烟曲霉sho1、pbs2基因额外拷贝对几种应激能力的影响

目的构建烟曲霉额外拷贝菌株,了解额外拷贝烟曲霉sho 1、pbs 2基因能否增强菌株对高渗透压、过氧化氢(H2O2)、碱性pH、刚果红应激的抵抗能力,探讨HOG通路(high osmolarity glycerol pathway)参与的应激反应。方法用原生质体法构建分别含有烟曲霉sho 1、pbs 2基因的额外拷贝菌株,采用Real-time PCR方法检测额外拷贝株中sho 1、pbs 2的表达情况。观察并比较缺陷株、额外拷贝株对NaCl(1 mol/L)、H2O2(5 mmol/L)、刚果红(400 mg/L)及碱性pH(10.0)应激的反应。结果获得了含有烟曲霉sho 1、pbs 2基因的额外拷贝菌株MCsho1、MCpbs2,和含空白质粒的对照株Empty。额外拷贝株sho 1、pbs 2的表达水平增高,对NaCl(1 mol/L)、H2O2(5 mmol/L)、刚果红(400 mg/L)、碱性pH(10.0)应激的抵抗强于Empty。MCpbs2对这些应激的抵抗较MCsho1更显著。烟曲霉缺陷株△sho 1、△pbs 2对NaCl(1 mol/L)、H2O2(5mmol/L)、碱性pH(10.0)的敏感性高于野生株AF293。△sho 1对刚果红(400 mg/L)的敏感性高于野生株,△pbs 2对刚果红的敏感性与野生株比,无显著差别。结论额外拷贝烟曲霉sho 1或pbs 2基因能增强菌株对高渗透压、氧化压力、刚果红、碱性pH应激的抵抗能力。     

压力应激影响烟曲霉致病力的研究进展

烟曲霉是环境中广泛存在的腐生真菌,是重要的机会致病菌。近年来随着免疫受损人群的增多,烟曲霉引起侵袭性曲霉病不断增加,在深部丝状真菌感染中居于首位。由于宿主体内环境与自然环境存在诸多差异,使得烟曲霉孢子适应体内环境条件是其能够生存并致病的前提。研究发现烟曲霉适应宿主环境的能力,如温度、氧化应激、渗透压、缺氧、营养缺乏等,与其致病力密切相关。对压力应激与致病力关系的研究有助于解析烟曲霉的致病机制,可为研发烟曲霉感染诊治的新途径提供理论基础。本文对烟曲霉的压力应激与其致病力的关系进行了综述。     

烟曲霉af-fadO基因敲除株构建揭示该基因在恩波吡维胺与唑类协同抗烟曲霉中的作用

恩波吡维胺(pyrviniumpamoate,PP)是一种传统抗寄生虫药,与唑类联合具有协同抗烟曲霉作用。af-fadO(FADbindingoxidoreductase,FAD依赖性氧化还原酶)基因的编码产物在烟曲霉中推定为一种与FAD结合的氧化还原酶,可能参与真菌的氧化呼吸过程。通过敲除烟曲霉af-fadO基因,构建烟曲霉af-fadO基因的敲除株,揭示该基因及其编码蛋白在PP与唑类协同抗烟曲霉中的作用,并探索了其对烟曲霉药物敏感性和渗透压、氧化压力物质敏感性的影响。本研究采用同源重组构建出af-fadO基因的敲除盒,筛选出符合的烟曲霉af-fadO敲除株Δaf-fadO。利用纸片法和棋盘法观察该突变株与野生株WT(ku80,pyrG+)在PP与唑类(泊沙康唑、伊曲康唑、伏立康唑)联合抗烟曲霉中的敏感性变化。同时,观察该突变株与野生株WT对氧化压力(H2O2和甲萘醌)、渗透压物质(NaCl、D-山梨醇)敏感性的变化。体外联合药物敏感性试验结果显示Δaf-fadO对PP与泊沙康唑的分级抑菌浓度指数(fractional inhibitory concentration index, ...     

