分选酶的研究进展及其在生物技术中的应用

分选酶(sortase)普遍存在于革兰氏阳性细菌中,是一类膜结合的转肽酶,负责将表面蛋白共价结合到细胞壁的肽聚糖上。由于其独特的作用机制,分选酶在生物技术领域具有广阔的应用前景,可应用于革兰氏阳性菌的表面展示、蛋白质工程等。     

新抗菌靶点分选酶基因（srtA）在两种原核载体中的克隆表达

【目的】革兰氏阳性菌的表面蛋白在病原菌致病性方面具有重要作用,表面蛋白锚定到细胞壁过程的关键酶—分选酶成为抗感染的新靶点。【方法】本文利用GenBank中的分选酶A基因（srtA）序列设计特异性引物,以金黄色葡萄球菌基因组DNA为模板进行PCR扩增,获得618 bp的DNA片段。按照常规分子克隆操作成功构建两种原核表达载体pet22-srtA和pTRX-srtA,转入大肠杆菌感受态BL21（DE3）中,在1 mmol/LIPTG诱导下进行表达。利用SDS-PAGE和western blot进行鉴定和分析,【结果】结果显示：(1)重组载体pet22-srtA和pTRX-srtA分别表达出相对分子量为约45 kDa和39 kDa的外源蛋白;【结论】（2）分子伴侣硫氧还蛋白（Trx）有利于分选酶A基因的可溶性表达。该实验为后续的分选酶酶学性质研究特别是抑制剂筛选研究奠定良好基础。     

分选酶A(SrtA)的GST融合表达、纯化及活性测定

构建GST/金黄色葡萄球菌分选酶A (SrtA)的原核表达载体,在大肠杆菌中表达、纯化分选酶,并利用展示在酵母表面的底物检测分选酶的活性.以pMD20-SrtA为模板,PCR扩增得到SrlA△N59基因,经BamH I和Xho I双酶切,连接到原核表达栽体pGEX-4T-1中,构建重组表达栽体pGEX-SrtA△N59,转化大肠杆菌BL21( DE3),IPTG诱导表达,GST亲和层析分离纯化得到SrtA△N59,与展示在酵母表面的底物序列QALPETGEE-linker-EGFP作用,产生游离的EGFP,通过酶标仪检测EGFP荧光强度确定分选酶的活性.结果显示,重组表达栽体pGEX-SrtA△N59经IPTG诱导,表达出相对分子质量约为42 kD的融合蛋白,SDS-PAGE分析,该融合蛋白是以可溶形式表达.分离纯化得到的分选酶与底物作用,其荧光强度由568.66±12.14增加至921.43±13.02.以上结果表明,成功构建了重组表达裁体pGEX-SrtA△N59,并在大肠杆菌中获得了可溶表达的有活性的分选酶.     

酵母表面展示分选酶底物用于分选酶活性检测

摘要：【目的】以EGFP标签检测分选酶底物QALPETGEE在毕赤酵母表面的表达,然后将酵母表面展示的底物与分选酶相互作用以检测分选酶活性。【方法】以pcDNA-myc-his-EGFP为模板,通过PCR技术将QALPETGEE-linker-EGFP基因连接到穿梭载体pKFS上,构建QALPETGEE-linker-EGFP酵母表面展示载体后转化至毕赤酵母（Pichia pastoris）GS115中。重组菌经培养,利用荧光显微镜检测重组酵母的荧光强度,然后通过荧光分光光度计检测分选酶与底物相互作用后产     

流式细胞分选技术在微生物表面展示文库筛选中的应用进展

流式细胞分选技术具有测量速度快、统计学精度高、可在分析的同时把具有指定特征的细胞分选出来等特点,已广泛应用到微生物(细菌、酵母、噬菌体和杆状病毒等)表面展示文库的筛选。筛选配基的类型涵盖抗原模拟表位、特异性单链抗体或单链T细胞受体、酶变异体以及蛋白酶抑制剂等。本文对这方面的研究进展作一综述。     

分选酶A在pET32a(+)原核表达载体中的表达和鉴定

旨在pET32a(+)原核表达载体中表达金黄色葡萄球菌(Staphylococcus aureus)中的转肽酶分选酶(SrtA)并进行鉴定.以含有pET22-srtA质粒为模板,设计并合成引物,PCR扩增得到SrtA△N24和SrtA△N59基因,经过BamH Ⅰ、Xho Ⅰ酶切,克隆入表达载体pET32a(+)中,构建重组载体pET32a-SrtA△N24及pET32a-SrtA△N59,并转化入大肠杆菌BL21(DE3).经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blotting对表达产物分别进行分析和鉴定.然后对重组质粒在大肠杆菌BL21(DE3)中的表达条件进行了优化.结果显示重组载体pET32a-SrtA△N24和pET32a-SrtA△N59分别表达出相对分子量为约42 kD和37 kD的融合蛋白,经SDS-PAGE和Westem blotting检测显示其分子量与预期的大小相符合.成功构建了重组质粒pET32a-SrtA△N24和pET32a-SrtA△N59,并且在大肠杆菌BL21(DE3)中获得了高效融合表达.     

