金黄色葡萄球菌双组分系统反应调节蛋白AgrA的原核表达、纯化及活性鉴定

AgrA作为金黄色葡萄球菌双组分信号转导系统(two-component signal transduction system,TCST)的反应调节因子,能调控细菌毒力因子的表达,在金黄色葡萄球菌致病过程中起着重要的作用。采用无限制克隆法构建AgrA表达载体,在AgrA蛋白的C端融合绿色荧光蛋白(GFP)标签,通过实时监测GFP的荧光强度来快速检测重组蛋白的表达水平。首先利用单因素实验,筛选出宿主菌株BL21-(DE3)-PlysS;其次,结合Box-Behnken试验设计,筛选出最优蛋白质表达条件:诱导时间为22h、转速为222r/min、诱导剂浓度为0.5mmol/L,AgrA产量达到5.56mg/L。最后,基于AgrA蛋白LytTR区域的非放射性凝胶阻滞实验(non-radioactive electrophoretic mobility shift assay,EMSA)验证了AgrA的生物活性。提出了反应调节蛋白AgrA在大肠杆菌高效可溶性表达的策略,为双组分信号转导系统的体外研究奠定基础,也为其他反应调节蛋白的可溶性表达与分离纯化提供了一个可行借鉴。     

金黄色葡萄球菌蛋白质相互作用网络及功能

【目的】金黄色葡萄球菌是一种革兰氏阳性菌,是目前最难以对付的病菌之一。它能引起多种感染,特别是在医院环境中。近年来,抗药性金黄色葡萄球菌传染更加严重,已成为公共卫生威胁。由于以前对于金黄色葡萄球菌的实验性研究大都是基于单个基因或者蛋白进行的,为了更好的研究这个物种,有必要从整体上把握金黄色葡萄球菌的蛋白作用机理。【方法】采用系统发生谱、操纵子法、基因融合法、基因邻近法、同源映射法等五种计算方法预测金黄色葡萄球菌蛋白质相互作用网络。【结果】从蛋白组的角度构建了金黄色葡萄球菌蛋白相互作用网络,并对网络进行功能分析。【结论】网络的分析表明金黄色葡萄球菌的蛋白质相互作用网络也服从scale-free属性,发现了SA0939、SA0868、rplD等重要的蛋白。通过对金黄色葡萄球菌的重要的细胞壁合成和信号转导调控蛋白局部网络分析,发现了一些对这两个系统十分重要的蛋白分子,这些信息将为更好的了解金黄色葡萄球菌的致病机理和开发新的药物靶点提供指导。     

九香虫抗菌肽CcAMP1的分离纯化和抗菌活性检测

【目的】从药用昆虫九香虫 Coridius chinensis 中分离纯化抗菌肽,为进一步开发九香虫抗菌肽资源及深入挖掘九香虫的药用功能奠定基础。【方法】用大肠杆菌Escherichia coli 和金黄色葡萄球菌 Staphylococcus aureus 混合物作诱导源刺激九香虫产生抗菌肽,对血淋巴进行提取、凝胶过滤层析、固相萃取及反相色谱纯化,活性组分经质谱测定。对分离得到的这种抗菌肽进行人工合成,并进行抗菌活性检测。【结果】本研究获得一种九香虫抗菌肽CcAMP1,由17个氨基酸残基组成,分子量为1 997.37 u,带1个正电荷,表面有5个疏水氨基酸。对人工合成的CcAMP1进行抗菌活性检测表明,该抗菌肽与九香虫血淋巴一样对金黄色葡萄球菌等革兰氏阳性菌和大肠杆菌等革兰氏阴性菌都有较好的抗菌活性,且对革兰氏阴性菌的抗菌活性更强。【结论】从九香虫中分离得到具有较强抗菌活性的阳离子抗菌肽CcAMP1,有较大的开发利用价值。     

