• 【皮肤隐球菌感染和艾滋病。12例报告及文献复习。】 复制标题 收藏 收藏
    DOI: 复制DOI
    作者列表:Murakawa GJ,Kerschmann R,Berger T
    BACKGROUND & AIMS: BACKGROUND:Cryptococcal infections occur in 6% to 13% of patients with acquired immunodeficiency syndrome (AIDS), most commonly infecting the central nervous system. Cutaneous lesions have been described morphologically as umbilicated papules, nodules, and violaceous plaques and can mimic molluscum contagiosum and Kaposi's sarcoma. Cutaneous lesions can present months prior to other signs of systemic infection.

    OBSERVATIONS:Cases of infection with cutaneous Cryptococcus and AIDS were reviewed and compared with cases reported in the literature. Among patients with Cryptococcus infection and AIDS seen at our institutions, 5.9% had skin lesions. All patients with cutaneous lesions had systemic involvement. Women were less commonly infected than men. There was no apparent predisposition associated with age, race, or human immunodeficiency virus infection risk factors. The median CD4 helper T-cell count was 0.024 X 10(9)/L (24/microL), and 44% (16/36) of the patients had previous opportunistic infections. Lesions were most commonly seen on the head and neck (78% [36/46]) and often mimicked molluscum contagiosum (54% [25/46]). The median serum and cerebrospinal fluid cryptococcal antigen titers were 1:32,768 and 1:512, respectively. Patients in our group did well with therapy (one death at 6 weeks, compared with 38% [13/34] mortality in the literature). There was no correlation between onset of lesions, number of lesions, CD4 helper T-cell count, or histopathologic characteristics.

    CONCLUSIONS:Disseminated Cryptococcus infection in AIDS presents with cutaneous lesions in up to 6% of cases. Clinicians need to be aware of the varied morphologic characteristics, since cutaneous lesions may present well in advance of other signs of systemic infection.

    背景与目标: 背景 : 隐球菌感染6% 13% 获得性免疫缺陷综合症 (AIDS) 患者,最常见的是感染中枢神经系统。皮肤病变在形态上被描述为脐带丘疹,结节和紫膜斑块,可以模仿传染性软疣和卡波西氏肉瘤。皮肤病变可以在其他全身感染迹象出现之前几个月出现。
    观察 : 回顾了皮肤隐球菌和艾滋病感染的病例,并将其与文献报道的病例进行了比较。在我们机构看到的隐球菌感染和艾滋病患者中,5.9% 有皮肤病变。所有皮肤病变患者均有全身受累。女性的感染率低于男性。没有与年龄,种族或人类免疫缺陷病毒感染危险因素相关的明显倾向。CD4辅助T细胞计数中位数为0.024 × 10(9)/L (24/microL),44% (16/36) 患者既往有机会性感染。病变最常见于头部和颈部 (78% [36/46]),通常模仿传染性软疣 (54% [25/46])。血清和脑脊液隐球菌抗原滴度中位数分别为1:32,768和1:512。我们组的患者在治疗方面做得很好 (6周时死亡1例,而文献中的死亡率为38% 例 [13/34])。病变的发作,病变的数量,CD4辅助T细胞计数或组织病理学特征之间没有相关性。
    结论 : 艾滋病的播散性隐球菌感染表现为皮肤病变多达6%。临床医生需要意识到各种形态特征,因为皮肤病变可能会在其他全身性感染迹象之前出现。
  • 【新型隐球菌在受感染啮齿动物中合成聚合黑色素。】 复制标题 收藏 收藏
    DOI:10.1128/iai.68.5.2845-2853.2000 复制DOI
    作者列表:Rosas AL,Nosanchuk JD,Feldmesser M,Cox GM,McDade HC,Casadevall A
    BACKGROUND & AIMS: :The ability of Cryptococcus neoformans to synthesize polymerized melanin in vitro has been associated with virulence, but it is unclear whether this fungus synthesizes polymerized melanin during infection. To study this question, we used two approaches: one involved the generation of monoclonal antibodies (MAbs) to melanin for use in immunohistochemical studies of C. neoformans-infected rodents, and the other sought to isolate fungal melanin from infected tissues. Digestion of in vitro-melanized C. neoformans cells with proteases, denaturant, and hot concentrated acid yields melanin particles that retain the shape of fungal cells and are therefore called melanin ghosts. BALB/c mice were immunized with melanin ghosts, and two immunoglobulin M MAbs to melanin were generated from the spleen of one mouse. Immunofluorescence analyses of lung and brain tissues of rodents infected with wild-type melanin-producing (Mel(+)) C. neoformans strains demonstrated binding of the MAbs to the fungal cell wall. No binding was observed when infections were performed with mutant albino (Mel(-)) C. neoformans strains. Particles with striking similarity to melanin ghosts were recovered after digestion of lung and brain tissues from Mel(+) C. neoformans-infected rodents and were reactive with the MAbs to melanin. No particles were recovered from tissues infected with Mel(-) C. neoformans. A Mel(+) C. neoformans strain grown on lung or brain homogenate agar became lightly pigmented and also yielded particles similar to melanin ghosts upon digestion, providing additional evidence that lung and brain tissues contain substrate for C. neoformans melanization. These results demonstrate that C. neoformans synthesizes polymerized melanin during infection, which has important implications for pathogenesis and antifungal drug development.