利用固定化金属亲和层析和质谱技术鉴定化脓性链球菌铜结合蛋白

化脓性链球菌是一种常见的高致病性革兰氏阳性菌,每年都在全球范围内引发大量的感染现象与病死案例。金属蛋白在微生物的代谢以及毒性构成中的作用已经逐渐得到重视。通过固定化金属离子亲合层析技术对化脓性链球菌中铜结合蛋白进行分离,配合质谱技术的使用,共鉴定出21个铜结合蛋白,这些蛋白分别在翻译、代谢、离子通道等各个生理进程中发挥相应作用。     

噬菌体裂解酶——现状与未来

噬菌体裂解酶是一种由DNA噬菌体基因编码的高特异性酶, 可高效消化细菌细胞壁。革兰氏阳性菌噬菌体裂解酶的结构域相似, 裂解效率高, 与抗生素具协同抗菌作用, 且不易产生耐受性菌株, 抗体等体液因子对裂解酶的裂解活性影响小, 裂解酶作为一种潜在抗感染药物具有重要的研究价值。目前已建立了多种病原菌裂解酶应用的动物模型, 在防控耐药性病原菌感染上取得重要进展。本文就噬菌体裂解酶的抗菌作用进行综述。     

革兰氏阳性菌群体感应系统研究进展

细菌利用群体感应系统进行细菌间以及细菌与宿主间的交流,革兰氏阳性与阴性菌的群体感应系统差异显著,阳性菌的群体感应系统主要由寡肽类信号分子和受体蛋白组成,对细菌致病性等相关生理特性具有重要作用。就常见的革兰氏阳性菌：蜡样芽孢杆菌、枯草芽孢杆菌、金黄色葡萄球菌和肺炎链球菌的群体感应系统的基因组成、信号分子及其调控机制特点的研究进行了总结,对群体感应系统在细菌营养吸收、生物膜形成、毒力因子和孢子产生等重要生理活动的调节机制进行了重点阐述,为革兰氏阳性菌群体感应的相关研究提供了有益参考。     

革兰氏阳性菌荚膜多糖研究进展*

细菌的荚膜多糖是生物膜的重要组成部分,在细菌的生长分裂、维持细胞壁形态、抵御外界环境以及免疫反应等方面都起到重要作用。在致病菌中,荚膜多糖常作为一种毒力因子发挥作用。在革兰氏阳性菌中,荚膜多糖的化学结构、生物合成过程及功能应用越来越受到关注。讨论了革兰氏阳性菌中部分致病菌的荚膜多糖与非致病菌表面多糖的分布位置、化学组成及其结构特异性。重点讨论三种具有代表性的革兰氏阳性致病菌及非致病菌株:肺炎链球菌(Streptococcus pneumonia)、金黄色葡萄球菌(Staphylococcus aureus)及乳酸乳球菌(Lactococcus lactis)。综述革兰氏阳性菌中荚膜多糖生物合成的三种方式:Wzx/Wzy-依赖通路、ABC转运蛋白(ABC transporter)途径及合酶依赖途径,并举例解释了相应多糖的合成过程及相关基因。介绍了革兰氏阳性菌荚膜多糖及表面多糖的生理功能,如屏障保护功能、胞间黏附功能以及参与宿主细胞的免疫反应等。结合荚膜多糖的生物学功能,概述其当前主要研究进展,如构建高耐受工程菌疫苗研制等。结合细菌荚膜多糖的特征差异,对其在医药与工业生产领域的广阔前景提出展望和建议。     

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.     