金黄色葡萄球菌arl双组分信号转导系统受体蛋白ArlS_(CA)的表达、纯化及活性研究

金黄色葡萄球菌是一类重要的病原菌,其毒力因子的表达及分泌过程由多种双组分信号转导系统(two component signal transduction system,TCSTS)共同调控,其中ArlRS双组分信号转导系统与细菌的生长和分裂密切相关。ArlRS双组份系统的信号传递通过组氨酸激酶ArlS磷酸化实现,ArlS的胞内域被认为是调控毒力因子表达的重要功能域,以ArlS蛋白的胞内域部分即ArlS_(CA)为目标蛋白进行相关的活性研究。首先,构建pProEX-HTa-arls和pProEX-HTa-arlr重组质粒,对目的蛋白进行诱导表达。其次,利用金属离子螯合层析、离子交换层析以及凝胶过滤层析方法对目的蛋白进行分离纯化,纯化后的ArlR蛋白纯度可达98%,产量约为25mg/L;纯化后的ArlS蛋白纯度可达90%,产量约为15mg/L。圆二色谱检测结果显示纯化后的目的蛋白有完整的二级结构,体外磷酸化结果显示,ArlS蛋白具有激酶活性,自磷酸化后可以将磷酸基团转移给反应调控蛋白ArlR。最后,利用定点突变的方法,构建了418位和420位氨基酸残基突变的表达载体pProEX-HTa-ArlS_(CAG418A)和pProEX-HTa-ArlS_(CAG420A)。ArlS_(CAG418A)和ArlS_(CAG420A)蛋白不具有激酶活性,说明418位和420位氨基酸残基在ArlS蛋白的自磷酸过程中起着关键作用。     

苏云金芽胞杆菌Sigma K在大肠杆菌中的表达与纯化

【目的】在大肠杆菌中表达纯化苏云金芽胞杆菌HD73的转录调控因子Sigma K(σK)。【方法】PCR扩增出苏云金芽胞杆菌HD73中sig K基因的ORF(Open reading frame)装载到带有His标签的表达载体p ET21b上,转入到表达菌株BL21(DE3)中获得重组菌株BL21(p ETsig K),通过SDS-PAGE、镍柱亲和纯化、阴离子交换纯化和凝胶迁移实验(EMSA)等方法对Sigma K蛋白进行提取、纯化和生物活性分析。【结果】正确表达出大小约为27 k D的His-Sigma K蛋白,并获得了纯化的蛋白。EMSA结果表明纯化的His-Sigma K蛋白可以与受其控制的cry1Ac基因启动子结合。【结论】表达和纯化了His-Sigma K蛋白,His-Sigma K具有与受其控制的启动子结合的功能。     

重组鹅β-防御素12蛋白的原核表达及生物学特性

【目的】研究重组鹅β-防御素12蛋白的原核表达并探究其生物学特性。【方法】采用His标签蛋白原核表达系统,将鹅防御素12(Av BD12)基因亚克隆到表达载体p Pro EX-HTa上,构建重组表达质粒。将重组表达质粒转化到大肠杆菌Rosseta感受态中,用IPTG进行诱导表达,并对该重组蛋白进行纯化。进一步采用菌落计数法测定其体外抗菌活性和盐离子稳定性。【结果】经Tricine-SDS-PAGE电泳分析,诱导表达的鹅Av BD12重组蛋白分子量约为12 k D,大部分以包涵体形式存在。该重组蛋白对大肠杆菌、鸡白痢沙门氏菌、金黄色葡萄球菌、四联球菌、枯草芽孢杆菌均具有抗菌活性,高浓度盐离子显著抑制重组蛋白的抗菌活性。此外,该重组蛋白对鸡红细胞没有溶血活性。【结论】该重组蛋白具有广谱抗菌活性,高浓度盐离子显著降低其抗菌活性,且该重组蛋白不具有溶解鸡红细胞的活性。     

吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的转录组影响分析

【背景】金黄色葡萄球菌是目前食品和临床引起感染的重要病原菌,迫切需要开发新型抗菌药物。【目的】分析吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的转录组影响和主要代谢信号通路。【方法】采用液体稀释法测定P-FAH-Cu-phen作用金黄色葡萄球菌的最低抑菌浓度(minimum inhibitory concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC)。将终浓度2 μg/mL的配合物分别作用于对数生长期的金黄色葡萄球菌30 min和2 h,进行转录组测序及分析。【结果】 P-FAH-Cu-phen作用金黄色葡萄球菌的MIC和MBC分别为2 μg/mL和4 μg/mL。与空白对照相比,配合物处理细菌30 min后,其差异基因共有356个,其中上调表达180个、下调表达176个;配合物处理细菌2 h后,其差异基因共有23个,其中上调表达3个、下调表达20个。差异基因功能主要富集于膜的组成部分、细胞质、质膜、ATP结合、发病机制、金属离子结合、组氨酸生物合成过程、DNA结合、水解酶活性、跨膜转运蛋白活性、硝酸盐同化、硝酸盐代谢过程、硝酸还原酶复合物、硝酸还原酶活性等。差异基因涉及的信号通路主要有双组分系统、群体感应、氮代谢、三羧酸循环、氨基酸代谢等。【结论】影响细菌质膜组成、毒素生成、生物膜形成、细胞壁合成、能量代谢等可能是吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的主要抑菌作用。研究为揭示吡唑啉酮铜配合物抑制金黄色葡萄球菌分子机制提供了理论依据。     

c-di-GMP抑制转录调控因子Clpxoo与葡聚糖酶基因启动子的结合北大核心CSCD

【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)c-di-GMP信号受体和转录调控因子Clpxoo对葡聚糖酶基因(engAxoo)的转录调控作用机制。【方法】通过基因表达载体的构建和转化、蛋白诱导表达及其Ni-NTA Resin亲和层析,进行了clpxoo基因的原核表达和产物纯化。通过荧光素(FAM)探针标记和凝胶阻滞试验(EMSA)对Clpxoo纯化蛋白与葡聚糖酶基因启动子(engAxoo-p)的结合活性及其c-diGMP信号分子的抑制作用进行了测定分析。【结果】在优化的诱导表达和纯化条件下,成功地获得了Clpxoo纯化蛋白。Clpxoo可与engAxoo-p序列发生阻滞现象,表明它具有与启动子特异性结合的活性。在反应体系中加入c-di-GMP可导致结合作用的消除。【结论】Clpxoo接受c-di-GMP信号后,可能通过构象的改变,从而与engAxoo-p的结合活性受到抑制;优化的Clpxoo蛋白纯化和EMSA方法可以用于后续的调控元大规模鉴定的研究。     

乳源耐甲氧西林金黄色葡萄球菌icaA/D基因缺失株的构建及其生物被膜形成能力与耐药性分析

【背景】耐甲氧西林金黄色葡萄球菌(Methicillin Resistant Staphylococcus aureus,MRSA)是一种具有多重耐药性的人畜共患病原菌,常引起奶牛乳房炎等疾病。形成生物被膜是MRSA重要的耐药机制之一。研究发现ica操纵子调控的胞间多糖黏附素(Polysaccharide Intercellular Adhesin,PIA)通过促进MRSA的黏附与聚集介导生物被膜形成,icaA和icaD基因共表达可显著提高MRSA的N-乙酰葡聚糖转移酶活性,但icaA/D蛋白对MRSA生物被膜形成和耐药性的影响仍不清楚。【目的】探讨icaA/D基因、MRSA生物被膜形成及耐药性三者之间的相关性,为寻找药物作用新靶点提供科学依据。【方法】以具有多重耐药性且生物被膜形成能力强的乳源MRSA M5分离株为研究对象,利用同源重组技术构建其icaA/D基因缺失株;利用FITC-ConA染色结合激光共聚焦显微镜观察野生株与icaA/D基因缺失株的生物被膜形成过程与能力;采用微量肉汤稀释法测定14种抗菌药物对野生株与icaA/D基因缺失株的最小抑菌浓度(MinimumInhibitoryConcentration,MIC)。【结果】构建了MRSA M5的icaA/D基因缺失株。激光共聚焦显微镜下观察到野生株在培养16 h后形成了一层厚的成熟生物被膜,随后开始解离,直至120 h完全解离;而icaA/D基因缺失株在培养后16 h仅形成一薄层生物被膜,48h完全解离。10种受试抗菌药物对缺失株的MIC较野生株减小,而且缺失株对8种药物的敏感性由原来的耐药或中介转变为中介或敏感。【结论】icaA/D基因缺失可明显降低MRSA的生物被膜形成能力与耐药性。     

