染料木素对MRSA外排蛋白表达的影响

[目的] 研究染料木素对耐甲氧西林金黄色葡萄球菌（MRSA）外排蛋白的影响。[方法] 通过联合药敏实验检测染料木素影响MRSA对环丙沙星的敏感性;利用等重同位素多标签相对定量蛋白质组学（iTRAQ）技术,检测染料木素作用MRSA41577后菌体蛋白表达量的变化;通过生物信息学方法对差异显著的蛋白进行系统分析;通过qPCR和尼罗红外排实验,探讨耐药相关的蛋白介导细菌耐药的作用机制。[结果] 联合药敏实验结果显示,染料木素能增强MRSA对环丙沙星的敏感性;通过iTRAQ技术检测到差异显著蛋白共有129个,包括60个表达上调的蛋白和69个表达下调的蛋白;生物信息学分析结果显示,与细菌耐药相关的蛋白约有14个,其中,通过主动外排系统介导细菌耐药的蛋白主要有PstB、PstS等;qPCR结果显示,与对照组相比,PstB、PstS的基因表达量分别下降了51.6%和78.6%;尼罗红外排实验结果显示,染料木素与尼罗红之间存在竞争关系,为MRSA41577的竞争性抑制剂。[结论] 染料木素可通过降低MRSA41577外排基因pstB和pstS的mRNA表达量,进而影响PstB、PstS外排蛋白的表达来逆转细菌耐药;此外,染料木素还是MRSA41577的竞争性外排抑制剂,可通过与底物竞争外排的方式,使抗菌药物留在菌体内发挥抗菌作用。     

植物菌根共生磷酸盐转运蛋白

大多数植物能和丛枝菌根（arbuscular mycorrhiza, AM）真菌形成菌根共生体。AM能够促进植物对土壤中矿质营养的吸收,尤其是磷的吸收。磷的吸收和转运由磷酸盐转运蛋白介导。总结了植物AM磷酸盐转运蛋白及其结构特征,分析其分类及系统进化,并综述了AM磷酸盐转运蛋白介导的磷的吸收和转运过程及其基因的表达调控。植物AM磷酸盐转运蛋白属于Pht1家族成员,它不仅对磷的吸收和转运是必需的,而且对AM共生也至关重要,为进一步了解菌根形成的分子机理及信号转导途径提供了理论基础。     

氯霉素和四环素阻碍细菌蛋白分泌研究进展

氯霉素和四环素发挥活性的一个途径就是阻碍细菌蛋白质的分泌,其分泌功能是由其氨基端的信号序列决定的,该序列能将蛋白质引导到由SecY,E,G和A组成的转运蛋白复合体上。蛋白的转运还取决于融合蛋白的折叠特点,蛋白质转运到周质后的错误折叠可导致毒素聚集体形成,快速折叠还会使转运复合体发生拥堵,使所有的蛋白质分泌都受到抑制,导致细胞死亡。抗生素氯霉素和四环素处理细菌后会导致转运复合体中SecY的降解,造成致命的蛋白拥堵。现就抗生素氯霉素和四环素的干扰细菌蛋白质合成的作用机制以及导致SecY的降解来发挥阻碍细菌蛋白质分泌活性的一个新模式进行概述,以期为探讨新的靶向细菌的治疗方法提供科学依据。     

乳腺癌耐药蛋白的研究进展

乳腺癌耐药相关蛋白(BCRP/ABCG2)是新近发现的ATP结合盒(Adenosine triphosphate-binding cassette,ABC)膜转运蛋白超家族成员。它作为细胞膜上的药物排出泵,可以将一系列细胞毒药物转运至胞外从而介导肿瘤细胞多药耐药。在很多血液肿瘤和实体瘤中均检测到ABCG2表达。ABCG2在肿瘤的多药耐药上发挥重要作用。本文对ABCG2的发现、基因表达特征、与造血干细胞分化的关系、转运底物及其耐药逆转和临床意义等方面的研究进展进行综述。     

