysis, we used native chemical ligation to generate semi-synthetic SrtA in which we replaced Arg-197 with citrulline, a non-ionizable analog. This change resulted in a decrease of <3-fold in k(cat)/K(m), indicating that Arg-197 utilizes a hydrogen bond, rather than an electrostatic interaction. Our results are consistent with a model for LPXTG recognition wherein the Leu-Pro sequence is recognized primarily by hydrophobic contacts with SrtA Val-168 and Leu-169, as well as a hydrogen bond from Arg-197. This model contradicts the previously proposed mechanism of binding predicted by the x-ray crystal structure of SrtA.     

Inhibition of sortase-mediated Staphylococcus aureus adhesion to fibronectin via fibronectin-binding protein by sortase inhibitors

The sortase enzymes are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus, deletion of the sortase isoforms results in marked reduction in virulence and infection potential, making it an important antivirulence target. Recombinant sortase A (SrtA) and sortase B (SrtB) were incubated with peptide substrate containing either the LPETG or NPQTN motifs. (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile, β-sitosterol-3-O-glucopyranoside, berberine chloride, and psammaplin A1 showed potent inhibitory activity against SrtA and SrtB. These compounds also exhibited potent inhibitory activity against S. aureus cell adhesion to fibronectin. The fibronectin-binding activity data highlight the potential of these compounds for the treatment of S. aureus infections via inhibition of sortase activity.     

Inhibition of the bacterial surface protein anchoring transpeptidase sortase by isoquinoline alkaloids

The inhibitory activity of Coptis chinensis rhizome-derived material was evaluated against sortase, a bacterial surface protein anchoring transpeptidase, from Staphylococcus aureus ATCC 6538p and compared to that of four commercially available isoquinoline alkaloids. The biologically active constituent of C. chinensis extract was characterized as the isoquinoline alkaloid, berberine chloride, by spectral analysis. The isolate was a potent inhibitor of sortase, with an IC50 value of 8.7 microg/ml and had antibacterial activity against Gram-positive bacteria with a minimum inhibitory concentration (MIC) in the range of 50-400 microg/ml. Among the four isoquinoline alkaloids tested, berberine chloride had strong inhibitory activity. These results indicate that berberine is a possible candidate for the development of a bacterial sortase inhibitor.     

Inhibition of the bacterial surface protein anchoring transpeptidase sortase by medicinal plants

Inhibition by medicinal plant extracts of a recombinant sortase was evaluated for antibacterial drug discovery. The coding region of sortase, a transpeptidase that cleaves surface proteins of gram-positive bacteria, was amplified by PCR from the chromosome of Staphylococcus aureus ATCC 6538p with the exception of an N-terminal membrane anchor sequence, expressed in Escherichia coli, and purified by metal chelate affinity chromatography. The purified sortase had maximum activity at pH 7.5 and was stable at 20-45 degrees C for the cleavage of a synthetic fluorophore substrate. The enzyme inhibitory activity in medicinal plants was also evaluated for antibacterial drug discovery. Among 80 medicinal plants tested, Cocculus trilobus, Fritillaria verticillata, Liriope platyphylla, and Rhus verniciflua had strong inhibitory activity. The extract with the greatest activity was the ethyl acetate fraction derived from the rhizome of Cocculus trilobus (IC50 = 1.52 microg/ml).     

Probing of the cis-5-phenyl proline scaffold as a platform for the synthesis of mechanism-based inhibitors of the Staphylococcus aureus sortase SrtA isoform

cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile. 4-Vinylsulfonyl 5-phenyl prolinates inhibit Staphylococcus aureus sortase SrtA irreversibly by modification of the enzyme Cys184 and could be used as hits for the development of antibacterials and antivirulence agents.     

The structure of sortase B, a cysteine transpeptidase that tethers surface protein to the Staphylococcus aureus cell wall

Many surface proteins of Gram-positive bacteria, which play important roles during the pathogenesis of human infections, are anchored to the cell wall envelope by a mechanism requiring sortases. Sortase B, a cysteine transpeptidase from Staphylococcus aureus, cleaves the C-terminal sorting signal of IsdC at the NPQTN motif and tethers the polypeptide to the pentaglycine cell wall cross-bridge. During catalysis, the active site cysteine of sortase and the cleaved substrate form an acyl intermediate, which is then resolved by the amino group of pentaglycine cross-bridges. We report here the crystal structures of SrtBDeltaN30 in complex with two active site inhibitors, MTSET and E64, and with the cell wall substrate analog tripleglycine. These structures reveal, for the first time, the active site disposition and the unique Cys-Arg catalytic machinery of the cysteine transpeptidase, and they also provide useful information for the future design of anti-infective agents against sortases.     