A novel sortase, SrtC2, from Streptococcus pyogenes anchors a surface protein containing a QVPTGV motif to the cell wall

The important human pathogen Streptococcus pyogenes (group A streptococcus GAS), requires several surface proteins to interact with its human host. Many of these are covalently linked by a sortase enzyme to the cell wall via a C-terminal LPXTG motif. This motif is followed by a hydrophobic region and charged C terminus, which are thought to retard the protein in the cell membrane to facilitate recognition by the membrane-localized sortase. Previously, we identified two sortase enzymes in GAS. SrtA is found in all GAS strains and anchors most proteins containing LPXTG, while SrtB is present only in some strains and anchors a subset of LPXTG-containing proteins. We now report the presence of a third sortase in most strains of GAS, SrtC. We show that SrtC mediates attachment of a protein with a QVPTGV motif preceding a hydrophobic region and charged tail. We also demonstrate that the QVPTGV sequence is a substrate for anchoring of this protein by SrtC. Furthermore, replacing this motif with LPSTGE, found in the SrtA-anchored M protein of GAS, leads to SrtA-dependent secretion of the protein but does not lead to its anchoring by SrtA. We conclude that srtC encodes a novel sortase that anchors a protein containing a QVPTGV motif to the surface of GAS.     

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.     

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.     

Aaptamines as sortase A inhibitors from the tropical sponge Aaptos aaptos

Four aaptamines (1-4), 1H-benzo[de][1,6]-naphthyridine alkaloids, were isolated from the marine sponge Aaptos aaptos and their inhibitory activities against sortase A (SrtA), an enzyme that plays a key role in cell wall protein anchoring and virulence in Staphylococcus aureus, were evaluated. Isoaaptamine (2) was a potent inhibitor of SrtA, with an IC(50) value of 3.7+/-0.2 microg/mL. The suppression of fibronectin-binding activity by isoaaptamine (2) highlights its potential for the treatment of S. aureus infections via inhibition of SrtA activity. Our studies have identified a series of SrtA inhibitors, providing the basis for further development of potent inhibitors.     

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.     

Synthetic analogs of indole-containing natural products as inhibitors of sortase A and isocitrate lyase

Guided by the inhibitory activities of indole-containing natural products against isocitrate lyase (ICL) from Candida albicans and sortase A (SrtA) from Staphylococcus aureus, a series of compounds structurally analogous to natural products were synthesized. Eight SrtA inhibitors and an ICL inhibitor having higher activities than the natural products were discovered by screening the enzyme inhibitory activities of synthesized compounds. Among the SrtA inhibitors discovered, six exhibited higher activities than p-hydroxymercuribenzoic acid, which suggests that these compounds have great potential as alternative antibacterial agents.     

New method for site-specific modification of liposomes with proteins using sortase A-mediated transpeptidation

A new method was developed for site-specific modifications of liposomes by proteins via sortase A (SrtA)-mediated transpeptidation reactions. In this regard, the enhanced green fluorescent protein (eGFP) was biologically engineered to carry at its polypeptide C-terminus the LPATG motif recognized by SrtA and used as the protein donor for linking to liposomes that were decorated with phospholipids carrying a diglycine motif as the other SrtA substrate and the eGFP acceptor. Under the influence of SrtA, eGFP was efficiently attached to liposomes, as proved by analyzing the enzymatic reaction products and the resultant fluorescent liposomes. It was observed that increasing the concentration and the distance of the diglycine motif on and from the liposome surface could significantly improve the efficiency of liposome modification by proteins. It is anticipated that this strategy can be widely useful for the modification of liposomes by other proteins.     

Housekeeping sortase facilitates the cell wall anchoring of pilus polymers in Corynebacterium diphtheriae

Many surface proteins in Gram-positive bacteria are covalently linked to the cell wall through a transpeptidation reaction catalysed by the enzyme sortase. Corynebacterium diphtheriae encodes six sortases, five of which are devoted to the assembly of three distinct types of pilus fibres--SrtA for the SpaA-type pilus, SrtB/SrtC for the SpaD-type pilus, and SrtD/SrtE for the SpaH-type pilus. We demonstrate here the function of SrtF, the so-called housekeeping sortase, in the cell wall anchoring of pili. We show that a multiple deletion mutant strain expressing only SrtA secretes a large portion of SpaA polymers into the culture medium, with concomitant decrease in the cell wall-linked pili. The same phenotype is observed with the mutant that is missing SrtF alone. By contrast, a strain that expresses only SrtF displays surface-linked pilins but no polymers. Therefore, SrtF can catalyse the cell wall anchoring of pilin monomers as well as pili, but it does not polymerize pilins. We show that SrtA and SrtF together generate wild-type levels of the SpaA-type pilus on the bacterial surface. Furthermore, by regulating the expression of SpaA in the cell, we demonstrate that the SrtF function becomes critical when the SpaA level is sufficiently high. Together, these findings provide key evidence for a two-stage model of pilus assembly: pilins are first polymerized by a pilus-specific sortase, and the resulting fibre is then attached to the cell wall by either the cognate sortase or the housekeeping sortase.