SakA对马尔尼菲篮状菌药物应激及致病力的影响

目的探讨SakA对马尔尼菲篮状菌(Talaromyces marneffei,TM)致病力及药物应激的影响。方法构建SakA敲除株(ΔsakA),观察孢子与巨噬细胞共培养;比较野生株及ΔsakA在药物压力下的生长状况;比较野生株和ΔsakA分别感染小鼠的死亡率、菌载量。结果被巨噬细胞吞噬24h后,野生株在胞内可见腊肠样酵母细胞,而ΔsakA以孢子形态存在。ΔsakA共培养裂解物形成的菌落数少于野生株。与野生株比较,ΔsakA对棘白霉素类药物更敏感。ΔsakA感染小鼠死亡率、菌载量较野生株明显降低。结论 SakA与TM酵母相对棘白霉素不敏感相关,在其致病过程中发挥重要作用。     

烟曲霉再鉴定、标准化CSP分型及体外药物敏感性

目的 了解烟曲霉复合体临床株菌种分布,经典烟曲霉临床株CSP基因型及对常见抗真菌药物敏感性状况.方法 菌株来源:北京大学真菌和真菌病研究中心保存分离自125名患者的162株烟曲霉复合体菌株.通过形态学,最高生长温度及分子生物学测序分步鉴定;对CSP基因进行扩增、测序,采用国际化命名体系进行CSP分型;采用微量液基稀释法测定经典烟曲霉对伊曲康唑(ITC)、两性霉素B(AMB)、伏立康唑(VRC)及卡泊芬净(CAS)的敏感性.结果 所有烟曲霉复合体菌株均为经典烟曲霉;共分为16个CSP基因型,最常见为t04A、t03和t01;分离自4名患者的13株菌对ITC的MICs≥4 μg/mL,其中2株菌AMB和VRC的MICa分别为4μg/mL和16 μg/mL.CAS的MECs最高为4μg/mL,仅1株.结论 未检出烟曲霉相关新种;经典烟曲霉临床株共16个CSP基因型,分布与国际研究结果基本一致,其中5个为新型.我国经典烟曲霉临床株ITC耐药率为3.2％,个别菌株AMB,VRC和CAS耐药.     

金属还原酶FreB2对烟曲霉铁吸收及氧化压力应答的影响

利用构建的烟曲霉金属还原酶基因(AFUA-1G00350,Fre B2)缺失突变株,对烟曲霉金属还原酶基因Fre B2功能进行初步研究,为揭示该基因与烟曲霉的致病关系提供依据。比较野生株和基因缺失突变株在AMM和无铁AMM液体培养基中生长时高铁还原酶的活性,绘制不同时间野生株和基因缺失突变株在AMM和无铁AMM液体培养基中生长时高铁还原酶活性曲线。利用Real-Time PCR方法分析Sre A、Sid A、Fet C、Ftr A和Fre B这些与铁的吸收相关基因的mRNA的表达量变化。测定野生株和基因缺失突变株对氧化压力的敏感性及胞内活性氧物质含量。不论在AMM液体培养基中还是在无铁AMM液体培养基中培养时,突变株高铁还原酶的活性都明显高于野生株高铁还原酶活性。与野生株相比培养60 h时,突变株Sre A、Sid A、Fet C、Ftr A和Fre B这些与铁的吸收相关基因的表达量出现明显上调。氧化压力敏感性实验显示,基因缺失突变株对H2O2的敏感性显著增强,同时胞内活性氧物质含量明显增多。金属还原酶基因Fre B2在烟曲霉铁吸收及氧化压力应答过程中发挥作用;烟曲霉与铁吸收相关基因之间存在功能互补效应。     

分离自1例慢性阻塞性肺疾病患者的Aspergillus lentulus在蜡螟模型上的毒力初探

目的建立Aspergillus lentulus(A.lentulus或A.L)感染动物模型,借动物模型初步探究A.lentulus的毒力。方法将125只蜡螟随机分成5组,以Aspergillus lentulus临床株、Aspergillus lentulus标准株作为实验组,烟曲霉、白念珠菌为对照组,PBS为空白对照组。实验组及对照组菌株分别制成10~6 CFU/mL孢子悬液,感染各组蜡螟。记录72 h内蜡螟的生存情况并制作生存曲线,24 h后提取蜡螟肠道组织,HE染色组织切片观察肠道组织损伤情况,用组织匀浆法,测定蜡螟肠道内真菌载量及真菌逆培养阳性率,用真菌荧光染色法观察肠道培养真菌镜下形态。结果 A.lentulus临床株和A.lentulus标准株的蜡螟存活数与对照组之间差异有统计学意义(P0.05);结果显示A.lentulus临床株和A.lentulus标准株肠壁结构大致正常,局部可见水肿少量菌丝、孢子及炎症细胞浸润,对照组肠道结构破坏严重,可见菌丝、孢子及大量炎症细胞浸润;不同菌种感染蜡螟幼虫各组肠道载菌量及真菌逆培养阳性率比较,差异具有统计学意义(P0.05);真菌荧光显微镜观察,A.lentulus临床株和标准株菌丝多、孢子少,白念珠菌和烟曲霉组孢子多。结论与烟曲霉和白念珠菌相比,A.lentulus菌株对蜡螟毒力和肠道损伤能力较弱且致死率低。     