金黄色葡萄球菌D试验和耐药性分析

目的 了解本地区金黄色葡萄球菌中红霉素对克林霉素诱导耐药表型及其对常用抗菌药物的耐药性.方法 用K-B琼脂扩散法检测耐甲氧西林的金黄色葡萄球菌(MRSA)及双纸片法(D试验)检测红霉素对克林霉素诱导耐药表型,用VITEK 2 Compact进行菌株鉴定和药敏试验.结果 156株金黄色葡萄球菌中MRSA有51株,占32.7％.红霉素对克林霉素诱导耐药共29株,占18.6％,其中MRSA有10株,甲氧西林敏感的金黄色葡萄球菌(MRSS)有19株.金黄色葡萄球菌对多种抗菌药物具有不同的耐药性,对青霉素的耐药性超过95％,对万古霉素、奎努普汀/达福普汀、利奈唑烷和替加环素敏感.结论 临床微生物实验室应加强对金黄色葡萄球菌中克林霉素诱导耐药的检测,临床治疗中也应加强抗菌药物的合理应用以防多耐药菌株的产生.     

Transmembrane topology and histidine protein kinase activity of AgrC, the agr signal receptor in Staphylococcus aureus

The agr P2 operon in Staphylococcus aureus codes for the elements of a density-sensing cassette made up of a typical two-component signalling system and its corresponding inducer. It is postulated that the autoinducer, a post-translationally modified octapeptide generated from the AgrD peptide, interacts with a receptor protein, coded by agrC , to transmit a signal via AgrA regulating expression of staphylococcal virulence genes through expression of agr RNA III. We show by analysis of PhoA fusions that AgrC is a transmembrane protein, and confirm using Western blotting that a 46 kDa protein corresponding to AgrC is present in the bacterial membrane. This protein is autophosphorylated on a histidine residue only in response to supernatants from an agr⁺ strain, and can also respond to the purified native octapeptide. A recombinant fusion protein where most of the N-terminal region of AgrC is replaced by the Escherichia coli maltose-binding protein is also autophosphorylated in response to stimulation by agr⁺ supernatants or purified octapeptide. We conclude that AgrC is the sensor molecule of a typical two-component signal system in S. aureus , and that the ligand-binding site of AgrC is probably located in the third extracellular loop of the protein.     

New insight into transmembrane topology of Staphylococcus aureus histidine kinase AgrC

Staphylococcus aureus accessory gene regulator (agr) locus controls the expression of virulence factors through a classical two-component signal transduction system that consists of a receptor histidine protein kinase AgrC and a cytoplasmic response regulator AgrA. An autoinducing peptide (AIP) encoded by agr locus activates AgrC, which transduces extracellular signals into the cytoplasm. Despite extensive investigations to identify AgrC–AIP interaction sites, precise signal recognition mechanisms remain unknown. This study aims to clarify the membrane topology of AgrC by applying the green fluorescent protein (GFP) fusion technique and the substituted cysteine accessibility method (SCAM). However, our findings were inconsistent with profile obtained previously by alkaline phosphatase. We report the topology of AgrC shows seven transmembrane segments, a periplasmic N-terminus, and a cytoplasmic C-terminus.     

Identification of the putative staphylococcal AgrB catalytic residues involving the proteolytic cleavage of AgrD to generate autoinducing peptide

The P2 operon of the staphylococcal accessory gene regulator (agr) encodes four genes (agrA, -B, -C, and -D) whose products compose a quorum sensing system: AgrA and AgrC resemble a two-component signal transduction system of which AgrC is a sensor kinase and AgrA is a response regulator; AgrD, a polypeptide that is integrated into the cytoplasmic membrane via an amphipathic alpha-helical motif in its N-terminal region, is the propeptide for an autoinducing peptide that is the ligand for AgrC; and AgrB is a novel membrane protein that involves in the processing of AgrD propeptide and possibly the secretion of the mature autoinducing peptide. In this study, we demonstrated that AgrB had endopeptidase activity, and identified 2 amino acid residues in AgrB (cysteine 84 and histidine 77) that might form a putative cysteine endopeptidase catalytic center in the proteolytic cleavage of AgrD at its C-terminal processing site. Computer analysis revealed that the cysteine and histidine residues were conserved among the potential AgrB homologous proteins, suggesting that the Agr quorum sensing system homologues might also exist in other Gram-positive bacteria.     