    背景与目标: : 新型隐球菌在体外合成聚合黑色素的能力与毒力有关,但尚不清楚这种真菌是否在感染过程中合成聚合黑色素。为了研究这个问题,我们使用了两种方法: 一种方法涉及产生针对黑色素的单克隆抗体 (mab),用于新共生菌感染的啮齿动物的免疫组织化学研究,另一种方法试图从感染的组织中分离真菌黑色素。用蛋白酶,变性剂和热浓酸消化体外黑色素化的C. neoformans细胞,产生保留真菌细胞形状的黑色素颗粒,因此被称为黑色素鬼。用黑色素鬼免疫BALB/c小鼠,从一只小鼠的脾脏中产生两个针对黑色素的免疫球蛋白M单克隆抗体。感染野生型黑色素 (Mel ()) C的啮齿动物的肺和脑组织的免疫荧光分析。neoformans菌株证明了mab与真菌细胞壁的结合。当使用突变的白化病 (Mel(-)) 新孢子虫菌株进行感染时,未观察到结合。从Mel () C. neoformans感染的啮齿动物消化肺和脑组织后,回收了与黑色素鬼具有惊人相似性的颗粒,并与mab对黑色素反应。未从感染Mel(-) 新生梭菌的组织中回收颗粒。在肺或脑匀浆琼脂上生长的Mel () C. neoformans菌株变浅,并且在消化后也产生类似于黑色素幽灵的颗粒,这提供了其他证据,表明肺和脑组织含有neoformans黑色素化的底物。这些结果表明,新生梭菌在感染过程中合成聚合的黑色素,这对发病机理和抗真菌药物开发具有重要意义。
  • 【新型隐球菌对四种抗真菌药敏试验的Etest和微量稀释法比较。】 复制标题 收藏 收藏
    DOI:10.1093/jac/46.6.997 复制DOI
    作者列表:Aller AI,Martín-Mazuelos E,Gutiérrez MJ,Bernal S,Chávez M,Recio FJ
    BACKGROUND & AIMS: :We performed a prospective study to compare the Etest and the microdilution method (NCCLS guidelines) for determining the MICs of fluconazole, itraconazole, flucytosine and amphotericin B for 35 strains of Cryptococcus neoformans. For the microdilution method (MDM) RPMI 1640 medium with 2% glucose was used for fluconazole, itraconazole and flucytosine, and Antibiotic Medium 3 for amphotericin B. For the Etest, RPMI 1640 medium with 2% glucose and solidified with 1.5% agar was used for the four antifungal agents. Amphotericin B was also tested on Antibiotic Medium 3 solidified with 1.5% agar. Fluconazole and flucytosine MICs by the Etest showed good correlation with the broth MDM (81.1 and 89.2% agreement within two dilutions, respectively). With the tested population of itraconazole- and amphotericin B-susceptible isolates, the MIC agreement for itraconazole was 54%; amphotericin B showed the lowest agreement (8.1% on Antibiotic Medium 3 and 13.5% on RPMI).