The role of Staphylococcus aureus sortase A and sortase B in murine arthritis

Gram-positive pathogenic bacteria display proteins on their surface that play important roles during infection. In Staphylococcus aureus, these surface proteins are anchored to the cell wall by two sortase enzymes, SrtA and SrtB, that recognize specific surface protein sorting signals. The role of sortase enzymes in bacterial virulence was examined using a murine septic arthritis model. Intravenous inoculation with any of the Delta(srtA), Delta(srtB) or Delta(srtAB) mutants resulted in significantly increased survival and significantly lower weight loss compared with the parental strain. Mice inoculated with the Delta(srtA) mutant did not express severe arthritis, while arthritis in mice inoculated with the Delta(srtB) mutant was not different from that seen in mice that were infected with the wild-type parent strain. Furthermore, persistence of staphylococci in kidneys and joints following intravenous inoculation of mice was more pronounced for wild-type and Delta(srtB) mutant strains than for Delta(srtA) or Delta(srtAB) variants. Together these results indicate that sortase B (srtB) plays a contributing role during the pathogenesis of staphylococcal infections, whereas sortase A (srtA) is an essential virulence factor for the establishment of septic arthritis.     

黄芩素抑制金黄色葡萄球菌生物被膜的形成

细菌生物被膜的形成是导致细菌耐药和引起持续性感染的主要原因之一。本文通过检测黄芩素对金黄色葡萄球菌26112菌株（Staphylococcus aureus 26112,SA26112）多糖细胞间黏附素（polysaccharide intercellular adhesion, PIA）的合成和胞外DNA（extracellular DNA,eDNA）释放量的影响,及其对icaA和cidA基因表达量的影响,探讨黄芩素对金黄色葡萄菌生物被膜形成的抑制作用及其机制。结果显示,黄芩素能抑制SA26112生物被膜的形成,其抑杀SA26112的最低抑菌浓度和最低杀菌浓度均为0.04 mg/mL。0.16 mg/mL黄芩素和256 μg/mL环丙沙星单独作用时,均不能杀死其成熟生物被膜内的SA26112细菌,而当二者联用时则可杀死成熟生物被膜内的细菌。黄芩素能显著抑制SA26112菌株PIA的合成、eDNA的释放量及icaA和cidA基因的相对表达量。其中,0.04 mg/mL黄芩素作用SA26112菌株24 h,与对照组相比,eDNA的释放量减少97%,icaA和cidA基因的相对表达量分别减少62%和41%。上述结果表明,黄芩素能抑制SA26112菌株生物被膜的形成,其作用机制可通过降低icaA和cidA的基因表达量,进而影响PIA的合成和eDNA的释放,来抑制金黄色葡萄球菌生物被膜的形成。     

香芹酚抑制金黄色葡萄球菌生物被膜的形成

【背景】生物被膜是细菌的一种自我保护形式,可以增强细菌对药物及宿主免疫应答的抵抗力,引起细菌耐药性和持续性感染。【目的】探究香芹酚对金黄色葡萄球菌生物被膜的作用机制,为开发新型抗生物被膜药物提供可靠的理论依据。【方法】通过结晶紫染色法检测香芹酚对供试菌株生物被膜形成的抑制和对成熟生物被膜的清除作用;使用刚果红平板法探究香芹酚对供试菌株生物被膜形成过程中细胞间多糖黏附素(polysaccharide intercellular adhesion,PIA)合成的作用;通过分光光度法检测香芹酚对胞外DNA (extracellular DNA,eDNA)分泌的抑制作用;利用RT-PCR技术检测香芹酚对供试菌株的生物被膜相关基因icaA、cidA和sarA转录水平的影响。【结果】香芹酚对生物被膜形成的抑制和生物被膜的清除均有较强作用效果。256μg/mL香芹酚抑制PIA合成和e DNA释放的效果显著。香芹酚可通过抑制相关基因转录从而抑制生物被膜的形成,当64μg/mL的香芹酚作用后,sarA的转录水平降低了60.44%±2.91%,cidA的转录水平降低了76.48%±1.67%,icaA的转...     

Crystal structures of Staphylococcus aureus sortase A and its substrate complex

The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys(184) and Arg(197) residues and at a greater distance from the imidazolium side chain of His(120). All three residues, His(120), Cys(184), and Arg(197), are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria.     

Staphylococcus aureus sortase A exists as a dimeric protein in vitro

We report the first direct observation of the self-association behavior of the Staphylococcus aureus sortase A (SrtA) transpeptidase. Formation of a SrtA dimer was observed under native conditions by polyacrylamide gel electrophoresis and fast protein liquid chromatography (FPLC). Subsequent peptide mass fingerprinting and protein sequencing experiments confirmed the dimeric form of the SrtA protein. Furthermore, SrtA can be selectively cross-linked both in vitro and in Escherichia coli. Multiple samples of enzyme were subjected to analytical sedimentation equilibrium ultracentrifugation to obtain an apparent Kd for dimer formation of about 55 microM. Finally, enzyme kinetic studies suggested that the dimeric form of SrtA is more active than the monomeric enzyme. Discovery of SrtA dimerization may have significant implications for understanding microbial physiology and developing new antibiotics.     