OsPT8过量表达提高转基因烟草的耐低磷能力

高亲和磷转运蛋白负责植物在低磷条件下吸收和转运磷酸盐,对植物的生长发育至关重要。将水稻中关键的高亲和磷转运蛋白基因OsPT8(A high affinity phosphate transporter gene OsPht1;8,以下简称OsPT8)通过农杆菌介导的方法转入烟草云烟87,以转基因烟草和野生型(云烟87)为材料,设置正常供磷(1 mmol/L Pi)和低磷(0.1 mmol/L Pi)两个处理的沙培试验,检测烟株地上部和地下部的生物量、全磷及有效磷的含量,分析烟草高亲和磷转运蛋白家族基因(NtPT1和NtPT2)的表达差异。结果显示,低磷条件下,OsPT8过量表达转基因株系生物量均显著高于野生型;在正常供磷和低磷条件下,OsPT8过量表达烟草株系全磷含量和有效磷含量均显著高于野生型,这表明高亲和磷转运蛋白基因OsPT8可以提高转基因烟草的耐低磷能力。RT-PCR和Q-PCR结果显示,转基因株系显著提高了烟草高亲和磷转运蛋白基因NtPT1和NtPT2的表达量,表明OsPT8对烟草磷吸收和转运的影响是通过OsPT8基因和烟草NtPT1、NtPT2基因等一个复杂的过程起作用的。     

非典型应答调控蛋白活性调控机制

双组分信号转导系统是生物中广泛存在的调控系统,通常由组氨酸激酶和应答调控蛋白(Responseregulator,RR)两个组分构成。典型的RR通过一个磷酸化机制调控活性。非典型应答调控蛋白在细菌中广泛存在,并调控细菌的生长发育、抗生素合成、Fe的转运等多种生理功能。以下主要综述目前研究比较清楚的非典型应答调控蛋白的结构和功能方面的进展,并以链霉菌中杰多霉素生物合成途径中的非典型应答调控蛋白JadR1为例,阐明调控蛋白活性调控的新机制。     

肿瘤多药耐药性中的ABC转运蛋白及其调节

ABC转运蛋白是一类利用ATP水解能量,逆浓度方向将一系列化合物转运通过膜结构的膜蛋白,这一类蛋白能转运离子,糖,氨基酸,维生素,多肽,多糖,激素,脂类及生物异源物质.P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP)和乳腺癌耐药蛋白(BCRP)等ABC转运蛋白还具有转运抗癌药物的能力,因此对化疗的有效性有负面的影响.近年来,许多研究涉及到如何逆转由ABC转 运蛋白引起的肿瘤多药耐药性.本文概述了近年来在蛋白水平,mRNA水平或DNA水平上对ABC转运蛋白调控的研究.     

盐爪爪Na^＋/H^＋逆向转运蛋白和焦磷酸酶的亚细胞定位

将盐爪爪Na+/H+逆向转运蛋白基因(KfNHX1)和焦磷酸酶基因(KfVP1)分别构建至植物表达载体,利用基因枪介导的方法转化洋葱表皮细胞,通过荧光显微镜观察研究其亚细胞定位.结果表明,转化了KfNHX1(或KfVP1)-GFP融合蛋白的洋葱表皮细胞仅膜系统散发荧光,而对照组即未转入KfNHX1(或KfVP1)基因的细胞则整体均匀发出荧光.说明KfNHX1和KfVP1可能定位于细胞的膜系统,作为跨膜转运蛋白在离子的调控运输中发挥重要作用.     