    背景与目标: : 我们进行了一项前瞻性研究,以比较Etest和微量稀释法 (NCCLS指南) 来确定35株新型隐球菌的氟康唑,伊曲康唑,氟胞嘧啶和两性霉素b的mic。对于微量稀释法 (MDM),将含有2% 葡萄糖的RPMI 1640培养基用于氟康唑,伊曲康唑和氟胞嘧啶,将抗生素培养基3用于两性霉素b。对于Etest,将具有2% 葡萄糖并用1.5% 琼脂固化的RPMI 1640培养基用于四种抗真菌剂。还在用1.5% 琼脂固化的抗生素培养基3上测试两性霉素b。Etest的氟康唑和氟胞嘧啶mic与肉汤MDM显示出良好的相关性 (分别在两个稀释液中81.1和89.2% 一致)。在测试的伊曲康唑和两性霉素b敏感分离株的群体中,伊曲康唑的MIC一致性为54%; 两性霉素b显示出最低的一致性 (在抗生素培养基3上8.1%,在RPMI上13.5%)。
  • 【甾醇24-C-甲基转移酶: 破坏新型隐球菌麦角固醇生物合成和稳态的酶促靶标。】 复制标题 收藏 收藏
    DOI:10.1016/j.abb.2008.11.003 复制DOI
    作者列表:Nes WD,Zhou W,Ganapathy K,Liu J,Vatsyayan R,Chamala S,Hernandez K,Miranda M
    BACKGROUND & AIMS: :Growth of Cryptococcus neoformans was inhibited by nine nitrogen and sulfur-containing sterols with a heteroatom positioned at C3, C7, C24, C25 or C32 in the lanostane frame. Analysis of the sterol composition of control and treated cells by GC-MS and (1)H NMR has proven that the C-methylation reaction catalyzed by the sterol 24-C-methyltransferase (24-SMT) is the crucial first step in a kinetically favored pathway that fails to include obtusifoliol or zymosterol as intermediates. Cultures fed [methyl-(2)H(3)]methionine led to two deuterium atoms into each of the newly biosynthesized sterols forming a route lanosterol, eburicol (24(28)-methylene-24,25-dihydrolanosterol), 32-noreburicol and ergost-7-enol to ergosterol. Examination of the substrate specificity of a soluble 24-SMT from C. neoformans showed lanosterol to be the optimal acceptor molecule. Incubation with the test compounds generated induced amounts of lanosterol, eburicol or 32-noreburicol concurrent with a decrease of ergosterol. Among them 24(R,S),25-epiminolanosterol (inhibitor of 24-SMT) showed the most potent in vitro antifungal activity comparable to those of itraconazole (inhibitor of the 14-demethylase). Taken together, these data indicate that treatment with substrate-based inhibitors of 24-SMT, a catalyst not found in humans, can disrupt ergosterol homeostasis involved with fungal growth and therefore these compounds can provide leads for rational drug design of opportunistic pathogens.
    背景与目标: : 新隐球菌的生长被九种含氮和硫的固醇抑制,其杂原子位于羊毛烷框架中的C3,C7,C24,C25或C32。通过gc-ms和 (1)H NMR分析对照和处理过的细胞的固醇组成已证明,由固醇24-C-甲基转移酶 (24-SMT) 催化的C-甲基化反应是至关重要的第一步。动力学上偏爱的途径未能包括obtusifoolol或zymosterol作为中间体。饲喂 [甲基-(2)H(3)] 甲硫氨酸的培养物导致两个氘原子进入每个新生物合成的甾醇中,形成途径羊毛甾醇,伊布里考 (24(28)-亚甲基-24,25-二氢羊毛甾醇),32-去甲纤维醇和ergost-7-enol麦角固醇。对来自新孢子虫的可溶性24-smt的底物特异性的检查表明,羊毛甾醇是最佳受体分子。与测试化合物一起孵育会产生诱导量的羊毛甾醇,埃布里考或32-去甲布里考,同时麦角固醇降低。其中24(R,S),25-表氨基醇 (24-smt抑制剂) 显示出与伊曲康唑 (14-脱甲基酶抑制剂) 相当的最有效的体外抗真菌活性。综上所述,这些数据表明,用基于底物的24-SMT抑制剂 (一种在人类中未发现的催化剂) 治疗可以破坏与真菌生长有关的麦角固醇稳态,因此这些化合物可以为机会性病原体的合理药物设计提供线索。
  • 【来自人类病原体新型隐球菌的壳聚糖脱乙酰基酶的独特亚位点特异性和潜在的自然功能。】 复制标题 收藏 收藏
    DOI:10.1073/pnas.1915798117 复制DOI
    作者列表:Hembach L,Bonin M,Gorzelanny C,Moerschbacher BM
    BACKGROUND & AIMS: :Cryptococcus neoformans is an opportunistic fungal pathogen that infects ∼280,000 people every year, causing >180,000 deaths. The human immune system recognizes chitin as one of the major cell-wall components of invading fungi, but C. neoformans can circumvent this immunosurveillance mechanism by instead exposing chitosan, the partly or fully deacetylated form of chitin. The natural production of chitosans involves the sequential action of chitin synthases (CHSs) and chitin deacetylases (CDAs). C. neoformans expresses four putative CDAs, three of which have been confirmed as functional enzymes that act on chitin in the cell wall. The fourth (CnCda4/Fpd1) is a secreted enzyme with exceptional specificity for d-glucosamine at its -1 subsite, thus preferring chitosan over chitin as a substrate. We used site-specific mutagenesis to reduce the subsite specificity of CnCda4 by converting an atypical isoleucine residue in a flexible loop region to the bulkier or charged residues tyrosine, histidine, and glutamic acid. We also investigated the effect of CnCda4 deacetylation products on human peripheral blood-derived macrophages, leading to a model explaining the function of CnCda4 during infection. We propose that CnCda4 is used for the further deacetylation of chitosans already exposed on the C. neoformans cell wall (originally produced by CnChs3 and CnCda1 to 3) or released from the cell wall as elicitors by human chitinases, thus making the fungus less susceptible to host immunosurveillance. The absence of CnCda4 during infection could therefore promote the faster recognition and elimination of this pathogen.
