信号分子AI-2的体外合成及其对副溶血性弧菌四环素耐药性的调控作用

【背景】抗菌药的过度使用引起细菌耐药性日益严重,作为重要的食源性致病菌,副溶血性弧菌也表现出一定程度的耐药性。群体感应系统可以调控细菌的耐药性,为研究副溶血性弧菌的耐药机制和控制技术提供新的途径。【目的】探讨群体感应信号分子AI-2 (autoinducer-2)对海产品中分离的副溶血性弧菌四环素耐药性的调控作用。【方法】通过原核表达制备AI-2合成关键酶——S-核糖同型半胱氨酸酶(S-ribosylhomocysteinase, LuxS)和S-腺苷同型半胱氨酸核苷酶(S-adenosylhomocysteinenucleosidase,Pfs),体外合成AI-2,通过菌落计数法分析AI-2对副溶血性弧菌在四环素亚抑菌浓度下耐受性的影响,采用逆转录实时荧光定量PCR法测定不同浓度AI-2对副溶血性弧菌四环素耐药基因转录水平的影响。【结果】通过原核表达获得LuxS和Pfs,作用于底物S-腺苷同型半胱氨酸能合成具有生物活性的AI-2,其荧光强度约为阳性对照的6倍。在四环素亚抑菌浓度下,AI-2能显著促进副溶血性弧菌的生长,6、15、30μmol/L浓度AI-2能不同程度地提高副溶血性弧菌四环素耐药基因的转录水平。【结论】AI-2能增强副溶血性弧菌对四环素的耐受作用,为解析副溶血性弧菌的耐药机制、研制以AI-2为靶点的副溶血性弧菌耐药性控制技术提供基础。     

嗜水气单胞菌群体感应信号分子AI-2的细胞外生物 合成及活性检测

【目的】对嗜水气单胞菌群体感应信号分子AI-2进行细胞外生物合成及活性检测。【方法】对LuxS、MtnN-1、MtnN-2蛋白进行氨基酸序列分析、表达及纯化。以S-腺苷同型半胱氨酸(SAH)为底物,利用纯化的LuxS分别与MtnN-1及MtnN-2蛋白共同作用合成AI-2,并利用哈维氏弧菌报告菌株BB170检测AI-2活性。【结果】嗜水气单胞菌培养液上清中AI-2活性在8 h达到空白对照的16.96倍。氨基酸序列分析表明,嗜水气单胞菌与水生病原菌哈维式弧菌和迟钝爱德华氏LuxS一致性达到76%以上,MtnN-1与MtnN-2氨基酸序列一致性为26.37%,其中MtnN-2与哈维氏弧菌和迟钝爱德华氏菌Pfs一致性达到53%以上。成功表达及纯化了LuxS、MtnN-1和MtnN-2蛋白,细胞外LuxS和MtnN-1共同作用合成的AI-2活性是空白对照的45.04倍,LuxS和MtnN-2共同作用合成的AI-2活性是空白对照的63.62倍。【结论】嗜水气单胞菌能够合成信号分子AI-2。MtnN-1和MtnN-2氨基酸序列尽管存在较大差异,但两者均能与LuxS共同催化AI-2的细胞外生物合成。     

高产信号分子AI-2乳酸菌的筛选及其Pfs基因的克隆和表达

【目的】从锡盟地区酸马奶酒分离的乳酸菌中筛选出高产信号分子自体诱导物2(Autoinducer-2,AI-2)的乳酸菌,通过优化其重组蛋白Pfs的诱导条件体外合成信号分子AI-2。【方法】利用生物学发光法对不同乳酸菌产信号分子AI-2的产量进行比较,以高产信号分子AI-2乳酸菌基因组DNA为模板,扩增其S-腺苷高半胱氨酸核苷酶(S-adenosylhomocysteine nucleosidase,Pfs)基因,构建原核表达载体。利用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)进行重组蛋白的诱导表达,通过优化培养基、诱导温度、诱导前菌体密度、IPTG浓度以及诱导时间得到高表达的Pfs蛋白,使其与底物作用最终体外合成信号分子AI-2。【结果】10株乳酸菌均可产信号分子AI-2,其中屎肠球菌8-3分泌信号分子AI-2的产量明显高于其他菌株;重组蛋白的最佳诱导条件为:选取SOC(Super optimal broth with catabolite repression)作为诱导表达培养基,菌液OD600为0.5–0.7时加入终浓度为0.1 mmol/L的IPTG,37°C诱导12 h;利用最优诱导条件获得了浓度为4.08 g/L的纯化Pfs蛋白,体外合成了信号分子AI-2。【结论】酸马奶酒中分离出的10株乳酸菌均可产生信号分子AI-2,且屎肠球菌8-3可通过Pfs基因的作用生成信号分子AI-2。     