介导多药耐药的ABC转运蛋白超家族与MTX耐药性的关系研究

细胞耐药性的产生是导致肿瘤化疗失败的重要因素, 尤其是多药耐药是目前研究的一个重点。ABC转运蛋白超家族成员介导药物的外排, 与多药耐药密切相关。为了解该家族成员与MTX耐药的相关性, 进一步探讨MTX的耐药机制, 应用SuperArray基因芯片对MTX耐药前后编码ABC转运蛋白超家族成员的mdr1、mrp1、mrp2、mrp3、mrp5、mrp6和abcg2 7个基因进行检测, 并对MRP1和MRP5蛋白表达进行了验证。结果显示, 与MTX耐药性相关的ABC转运蛋白超家族成员主要为多药耐药相关蛋白, 其中mrp1和mrp5呈现高表达, 并且, 在MTX抗性细胞中, MRP5在mRNA及蛋白水平的表达均明显增强, 提示其在MTX耐药机制中起重要作用, 可能为潜在的药物作用靶点。     

浅黄根须腹菌磷酸盐转运蛋白基因异源表达分析

为探究外生菌根真菌对油松磷吸收作用的分子机理,以油松优良乡土外生菌根真菌——浅黄根须腹菌Rhizopogon luteolus的磷酸盐转运蛋白基因（RlPT）为对象,在缺陷酵母MB192中进行了异源表达研究。结果显示,该cDNA编码的蛋白质能够互补高亲和力磷酸盐转运蛋白pho84的功能;由不同pH条件下生长试验可知,该蛋白是一个与质子相偶联的运输蛋白;RlPT测算的K_m值为57.90μmol/L磷酸盐;通过酸性磷酸酶的活性检测,进一步验证该基因是具有高亲和力磷酸盐转运蛋白功能的基因。激光共聚焦显微观察表明,该蛋白在低磷条件下多定位于酵母细胞膜上发挥其功能。     

Molecular aspects of phosphate transport in Escherichia coli

Escherichia coli transports inorganic phosphate (Pi) by the low-affinity transport system, Pit. When the level of the external Pi is lower than 20 microM, another transport system, Pst, is induced with a Kt of 0.25 microM. An outer-membrane porin, PhoE, with a Km of about 1 microM is also induced. The outer membrane allows the intake of organic phosphates which are degraded to Pi by phosphatases in the periplasm. The Pi-binding protein will capture the free Pi produced in the periplasm and direct it to the transmembrane channel of the cytoplasmic membrane. The channel consists of two proteins, PstA and PstC, which have six and five transmembrane helices, respectively. On the cytoplasmic side of the membrane the channel is linked to the PstB protein, which carries a nucleotide (probably ATP)-binding site. PstB probably provides the energy required by the channel to free Pi. The Pst system has two functions in E. coli: (i) the transport of Pi, and (ii) the negative regulation of the phosphate regulon (a complex of 20 proteins mostly related to organic phosphate transport). It is remarkable that these two functions are not related, since the repressibility of the regulon depends on the integral structure of Pst (PiBP + PstA + PstC + PstB) and not on the Pi transported. Another gene of the pst operon, phoU, produces a protein involved in the negative regulation of the Pho regulon, but the mechanism of this function has not been explained. Thus the regulatory function of the Pst system remains obscure. Its basal level, present when Pi is abundant, is sufficient to repress the Pho regulon but the negative regulatory function is lost upon Pi starvation.     

From cell membrane to nucleotides: the phosphate regulon in Escherichia coli

Most of the essential cellular components, like nucleic acids, lipids and sugars, are phosphorylated. The phosphate equilibrium in Escherichia coli is regulated by the phosphate (Pi) input from the surrounding medium. Some 90 proteins are synthesized at an increased rate during Pi starvation and the global control of the cellular metabolism requires cross-talk with other regulatory mechanisms. Since the Pi concentration is normally low in E. coli's natural habitat, these cells have devised a mechanism for synthesis of about 15 proteins to accomplish two specific functions: transport of Pi and its intracellular regulation. The synthesis of these proteins is controlled by two genes (the phoB-phoR operon), involving both negative and positive functions. PhoR protein is a histidine protein kinase, induced in Pi starvation and is a transmembrane protein. It phosphorylates the regulator protein PhoB which is also Pi starvation-induced. The PhoB phosphorylated form binds specifically to a DNA sequence of 18 nucleotides (the pho Box), which is part of the promoters of the Pho genes. The genes controlled by phoB constitute the Pho regulon. The repression of phoA (the gene encoding alkaline phosphatase) by high Pi concentrations in the medium requires the presence of an intact Pst operon (pstS, pstC, pstA, pstB and phoU) and phoR. The products of pstA and pstC are membrane bound, whereas the product of pstS is periplasmic and PstB and PhoU proteins are cytoplasmic. The function of the PhoU protein may be regulated by cofactor nucleotides and may be involved in signaling the activation of the regulon via PhoR.     