    背景与目标: : 新型隐球菌是一种机会性真菌病原体,每年感染约280,000人,导致> 180,000人死亡。人类免疫系统将几丁质识别为入侵真菌的主要细胞壁成分之一,但是新生梭菌可以通过暴露几丁质 (几丁质的部分或完全脱乙酰化形式) 来规避这种免疫监视机制。壳聚糖的自然产生涉及几丁质合酶 (CHSs) 和几丁质脱乙酰基酶 (CDAs) 的顺序作用。新孢子虫表达四种假定的cda,其中三种已被确认为作用于细胞壁中几丁质的功能酶。第四种 (CnCda4/Fpd1) 是一种分泌酶,在its -1亚位点上对d-氨基葡萄糖具有特殊的特异性,因此比几丁质更喜欢壳聚糖作为底物。我们使用位点特异性诱变通过将柔性环区中的非典型异亮氨酸残基转化为更大或带电荷的残基酪氨酸,组氨酸和谷氨酸来降低CnCda4的亚位点特异性。我们还研究了CnCda4去乙酰化产物对人外周血来源的巨噬细胞的影响,从而建立了解释CnCda4在感染过程中的功能的模型。我们建议将CnCda4用于已经暴露在新生梭菌细胞壁上的壳聚糖 (最初由CnChs3和CnCda1至3产生) 或由人几丁质酶作为引发剂从细胞壁释放的进一步去乙酰化,从而使真菌不易受到宿主免疫监视。因此,在感染过程中不存在CnCda4可以促进更快地识别和消除该病原体。
  • 【吲哚-3-乙酸增强了月桂隐球菌对梨果实的扩展青霉和灰葡萄孢的生物防治。】 复制标题 收藏 收藏
    DOI:10.1111/j.1567-1364.2006.00171.x 复制DOI
    作者列表:Yu T,Zheng XD
    BACKGROUND & AIMS: :This study evaluated the efficacy of indole-3-acetic acid (IAA) alone or with a biocontrol yeast, Cryptococcus laurentii, in the inhibition of blue and gray mold diseases (Penicillium expansum and Botrytis cinerea) on pear fruit. The results demonstrated that a combination of C. laurentii with IAA at 100 microg mL(-1) was more effective in suppressing blue and gray mold infections on pear fruit than application of C. laurentii alone. IAA alone or with C. laurentii stimulated catalase, peroxidase and polyphenol oxidase activities of pear fruit, indicating that IAA can induce fruit-mediated resistance, although this agent alone had no direct antifungal activity.
    背景与目标: : 这项研究评估了吲哚-3-乙酸 (IAA) 单独或与生物防治酵母laurentii隐球菌在抑制梨果实上的蓝色和灰霉病 (青霉和灰霉病) 中的功效。结果表明,与单独施用劳伦蒂斯菌相比,劳伦蒂斯菌与100微克毫升 (-1) 的IAA组合在抑制梨果实上的蓝色和灰色霉菌感染方面更有效。单独使用IAA或与C. laurentii刺激了梨果实的过氧化氢酶,过氧化物酶和多酚氧化酶活性,表明IAA可以诱导果实介导的抗性,尽管单独使用该药物没有直接的抗真菌活性。
  • 【哺乳动物血清和CO(2) 诱导新生隐球菌的胶囊生长。】 复制标题 收藏 收藏
    DOI:10.1128/iai.71.11.6155-6164.2003 复制DOI
    作者列表:Zaragoza O,Fries BC,Casadevall A
    BACKGROUND & AIMS: :The pathogenic fungus Cryptococcus neoformans has a polysaccharide capsule that is essential for virulence in vivo. Capsule size is known to increase during animal infection, and this phenomenon was recently associated with virulence. Although various conditions have been implicated in promoting capsule growth, including CO(2) concentration, osmolarity, and phenotypic switching, it is difficult to reproduce the capsule enlargement effect in the laboratory. In this study, we report that serum can induce capsule growth, and we describe the conditions that induce this effect, not only by serum but also by CO(2). Capsule enlargement was dependent on the medium used, and this determined whether the strain responded to serum or CO(2) efficiently. Serum was most effective in inducing capsule growth under nutrient-limited conditions. There was considerable variability between strains in their response to either serum or CO(2), with some strains requiring both stimuli. Sera from several animal sources were each highly efficient in inducing capsule growth. The cyclic AMP (cAMP) pathway and Ras1 were both necessary for serum-induced capsule growth. The lack of induction in the ras1 mutant was not complemented by exogenous cAMP, indicating that these pathways act in parallel. However, both cAMP and Ras1 were dispensable for inducing a partial capsule growth by CO(2), suggesting that multiple pathways participate in this process. The ability of serum to induce capsule growth suggests a mechanism for the capsular enlargement observed during animal infection.