小GTPase蛋白在烟曲霉耐药性和致病力中作用的研究进展

<正>烟曲霉是广泛存在于自然环境中的腐生真菌,其产生大量的分生孢子易被人体吸入肺中,在一定条件下可造成艾滋病患者和器官移植患者等免疫低下人群的感染。已有研究显示,临床中侵袭性曲霉感染致死率达50%以上,而造成这种高致死率的原因与烟曲霉菌丝极性生长、压力应激和免疫逃逸等过程有关^[1-2]。此外,临床上也面临烟曲霉对抗真菌药物(如三唑类和棘白菌素类等)耐受水平不断升高的棘手难题,这其中重要的耐药分子机制包括药物靶点突变/靶点高表达、药物应激反应等^[3-4]。因此,阐明烟曲霉在药物耐受和致病力等方面的重要分子机制,有助于临床和科研工作中防治烟曲霉感染和开发新型的靶点药物。目前的证据显示,小GTPase蛋白可能是参与调控致病真菌耐药性和致病力的关键分子基础^[5]。该蛋白在真菌细胞中多以单体形式(21～30kD)保守存在,可通过水解GTP实现开关模式转换,并以此参与细胞生长和分化、应激外界压力刺激等关键生命过程^[5]。小GTPase蛋白根据分子特征和功能等分为Rab、Ras、Arf、Rho和Ran...     

烟曲霉rRNA基因ITS区的克隆测序分析

对烟曲霉rRNA基因内转录间区(ITS区)进行了克隆测序,并将之与其他几种常见曲霉的相应序列进行了比较.发现3株烟曲霉的ITSⅠ区完全相同,而其中1株烟曲霉的ITSⅡ区与一条已知相应序列仅有2个碱基的变异.提示烟曲霉rRNA基因的两个ITS区序列均十分保守,而且与黄曲霉、黑曲霉、土曲霉及构巢曲霉的相应序列相比较,均有一定程度的变异.     

耐氟康唑念珠菌和耐伊曲康唑烟曲霉对泊沙康唑的敏感性测定

目的：测定耐氟康唑念珠菌和耐伊曲康唑烟曲霉临床分离株对泊沙康唑的敏感性。方法参照美国临床实验室标准化研究所制定的 M27-A3和 M38-A2方案,测定从临床获得的11株耐氟康唑的念珠菌和3株耐伊曲康唑烟曲霉对泊沙康唑的 MIC 值。结果对于氟康唑耐药的念珠菌,泊沙康唑的 MIC 范围是0.125-1μg/ mL。对于伊曲康唑耐药烟曲霉,泊沙康唑的 MIC 范围是0.06-0.5μg/ mL。结论11株耐氟康唑的念珠菌和3株耐伊曲康唑烟曲霉均对泊沙康唑有效。     

Cooperative regulation of DOG2, encoding 2-deoxyglucose-6-phosphate phosphatase, by Snf1 kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade in stress responses of Saccharomyces cerevisiae

We screened the genome of Saccharomyces cerevisiae for the genes responsive to oxidative stress by using the lacZ transposon-insertion library. As a result, we found that expression of the DOG2 gene coding for 2-deoxyglucose-6-phosphate phosphatase was induced by oxidative stress. The expression of DOG2 was also induced by osmotic stress. We found a putative cis element (STRE, a stress response element) in the DOG2 promoter adjacent to a consensus sequence to which the Mig1p repressor is known to bind. The basal levels of DOG2 gene expression were increased in a mig1Delta mutant, while the derepression of DOG2 was not observed in a snf1Delta mutant under glucose-deprived conditions. Induction of the DOG2 gene expression by osmotic stress was observed in any of the three disruptants pbs2Delta, hog1Delta, and snf1Delta. However, the osmotic induction was completely abolished in both the snf1Delta pbs2Delta mutant and the snf1Delta hog1Delta mutant. Additionally, these single mutants as well as double mutants failed to induce DOG2 expression by oxidative stress. These results suggest that Snf1p kinase and the high-osmolarity glycerol-mitogen-activated protein kinase cascade are likely to be involved in the signaling pathway of oxidative stress and osmotic stress in regulation of DOG2.     

Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants.

Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1delta could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.     