重组蛋白AgrC的表达纯化及人工超分子体系的构建

目的:克隆表达金黄色葡萄球菌agr系统的受体蛋白AgrC,构建人工超分子体系。方法:构建基因工程菌E.coli TOP10-pBAD-AgrC,对诱导条件进行优化,对蛋白的提取方法进行分析优化。表达产物使用Western blot鉴定,His-tag镍柱亲和层析纯化。使用肽脂质N+C5Gly2C16为膜材料制备囊泡作为载体,将纯化后的蛋白在囊泡上镶嵌。结果:构建的基因工程菌在18℃,0.002%的阿拉伯糖浓度下具有最高的表达效率,以内膜形式存在的AgrC蛋白在表面活性剂BDH中溶解效率最佳。结论:成功诱导表达并纯化了重组蛋白AgrC,成功利用其构建了人工超分子体系。     

Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus

The YycG/YycF essential two-component system (TCS), originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low-G+C gram-positive bacteria, including several pathogens such as Staphylococcus aureus. By studying growth of S. aureus cells where the yyc operon is controlled by an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoter, we have shown that this system is essential in S. aureus during growth at 37 degrees C and that starvation for the YycG/YycF regulatory system leads to cell death. During a previous study of the YycG/YycF TCS of B. subtilis, we defined a potential YycF consensus recognition sequence, consisting of two hexanucleotide direct repeats, separated by five nucleotides [5'-TGT(A/T)A(A/T/C)-N(5)-TGT(A/T)A(A/T/C)-3']. A detailed DNA motif analysis of the S. aureus genome indicates that there are potentially 12 genes preceded by this sequence, 5 of which are involved in virulence. An in vitro approach was undertaken to determine which of these genes are controlled by YycF. The YycG and YycF proteins of S. aureus were overproduced in Escherichia coli and purified. Autophosphorylation of the YycG kinase and phosphotransfer to YycF were shown in vitro. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the promoter region of the ssaA gene, encoding a major antigen and previously suggested to be controlled by YycF. YycF was also shown to bind specifically to the promoter regions of two genes, encoding the IsaA antigen and the LytM peptidoglycan hydrolase, in agreement with the proposed role of this system in controlling virulence and cell wall metabolism.     

RF克隆结合巢式PCR构建金黄色葡萄球菌组氨酸蛋白激酶AgrC表达载体

AgrC蛋白是位于金黄色葡萄球菌细胞膜上的组氨酸激酶,能够感受细胞外的化学信号并将信号传递到细胞内,进而调控细胞内与致病性相关的一系列基因的表达.利用无限制克隆法(RF克隆),并结合巢式PCR成功构建了AgrC表达载体pET-28a-AgrC.将表达载体电转入大肠杆菌中,并利用IPTG进行诱导表达AgrC蛋白.再经过低温超高压破碎、超高速离心、金属螯合层析与尺寸排阻层析等过程,分离纯化得到AgrC蛋白.     

Anti-MRSA agent discovery using Caenorhabditis elegans -based high-throughput screening

Staphylococcus aureus is a leading cause of hospital- and community-acquired infections. Despite current advances in antimicrobial chemotherapy, the infections caused by S. aureus remain challenging due to their ability to readily develop resistance. Indeed, antibiotic resistance, exemplified by methicillin-resistant S. aureus (MRSA) is a top threat to global health security. Furthermore, the current rate of antibiotic discovery is much slower than the rate of antibiotic-resistance development. It seems evident that the conventional in vitro bacterial growth-based screening strategies can no longer effectively supply new antibiotics at the rate needed to combat bacterial antibiotic-resistance. To overcome this antibiotic resistance crisis, screening assays based on host–pathogen interactions have been developed. In particular, the free-living nematode Caenorhabditis elegans has been used for drug screening against MRSA. In this review, we will discuss the general principles of the C. elegans-based screening platform and will highlight its unique strengths by comparing it with conventional antibiotic screening platforms. We will outline major hits from high-throughput screens of more than 100,000 small molecules using the C. elegans–MRSA infection assay and will review the mode-of-action of the identified hit compounds. Lastly, we will discuss the potential of a C. elegans-based screening strategy as a paradigm shift screening platform.