Crystal structure of the Mycobacterium tuberculosis phosphate binding protein PstS3

Mycobacterium tuberculosis evades host immune responses by colonizing macrophages. Intraphagosomal M. tuberculosis is exposed to environmental stresses such as reactive oxygen and nitrogen intermediates as well as acid shock and inorganic phosphate (Pi) depletion. Experimental evidence suggests that expression levels of mycobacterial protein PstS3 (Rv0928) are significantly increased when M. tuberculosis bacilli are exposed to Pi starvation. Hence, PstS3 may be important for survival of Mtb in conditions where there is limited supply of Pi. We report here the structure of PstS3 from M. tuberculosis at 2.3‐Å resolution. The protein presents a structure typical for ABC phosphate transfer receptors. Comparison with its cognate receptor PstS1 showed a different pattern distribution of surface charges in proximity to the Pi recognition site, suggesting complementary roles of the two proteins in Pi uptake. Proteins 2014; 82:2268–2274. © 2014 Wiley Periodicals, Inc.     

PstB protein of the phosphate-specific transport system of Escherichia coli is an ATPase.

The PstB protein of the phosphate-specific transport (Pst) system of Escherichia coli bound and hydrolyzed ATP, producing ADP. Urea-treated denatured PstB did not bind ATP. The N-terminal amino acid sequence of the immune serum-precipitable PstB protein was determined, and it corresponded to that deduced from the DNA sequence.     

The DING family of proteins: ubiquitous in eukaryotes,but where are the genes?

PstS and DING proteins are members of a superfamily of secreted, high‐affinity phosphate‐binding proteins. Whereas microbial PstS have a well‐defined role in phosphate ABC transporters, the physiological function of DING proteins, named after their DINGGG N termini, still needs to be determined. PstS and DING proteins co‐exist in some Pseudomonas strains, to which they confer a highly adhesive and virulent phenotype. More than 30 DING proteins have now been purified, mostly from eukaryotes. They are often associated with infections or with dysregulation of cell proliferation. Consequently, eukaryotic DING proteins could also be involved in cell–cell communication or adherence. The ubiquitous presence in eukaryotes of proteins structurally and functionally related to bacterial virulence factors is intriguing, as is the absence of eukaryotic genes encoding DING proteins in databases. DING proteins in eukaryotes could originate from unidentified commensal or symbiotic bacteria and could contribute to essential functions. Alternatively, DING proteins could be encoded by eukaryotic genes sharing special features that prevent their cloning. Both hypotheses are discussed.     

Three different putative phosphate transport receptors are encoded by the Mycobacterium tuberculosis genome and are present at the surface of Mycobacterium bovis BCG.