    背景与目标: : 致病真菌新型隐球菌具有体内毒力必不可少的多糖胶囊。已知动物感染期间胶囊的大小会增加,并且这种现象最近与毒力有关。尽管在促进胶囊生长方面涉及各种条件,包括CO(2) 浓度,渗透压和表型转换,但在实验室中很难重现胶囊的扩大效应。在这项研究中,我们报告了血清可以诱导胶囊生长,并且我们描述了不仅通过血清而且通过CO(2) 诱导这种作用的条件。胶囊的扩大取决于所使用的培养基,这决定了菌株是否有效地对血清或CO(2) 做出反应。在营养受限的条件下,血清最有效地诱导胶囊生长。菌株对血清或CO(2) 的反应之间存在相当大的差异,有些菌株需要两种刺激。来自几种动物来源的血清在诱导胶囊生长方面均非常有效。循环AMP (cAMP) 途径和Ras1对于血清诱导的胶囊生长都是必需的。ras1突变体缺乏诱导,没有得到外源cAMP的补充,表明这些途径平行作用。然而,cAMP和Ras1对于通过CO(2) 诱导部分胶囊生长都是必不可少的,这表明该过程有多种途径参与。血清诱导胶囊生长的能力提示了动物感染期间观察到的荚膜增大的机制。
  • 【mRNA合成和降解的解偶联会损害新型隐球菌对宿主温度的适应性。】 复制标题 收藏 收藏
    DOI:10.1111/mmi.12258 复制DOI
    作者列表:Bloom AL,Solomons JT,Havel VE,Panepinto JC
    BACKGROUND & AIMS: :The pathogenic fungus Cryptococcus neoformans must overcome multiple stressors to cause disease in its human host. In this study, we report that C. neoformans rapidly and transiently repressed ribosomal protein (RP) transcripts during a transition from 30°C to host temperature. This repression was accompanied by accelerated mRNA degradation mediated by the major deadenylase, Ccr4, and influenced by the dissociable RNA polymerase II subunit, Rpb4. Destabilization and deadenylation of RP transcripts were impaired in an rpb4Δ mutant, suggesting that Rpb4 may be involved in host temperature-induced Ccr4-mediated decay. Accelerated decay of ER stress transcripts 1 h following a shift to host temperature was also impaired in the rpb4Δ mutant. In response to host temperature, Rpb4 moved from the nucleus to the cytoplasm, supporting a role for Rpb4 in coupling transcription and degradation. The PKH signalling pathway was implicated as a regulator of accelerated degradation of the RP transcripts, but not of the ER stress transcripts, revealing a further level of specificity. When transcription and degradation were uncoupled by deletion of Rpb4, growth at host temperature was impaired and virulence was attenuated. These data suggest that mRNA synthesis and decay are coupled in C. neoformans via Rpb4, and this tight coordination promotes host-temperature adaptation and pathogenicity.
    背景与目标: : 致病真菌新型隐球菌必须克服多种应激源才能在其人类宿主中引起疾病。在这项研究中,我们报告了C. 型新生细胞在从30 °C到宿主温度的转变过程中迅速且短暂地抑制了核糖体蛋白 (RP) 转录本。这种抑制作用伴随着主要的去死酶Ccr4介导的加速mRNA降解,并受到可分离的RNA聚合酶II亚基rpb4的影响。rpb4Δ 突变体中RP转录物的去稳定化和去烯化作用受损,表明Rpb4可能参与宿主温度诱导的Ccr4-mediated衰变。在rpb4Δ 突变体中,随着宿主温度的变化,ER胁迫转录本的加速衰减也受到损害。响应宿主温度,Rpb4从细胞核移动到细胞质,支持Rpb4在偶联转录和降解中的作用。PKH信号通路被认为是RP转录本加速降解的调节剂,但不是ER应激转录本的调节剂,揭示了进一步的特异性水平。当转录和降解因Rpb4缺失而解偶联时,宿主温度下的生长受到损害,毒力减弱。这些数据表明,新生梭菌的mRNA合成和衰变通过Rpb4偶联,这种紧密的协调促进了宿主-温度的适应性和致病性。
  • 【CRZ1/SP1-like基因将病原酵母新型隐球菌在有限曝气下的存活,细胞完整性和生物膜形成联系起来。】 复制标题 收藏 收藏
    DOI:10.5507/bp.2013.024 复制DOI
    作者列表:Moranova Z,Virtudazo E,Hricova K,Ohkusu M,Kawamoto S,Husickova V,Raclavsky V
    BACKGROUND & AIMS: AIMS:Limited aeration has been demonstrated to cause slowdown in proliferation and delayed budding, resulting eventually in a unique unbudded G2-arrest in the obligate aerobic pathogenic yeast Cryptococcus neoformans. Also, the ability to adapt to decreased oxygen levels during pathogenesis has been identified as a virulence factor in C. neoformans. The aim of this study was to identify and characterize genes that are necessary for the proliferation slowdown and G2-arrest caused by limited aeration. METHODS:Random mutants were prepared and screened for lack of typical slowdown of proliferation under limited aeration. The CNAG_00156.2 gene coding for a zinc-finger transcription factor was identified in mutants showing most distinctive phenotype. Targeted deletion strain and reconstituted strain were prepared to characterize and confirm the gene functions. This gene was also identified in a parallel studies as homologous both to calcineurin responsive (Crz1) and PKC1-dependent (SP1-like) transcription factors. RESULTS:We have confirmed the role of the cryptococcal homologue of CRZ1/SP1-like transcription factor in cell integrity, and newly demonstrated its role in slowdown of proliferation and survival under reduced aeration, in biofilm formation and in susceptibility to fluconazole. CONCLUSIONS:Our data demonstrate a tight molecular link between slowdown of proliferation during hypoxic adaptation and maintenance of cell integrity in C. neoformans and present a new role for the CRZ1 family of transcription factors in fungi. The exact positioning of this protein in cryptococcal signalling cascades remains to be clarified.