Hyperosmotic stress response and regulation of cell wall integrity in Saccharomyces cerevisiae share common functional aspects

The osmosensitive phenotype of the hog1 strain is suppressed at elevated temperature. Here, we show that the same holds true for the other commonly used HOG pathway mutant strains pbs2 and sho1ssk2ssk22, but not for ste11ssk2ssk22. Instead, the ste11ssk2ssk2 strain displayed a hyperosmosensitive phenotype at 37 degrees C. This phenotype is suppressed by overexpression of LRE1, HLR1 and WSC3, all genes known to influence cell wall composition. The suppression of the temperature-induced hyperosmosensitivity by these genes prompted us to investigate the role of STE11 and other HOG pathway components in cellular integrity and, indeed, we were able show that HOG pathway mutants display sensitivity to cell wall-degrading enzymes. LRE1 and HLR1 were also shown to suppress the cell wall phenotypes associated with the HOG pathway mutants. In addition, the isolated multicopy suppressor genes suppress temperature-induced cell lysis phenotypes of PKC pathway mutants that could be an indication for shared targets of the PKC pathway and high-osmolarity response routes.     

Mutant U2AF1-induced differential alternative splicing causes an oxidative stress in bone marrow stromal cells

Alternative splicing (AS) is a critical regulatory process of gene expression. In bone marrow microenvironment, AS plays a critical role in mesenchymal stem cells fate determination by forming distinct isoforms of important regulators. As a spliceosome factor, U2AF1 is essential for the catalysis of pre-mRNA splicing, and its mutation can cause differential AS events. In the present study, by forced expression of mutant U2AF1 (U2AF1S34F) in the mouse bone marrow stroma OP9 cells, we determine AS changes in U2AF1S34F transduced OP9 cells and investigate their role in stroma cell biological functions. We find that abundant differential RNA splicing events are induced by U2AF1S34F in OP9 cells. U2AF1S34F causes increased generation of hydrogen peroxide, promotes production of cytokines and chemokines. U2AF1S34F transduced OP9 cells also exhibit dysfunction of mitochondria. RNA-seq data, gene ontology (GO), and gene set enrichment analysis reveal that differentially expressed genes downregulated in response to U2AF1S34F are enriched in peroxisome component and function. U2AF1S34F can also cause release of hydrogen peroxide from OP9 cells. Furthermore, we investigate the influence of U2AF1S34F-induced oxidative stress in stromal cells on hematopoietic cells. When co-culturing mouse bone marrow mononuclear cells with OP9 cells, the U2AF1S34F expressing OP9 cells induce phosphorylation of histone H2AX in hematopoietic cells. Collectively, our results reveal that mutant U2AF1-induced differential AS events cause oxidative stress in bone marrow stromal cells and can further lead to DNA damage and genomic instability in hematopoietic cells.     

Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function

As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen. Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding. mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm. Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX. When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced. TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1. Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress. To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells. When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells. In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated. Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.     

热胁迫下沙门菌细胞结构和特性变化研究进展

沙门菌(Salmonella)是一种非常重要的食源性致病菌.由于食品基质的保护作用,有些沙门菌可以抵抗热胁迫而存活下来.存活细胞往往因为热胁迫或应激而导致细胞结构、生理特性、基因及蛋白表达发生变化,并会进一步对食品原料和加工环境造成持续污染.本文主要综述沙门菌在热胁迫前后细胞形态、菌体组分、细胞壁和细胞膜结构等方面的变...     

Identification of three putative signal transduction genes involved in R gene-specified disease resistance in Arabidopsis

The RPS5 disease resistance gene of Arabidopsis mediates recognition of Pseudomonas syringae strains that possess the avirulence gene avrPphB. By screening for loss of RPS5-specified resistance, we identified five pbs (avrPphB susceptible) mutants that represent three different genes. Mutations in PBS1 completely blocked RPS5-mediated resistance, but had little to no effect on resistance specified by other disease resistance genes, suggesting that PBS1 facilitates recognition of the avrPphB protein. The pbs2 mutation dramatically reduced resistance mediated by the RPS5 and RPM1 resistance genes, but had no detectable effect on resistance mediated by RPS4 and had an intermediate effect on RPS2-mediated resistance. The pbs2 mutation also had varying effects on resistance mediated by seven different RPP (recognition of Peronospora parasitica) genes. These data indicate that the PBS2 protein functions in a pathway that is important only to a subset of disease-resistance genes. The pbs3 mutation partially suppressed all four P. syringae-resistance genes (RPS5, RPM1, RPS2, and RPS4), and it had weak-to-intermediate effects on the RPP genes. In addition, the pbs3 mutant allowed higher bacterial growth in response to a virulent strain of P. syringae, indicating that the PBS3 gene product functions in a pathway involved in restricting the spread of both virulent and avirulent pathogens. The pbs mutations are recessive and have been mapped to chromosomes I (pbs2) and V (pbs1 and pbs3).