A gene encoding a protein homologous to the periplasmic ABC phosphate binding receptor PstS from Escherichia coli was cloned and sequenced from a lambda gt11 library of Mycobacterium tuberculosis by screening with monoclonal antibody 2A1-2. Its degree of similarity to the E. coli PstS is comparable to those of the previously described M. tuberculosis phosphate binding protein pab (Ag78, Ag5, or 38-kDa protein) and another M. tuberculosis protein which we identified recently. We suggest that the three M. tuberculosis proteins share a similar function and could be named PstS-1, PstS-2, and PstS-3, respectively. Molecular modeling of their three-dimensional structures using the structure of the E. coli PstS as a template and their inducibility by phosphate starvation support this view. Recombinant PstS-2 and PstS-3 were produced and purified by affinity chromatography. With PstS-1, these proteins were used to demonstrate the specificity of three groups of monoclonal antibodies. Using these antibodies in flow cytometry and immunoblotting analyses, we demonstrate that the three genes are expressed and their protein products are present and accessible at the mycobacterial surface as well as in its culture filtrate. Together with the M. tuberculosis genes encoding homologs of the PstA, PstB, and PstC components we cloned before, the present data suggest that at least one, and possibly several, related and functional ABC phosphate transporters exist in mycobacteria. It is hypothesized that the mycobacterial gene duplications presented here may be a subtle adaptation of intracellular pathogens to phosphate starvation in their alternating growth environments.     

细菌的信号转导系统及其在耐药中的作用

耐药菌的日益增多给临床治疗带来巨大的困难,揭示耐药机制成为遏制耐药菌的基本环节.细菌的信号系统是菌体之间信息交流的主要渠道,在调控细菌耐药性方面发挥重要的作用.本文梳理了细菌双组分系统、群体感应系统、第二信使、吲哚等细菌信号系统(分子)与细菌耐药性的关系,总结了各信号系统调控细菌耐药性的机制和途径,包括调控生物膜的形成...     

Characterization of two genetically separable inorganic phosphate transport systems in Escherichia coli.

Inorganic phosphate (Pi) transport by wild-type cells of Escherichia coli grown in excess phosphate-containing media involves two genetically separable transport systems. Cells dependent upon the high affinity-low velocity Pst (phosphate specific transport) system have a Km of 0.43 +/- 0.2 microM Pi and a Vmax of 15.9 +/- 0.3 nmol of Pi (mg [dry weight]-1min-1) and will grow in the presence of arsenate in the medium. However, cells dependent upon the low affinity-high velocity Pit (Pi transport) system have a Km of 38.2 +/- 0.4 microM and a Vmax of 55 +/- 1.9 nmol of Pi (mg [dry weight]-1min-1), and these cells cannot grow in the presence of an arsenate-to-Pi ratio of 10 in the medium. Pi transport by both systems was sensitive to the energy uncoupler 2,4-dinitrophenol and the sulfhydryl reagent N-ethylmaleimide, whereas only the Pst system was very sensitive to sodium cyanide. Evidence is presented that Pi is transported as Pi or a very labile intermediate and that accumulated Pi does not exit through the Pst or Pit systems from glucose-grown cells. Kinetic analysis of Pi transport in the wild-type strain containing both the Pst and Pit transport systems revealed that each system was not operating at full capacity. In addition, Pi transport in the wild-type strain was completely sensitive to sodium cyanide (a characteristic of the Pst system).     

RPS2, an Arabidopsis disease resistance locus specifying recognition of Pseudomonas syringae strains expressing the avirulence gene avrRpt2.

A molecular genetic approach was used to identify and characterize plant genes that control bacterial disease resistance in Arabidopsis. A screen for mutants with altered resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) expressing the avirulence gene avrRpt2 resulted in the isolation of four susceptible rps (resistance to P. syringae) mutants. The rps mutants lost resistance specifically to bacterial strains expressing avrRpt2 as they retained resistance to Pst strains expressing the avirulence genes avrB or avrRpm1. Genetic analysis indicated that in each of the four rps mutants, susceptibility was due to a single mutation mapping to the same locus on chromosome 4. Identification of a resistance locus with specificity for a single bacterial avirulence gene suggests that this locus, designated RPS2, controls specific recognition of bacteria expressing the avirulence gene avrRpt2. Ecotype Wü-0, a naturally occurring line that is susceptible to Pst strains expressing avrRpt2, appears to lack a functional allele at RPS2, demonstrating that there is natural variation at the RPS2 locus among wild populations of Arabidopsis.