    背景与目标:
  • 【Allergen1调节新型隐球菌的多糖结构。】 复制标题 收藏 收藏
    DOI:10.1111/mmi.12216 复制DOI
    作者列表:Jain N,Cordero RJ,Casadevall A,Fries BC
    BACKGROUND & AIMS: :Cryptococcus neoformans is an important human, fungal pathogen that sheds polysaccharide (exo-PS) into host tissues. While shed exo-PS mediates numerous untoward effects (including promoting increased intracranial pressure), little is known about the regulation of this phenomenon. Since downregulation of the Allergen 1 (ALL1) gene is associated with high ICP, we investigated the relationship between ALL1 expression and exo-PS structure using a variety of biophysical techniques. The Δall1 mutants of two serotypes produced a shorter exo-PS with less branching and structural complexity than the parental strains. Consistent with lower branching, these exo-PSs manifested higher intrinsic viscosity than the parental strains. The Δall1 mutant strains manifested differences in epitope expression and significant resistance to phagocytosis. Exo-PS of Δall1 mutant exhibited anti-phagocytic properties. Comparative transcriptome analysis of mutant and parental strain under iron-deprived conditions indicated a role of ALL1 in iron homeostasis, characterized by differential regulation of genes that mediate iron reduction and transport. Together, our results demonstrate a role of ALL1 in regulating conformational aspects of PS structure and iron homeostasis. These findings provide a mechanism to explain how changes in ALL1 expression influence virulence of switch variants and suggest that structural changes and polymer length are epigenetically regulated.
    背景与目标: : 新型隐球菌是一种重要的人类真菌病原体,可将多糖 (exo-PS) 分解到宿主组织中。虽然shed exo-PS介导了许多不良影响 (包括促进颅内压升高),但对这种现象的调节知之甚少。由于过敏原1 (ALL1) 基因的下调与高ICP有关,因此我们使用多种生物物理技术研究了ALL1表达与exo-PS结构之间的关系。与亲本菌株相比,两种血清型的 Δall1突变体产生的exo-ps较短,分支和结构复杂性较小。与较低的支化一致,这些exo-PSs表现出比亲本菌株更高的特性粘度。Δall1突变菌株表现出表位表达的差异和对吞噬作用的显着抗性。Δall1突变体的Exo-PS具有抗吞噬特性。在缺铁条件下对突变体和亲本菌株的比较转录组分析表明,ALL1在铁稳态中的作用,其特征是介导铁还原和转运的基因的差异调节。总之,我们的结果证明了ALL1在调节PS结构和铁稳态的构象方面的作用。这些发现提供了一种机制来解释ALL1表达的变化如何影响开关变体的毒力,并表明结构变化和聚合物长度受到表观遗传调控。
  • 【苯甲腈对月桂隐球菌,红孢子虫和谷花红豆菌对梨果实青霉侵染的影响。】 复制标题 收藏 收藏
    DOI:10.1016/j.ijfoodmicro.2013.04.012 复制DOI
    作者列表:Yu C,Zhou T,Sheng K,Zeng L,Ye C,Yu T,Zheng X
    BACKGROUND & AIMS: :The effect of biocontrol yeasts and pyrimethanil at low concentration on inhibition of blue mold rot caused by Penicillium expansum in pear fruit was investigated. Pyrimethanil at low concentration (40μg/mL) alone had little inhibitory activity against the P. expansum infection in pear fruit wounds although it was effective in inhibiting the survival of P. expansum on Asp-agar medium. Pyrimethanil at this low concentration significantly enhanced the efficacy of Cryptococcus laurentii at 1×10(7)CFU/mL in reducing blue mold rot in vivo compared with C. laurentii at 1×10(7)CFU/mL alone. However, there was no additive inhibitory activity when pyrimethanil was combined for application with biocontrol yeasts Rhodosporidium paludigenum or Rhodotorula glutinis. Combination of pyrimethanil and C. laurentii at low concentration also inhibited blue mold rot when P. expansum was inoculated into fruit wounds 12h before treatment and fruit was stored at low temperature (4°C). Pyrimethanil at 0.04 to 400μg/mL did not influence the survival of C. laurentii in vitro, and it only slightly reduced the population growth of C. laurentii after 48h of incubation in the pear fruit wounds. There was no significant difference in quality parameters including total soluble solids, titratable acidity and ascorbic acid of pear fruit wounds among all treatments after 5days of treatment at 25°C. Integration of C. laurentii and pyrimethanil at low concentration might be an effective and safe strategy to control P. expansum infection in pear fruit, especially in an integrated postharvest disease management strategy.