和厚朴酚抑制耐甲氧西林金黄色葡萄球菌生物被膜形成

【目的】研究和厚朴酚(HNK)抑制MRSA生物被膜(BF)形成的作用机制。【方法】使用TTC法测定了HNK对供试菌株BF的形成和成熟BF的抑制作用;刚果红平板法定性检测了HNK对PIA合成的影响;分光光度法测定了HNK对供试菌株eDNA释放量的影响;RT-PCR技术检测了HNK对供试菌株icaA、cidA以及agrA基因表达量的影响。【结果】HNK对MRSA 41573 BF的形成和成熟BF均有较强的抑制作用,其中,HNK抑制MRSA 41573 BF形成的MIC和MBC分别为10μg/mL和20μg/mL;抑制成熟BF的MIC和MBC分别为50μg/mL和100μg/mL。当用亚抑菌浓度的HNK与万古霉素联合作用后,可显著提高成熟BF对万古霉素的敏感性。HNK能显著抑制PIA的合成,且呈浓度剂量依赖。HNK能抑制供试菌株eDNA的释放量,其中1/8 MIC的HNK作用供试菌株16 h后,与对照组相比,e DNA的释放量降低了28.3%。HNK可抑制供试菌株BF形成的相关基因,其中1/2 MIC的HNK作用供试菌株16 h后,与对照相比,icaA的表达量降低了59.1%,cidA的表达量降低了56%,agrA的表达量降低了72.3%。【结论】HNK能显著抑制MRSA 41573 BF的形成,其作用机制主要是通过抑制icaA和cidA基因表达量,影响PIA和eDNA的合成,进而抑制BF的形成。此外HNK也可通过调控细菌的QS系统影响BF的形成。     

冬凌草甲素对金黄色葡萄球菌生物膜的抑制机制

【背景】金黄色葡萄球菌是一种常见的食源性致病菌,易在食品及加工器具表面形成生物膜,引起食品腐败和疾病的传播,威胁食品安全。【目的】研究冬凌草甲素抑制金黄色葡萄球菌生物膜形成的作用机制。【方法】使用结晶紫染色法和扫描电镜观察冬凌草甲素对金黄色葡萄球菌生物膜形成的抑制作用,刚果红平板法定性检测冬凌草甲素对细胞间多糖黏附素(polysaccharideintercellular adhesion,PIA)合成的影响,分光光度法测定冬凌草甲素对供试菌株胞外DNA (eDNA)释放量的影响,RT-PCR技术检测冬凌草甲素对供试菌株ica A、cid A、agr A和sar A基因表达量的影响。【结果】冬凌草甲素对金黄色葡萄球菌生物膜形成有较强的抑制作用;冬凌草甲素能显著抑制PIA的合成,且呈浓度剂量依赖;冬凌草甲素能抑制供试菌株e DNA的释放量,其中1/4最小抑菌浓度(minimum inhibitory concentration,MIC)的冬凌草甲素作用金黄色葡萄球菌16 h后,与对照组相比,e DNA的释放量降低了48.62%;冬凌草甲素可显著抑制金黄色葡萄球菌生物膜形成相关基因的表达,其中1/2MIC的冬凌草甲素作用金黄色葡萄球菌16 h后,ica A、cid A、agr A和sar A基因的表达量分别比对照降低了91.6%、94.7%、77.6%和70.4%。【结论】冬凌草甲素通过抑制ica A和cid A基因的表达,影响PIA的合成和eDNA的释放,进而干预生物膜的形成。     

Large scale modification of biomolecules using immobilized sortase A from Staphylococcus aureus

Recently, sortase A (SrtA) from Staphyloccus aureus moved into the focus of bioscience because of its ability to incorporate site specific modifications into proteins. The enzyme was mostly used to modify target proteins in an analytical scale, to study biomolecules in their cellular context. In this study, we show the applicability of SrtA mediated ligation for site specific modification of proteins in a large scale. Therefore, the reaction was first optimized using peptides and subsequently new reaction conditions were applied for the large scale biotinylation of interleukin-8. Furthermore, we established C-terminal immobilization of the SrtA on a PEG based resin and could demonstrate maintaining enzymatic activity. Immobilized SrtA significantly facilitates previous ligation protocols as the enzyme can be easily recycled. Also, the removal of excess reaction solution and the whole washing process is significantly accelerated, as centrifugation or filtration techniques can be applied instead of time-consuming chromatography steps.