禽致病性大肠杆菌安徽分离株luxS和pfs基因的克隆、表达与细胞外合成AI-2 活性检测

【目的】LuxS/AI-2型密度感应系统存在于革兰氏阴性和阳性菌中,可产生用于细菌种间交流的通用自诱导信号分子AI-2(Autoinducer-2,AI-2),细菌许多生理功能都受此系统的调节。本研究开展对禽致病性大肠杆菌(Avian Pathogenic Escherichia coli,APEC)自诱导信号分子AI-2的检测和建立体外合成、定量的方法,为进一步研究APEC的AI-2调控作用奠定基础。【方法】利用哈维弧菌BB170(Vibrio harveyi BB170)开展对APEC AI-2的检测;利用表达、纯化的LuxS和Pfs在体外催化S-腺苷同型半胱氨酸(Sadenosylhomocysteine,SAH),进行AI-2的体外合成。【结果】APEC能产生自诱导信号分子AI-2;成功表达可用于AI-2合成的可溶性重组蛋白LuxS和Pfs;纯化的重组蛋白LuxS和Pfs与SAH同时作用后,合成了浓度为300μmol/L的AI-2;运用哈维弧菌BB170对合成的AI-2活性检测表明,其活性是阴性对照的700倍。【结论】APEC存在LuxS/AI-2型密度感应系统,APEC的LuxS和Pfs可以在体外催化SAH生成有活性的AI-2分子。本研究为进一步研究APEC的AI-2的调控作用奠定基础。     

阻断消耗途径提高毕赤酵母工程菌S-腺苷甲硫氨酸产量

【背景】S-腺苷甲硫氨酸(S-adenosyl-L-methionine, SAM)作为所有生物体内的重要中间代谢物,不仅可作为膳食补充剂,还具有良好的临床应用价值。【目的】将毕赤酵母重组菌GS115/DS16的SAM消耗途径阻断,进一步提高SAM的产量。【方法】分别敲除毕赤酵母重组菌GS115/DS16的S-腺苷同型半胱氨酸水解酶基因sah1、S-腺苷甲硫氨酸脱羧酶基因spe2和L-甲硫氨酰tRNA合酶基因msm1,构建工程菌G/Dsah、G/Dspe和G/Dmsm。检测3个工程菌的生长和SAM产量,以及L-Met添加量对SAM积累的影响。【结果】与出发菌GS115/DS16相比,工程菌G/Dsah、G/Dspe和G/Dmsm的单位菌体SAM产量分别提高了29.3%、55.6%和24.8%,其生长无显著差异。L-Met添加量优化后(0.06%),G/Dsah和G/Dmsm单位菌体的SAM产量分别提高了26.4%和28.9%。【结论】构建的毕赤酵母工程菌可用于SAM的工业化生产,该代谢工程策略可用于改进其他化学品的生产。     

真菌Mucor amphibiorum RCS1中一个类S-腺苷-L-高半胱氨酸水解酶基因的蓝光诱导表达

【目的】确定真菌Mucor amphibiorum RCS1中一个类S-腺苷-L-高半胱氨酸水解酶基因(S-adenosyl-L-homocysteine hydrolase-like,sahhl)受蓝光诱导表达。【方法】以真菌M.amphibiorum RCS1为研究对象,在随机PCR过程中从中获得了一段555 bp长度的DNA序列。以地高辛对这段已知序列进行标记制备探针,通过Northern杂交检测M.amphibiorum RCS1菌丝体培养过程中,由黑暗到蓝光再到黑暗这一光照条件改变的情况下,sahhl基因的转录情况。同时结合应用real-time PCR方法进行分析检测。【结果】经过比对确定这段555bp序列与已经发表的人(Homo sapiens)、家鼠(Mus musculus)和部分真菌的S-腺苷-L-高半胱氨酸水解酶基因sahh有较高的同源性。因此,初步确认这段mRNA序列来自M.amphibiorum RCS1的一个类S-腺苷-L-高半胱氨酸水解酶基因。sahhl基因在黑暗预培养24 h的情况下,蓝光诱导24 h时通过Northern杂交和real-time PCR均可从菌丝体中检测到sahhl基因的大量转录。但sahhl基因在黑暗预培养48 h的情况下,通过real-time PCR没有检测到sahhl基因的大量表达。【结论】上述结果说明,蓝光可以诱导M.amphibiorum RCS1中生长旺盛的菌丝体中sahhl基因的表达。     