    背景与目标: : 研究了低浓度生防酵母和吡啶对梨果中青霉引起的蓝霉腐病的抑制作用。单独使用低浓度 (40 μ g/mL) 的吡啶胺对梨果实伤口中的P. expansum感染几乎没有抑制活性,尽管它可以有效抑制Asp-琼脂培养基上的P. expansum存活。与单独使用1 × 10(7)CFU/mL的劳伦蒂尼相比,在这种低浓度下的吡啶胺可显着提高1 × 10(7)CFU/mL的劳伦蒂尼隐球菌在体内减少蓝霉菌腐烂的功效。但是,当将苯甲胺与生物防治酵母红孢子菌或谷氨酸红酵母结合使用时,没有附加抑制活性。当在治疗前12小时将P. expansum接种到果实伤口中并将果实在低温 (4 °C) 下储存时,低浓度的吡啶胺和C. laurentii的组合也可以抑制蓝霉腐烂。0.04至400 μ g/mL的吡啶胺不会影响劳伦蒂尼的体外存活,并且在梨果实伤口中孵育48小时后,仅略微降低了劳伦蒂尼的种群增长。在25 °C下处理5天后,所有处理之间的质量参数 (包括梨果实伤口的总可溶性固形物,可滴定酸度和抗坏血酸) 均无显着差异。在低浓度下整合laurentii和pyrimethanil可能是控制梨果实中扩展P. expansum感染的有效且安全的策略,尤其是在采后疾病综合管理策略中。
  • 【毒力因子脲酶及其在新型隐球菌代谢中的作用。】 复制标题 收藏 收藏
    DOI:10.1093/femsyr/foaa031 复制DOI
    作者列表:Toplis B,Bosch C,Schwartz IS,Kenyon C,Boekhout T,Perfect JR,Botha A
    BACKGROUND & AIMS: :Cryptococcal urease is believed to be important for the degradation of exogenous urea that the yeast encounters both in its natural environment and within the human host. Endogenous urea produced by the yeast's own metabolic reactions, however, may also serve as a substrate for the urease enzyme. Using wild-type, urease-deletion mutant and urease-reconstituted strains of Cryptococcus neoformans H99, we studied reactions located up- and downstream from endogenous urea. We demonstrated that urease is important for cryptococcal growth and that, compared to nutrient-rich conditions at 26°C, urease activity is higher under nutrient-limited conditions at 37°C. Compared to cells with a functional urease enzyme, urease-deficient cells had significantly higher intracellular urea levels and also showed more arginase activity, which may act as a potential source of endogenous urea. Metabolic reactions linked to arginase were also affected, since urease-positive and urease-negative cells differed with respect to agmatinase activity, polyamine synthesis, and intracellular levels of proline and reactive oxygen species. Lastly, urease-deficient cells showed higher melanin levels at 26°C than wild-type cells, while the inverse was observed at 37°C. These results suggest that cryptococcal urease is associated with the functioning of key metabolic pathways within the yeast cell.