luxS基因缺失对禽致病性大肠杆菌O1、O2和O78三种血清型分离株的生物学特性影响

【目的】LuxS/AI-2型密度群体感应系统产生的自诱导信号分子AI-2(AI-2的产生需要luxS基因编码的Lux S蛋白参与)参与对细菌众多生理功能的调控。探讨luxS对不同血清型禽致病性大肠杆菌(Avian Pathogenicity Escherichia coli,APEC)生物学特性的影响。【方法】本研究以APEC优势血清型APECO_1(O_1血清型)、DE17(O_2血清型)、E940(O_(78)血清型)及其相应luxS缺失株为研究对象,对野生株和缺失株的生长特性、生物被膜形成、rdar(red,dry and rough)形态、运动性和耐药性等特性进行分析。【结果】luxS基因的缺失不影响APEC生长特性,但导致APEC不能产生AI-2;此外,luxS基因的缺失显著降低APECO_1和E940的生物被膜形成(P0.05),而DE17的生物被膜形成无显著变化。对各菌株的rdar形态和运动性检测结果表明,luxS基因的缺失改变了APECO_1的rdar形态,对DE17和E940并无影响;显著降低了APECO_1和DE17运动能力,对E940并无影响。荧光定量PCR检测结果表明,luxS基因的缺失显著降低APECO_1、DE17和E940与细菌运动性相关的鞭毛基因fli G和fli I的转录水平(P0.05)。此外,对各菌株的耐药性检测结果表明,luxS基因缺失导致APECO_1对头孢吡肟和丁胺卡那由耐药变为高敏,同时对氯霉素与E940相同由高敏变为耐药,但对DE17的耐药性无显著改变。【结论】luxS对APEC的生物学特性具有重要的调控作用,且这种调控具有菌株特异性。     

新生隐球菌半胱氨酸转运蛋白Mup1鉴定及其表达调控研究

【目的】鉴定新生隐球菌(Cryptococcus neoformans)的半胱氨酸转运蛋白及其对致病性的影响。【方法】构建候选基因敲除株,检测突变株以半胱氨酸为唯一硫源的生长情况;检测半胱氨酸转运蛋白Mup1对新生隐球菌毒力因子表达和不同胁迫条件下生长的影响;通过新生隐球菌大蜡螟(Galleria mellonella)和小鼠感染模型分析Mup1对致病性的影响;通过转录组分析和酵母单杂交研究硫代谢核心转录因子Cys3与Mup1的调控关系。【结果】Mup1具有转运半胱氨酸、胱氨酸、胱硫醚和同型半胱氨酸的能力。基因MUP1缺失不影响毒力因子表达和细胞对应激的反应。大蜡螟和小鼠隐球菌感染模型表明Mup1对新生隐球菌的致病性无显著影响。转录组分析和酵母单杂交实验显示Cys3可能间接调控MUP1的转录。【结论】新生隐球菌Mup1具有转运半胱氨酸、胱氨酸、胱硫醚和同型半胱氨酸的功能,但不影响致病性,基因转录可能受Cys3的间接调控。     

S-腺苷甲硫氨酸合成酶对埃博霉素生物合成的影响

【目的】研究S-腺苷甲硫氨酸合成酶(SAMs)对埃博霉素生物合成的影响。【方法】通过向发酵培养基中添加抑制剂和促进剂,比较分析纤维堆囊菌中SAMs的活性变化以及埃博霉素的产量变化。【结果】在埃博霉素的合成期,SAMs的活性较高。加入抑制剂吲哚乙酸(IAA)之后,SAMs的活性和埃博霉素的产量都不同程度的降低,而加入促进剂对甲苯磺酸钠(p-TSA-Na)之后,SAMs的活性和埃博霉素的产量在不同程度上都有提高。在纤维堆囊菌的次级代谢中,SAMs活性与埃博霉素的生物合成量呈正相关。【结论】S-腺苷甲硫氨酸合成酶在纤维堆囊菌的埃博霉素生物合成过程中发挥了重要的作用。     

同型半胱氨酸在心血管疾病中的免疫调节作用

高同型半胱氨酸血症是动脉粥样硬化的独立危险因子,但是其致病机制尚未完全阐明。本文将从体液免疫、单核巨噬细胞以及T细胞活性等几方面归纳总结同型半胱氨酸在心血管疾病中的免疫调节作用。同型半胱氨酸可以诱导单核细胞和T细胞分泌趋化因子和细胞因子,还可以直接刺激B细胞增殖及IgG分泌。此外,本文还总结了高同型半胱氨酸致炎作用的细胞内机制。同型半胱氨酸可以直接或间接导致氧化应激或者内质网应激,还可以降低一氧化氮的生物活性,影响包括S-腺苷蛋氨酸和S-腺苷同型半胱氨酸的水平,从而导致心血管疾病的发生。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.