    背景与目标: : 隐球菌尿素酶被认为对于酵母在其自然环境和人类宿主中遇到的外源尿素的降解很重要。然而,由酵母自身的代谢反应产生的内源性尿素也可以用作脲酶的底物。使用野生型,脲酶缺失突变体和脲酶重组的新型隐球菌H99菌株,我们研究了内源性尿素上游和下游的反应。我们证明了脲酶对于隐球菌的生长很重要,并且与26 °C的营养丰富条件相比,在37 °C的营养受限条件下,脲酶活性更高。与具有功能性脲酶的细胞相比,脲酶缺陷的细胞具有显着更高的细胞内尿素水平,并且还显示出更高的精氨酸酶活性,这可能是内源性尿素的潜在来源。与精氨酸酶相关的代谢反应也受到影响,因为尿素酶阳性和尿素酶阴性的细胞在agmatinase活性,多胺合成以及脯氨酸和活性氧的细胞内水平方面有所不同。最后,脲酶缺陷细胞在26 °C时显示出比野生型细胞更高的黑色素水平,而在37 °C时观察到相反。这些结果表明,隐球菌尿素酶与酵母细胞内关键代谢途径的功能有关。
  • 【在小鼠疫苗模型中,热杀死的隐球菌突变株可诱导宿主对多种侵袭性真菌病的保护。】 复制标题 收藏 收藏
    影响因子 :
    发表时间:2019-11-26
    来源期刊:mBio
    DOI:10.1128/mBio.02145-19 复制DOI
    作者列表:Wang Y,Wang K,Masso-Silva JA,Rivera A,Xue C
    BACKGROUND & AIMS: :Cryptococcus neoformans is a fungal pathogen that infects the lungs and then often disseminates to the central nervous system, causing meningitis. How Cryptococcus is able to suppress host immunity and escape the antifungal activity of macrophages remains incompletely understood. We reported that the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase, promotes Cryptococcus virulence by regulating host-Cryptococcus interactions. Our recent studies demonstrated that the fbp1Δ mutant elicited superior protective Th1 host immunity in the lungs and that the enhanced immunogenicity of heat-killed fbp1Δ yeast cells can be harnessed to confer protection against a subsequent infection with the virulent parental strain. We therefore examined the use of heat-killed fbp1Δ cells in several vaccination strategies. Interestingly, the vaccine protection remains effective even in mice depleted of CD4+ T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that vaccinating mice with heat-killed fbp1Δ induces significant cross-protection against challenge with diverse invasive fungal pathogens, including C. neoformans, C. gattii, and Aspergillus fumigatus, as well as partial protection against Candida albicans Thus, our data suggest that the heat-killed fbp1Δ strain has the potential to be a suitable vaccine candidate against cryptococcosis and other invasive fungal infections in both immunocompetent and immunocompromised populations.IMPORTANCE Invasive fungal infections kill more than 1.5 million people each year, with limited treatment options. There is no vaccine available in clinical use to prevent and control fungal infections. Our recent studies showed that a mutant of the F-box protein Fbp1, a subunit of the SCF(Fbp1) E3 ligase in Cryptococcus neoformans, elicited superior protective Th1 host immunity. Here, we demonstrate that the heat-killed fbp1Δ cells (HK-fbp1) can be harnessed to confer protection against a challenge by the virulent parental strain, even in animals depleted of CD4+ T cells. This finding is particularly important in the context of HIV/AIDS-induced immune deficiency. Moreover, we observed that HK-fbp1 vaccination induces significant cross-protection against challenge with diverse invasive fungal pathogens. Thus, our data suggest that HK-fbp1 has the potential to be a broad-spectrum vaccine candidate against invasive fungal infections in both immunocompetent and immunocompromised populations.
    背景与目标: : 新型隐球菌是一种真菌病原体,会感染肺部,然后经常传播到中枢神经系统,从而引起脑膜炎。隐球菌如何抑制宿主免疫并逃避巨噬细胞的抗真菌活性尚不完全清楚。我们报道了F-box蛋白Fbp1 (SCF(Fbp1) E3连接酶的一个亚基) 通过调节宿主-隐球菌相互作用来促进隐球菌的毒力。我们最近的研究表明,fbp1Δ 突变体在肺部引起了良好的Th1宿主保护性免疫,并且可以利用热杀死的fbp1Δ 酵母细胞增强的免疫原性来保护其免受随后的毒力亲本菌株的感染。因此,我们在几种疫苗接种策略中检查了热杀死的fbp1Δ 细胞的使用。有趣的是,即使在CD4 + T细胞耗尽的小鼠中,疫苗保护仍然有效。在艾滋病毒/艾滋病引起的免疫缺陷的背景下,这一发现尤其重要。此外,我们观察到用热杀死的fbp1Δ 给小鼠接种疫苗会引起对多种侵袭性真菌病原体 (包括新生梭菌,加蒂梭菌和烟曲霉) 的攻击的显着交叉保护,以及对白色念珠菌的部分保护。我们的数据表明,在免疫能力和免疫功能低下的人群中,热灭活的fbp1Δ 菌株有可能成为抗隐球菌病和其他侵袭性真菌感染的合适候选疫苗。侵袭性真菌感染每年杀死超过150万人,治疗选择有限。临床上没有可用的疫苗来预防和控制真菌感染。我们最近的研究表明,新型隐球菌中SCF(Fbp1) E3连接酶的亚基F-box蛋白Fbp1的突变体引起了良好的Th1宿主免疫力。在这里,我们证明了热杀死的fbp1Δ 细胞 (HK-fbp1) 可以被用来保护抵抗强毒力的亲本菌株的挑战,即使在CD4 T细胞耗尽的动物中也是如此。在艾滋病毒/艾滋病引起的免疫缺陷的背景下,这一发现尤其重要。此外,我们观察到HK-fbp1疫苗可诱导针对多种侵袭性真菌病原体的攻击的显着交叉保护。因此,我们的数据表明,在免疫能力和免疫功能低下的人群中,HK-fbp1有可能成为针对侵袭性真菌感染的广谱候选疫苗。
  • 【Β1整合素是介导新隐球菌的NK细胞杀伤所必需的。】 复制标题 收藏 收藏
    DOI:10.4049/jimmunol.1701805 复制DOI
    作者列表:Xiang RF,Li S,Ogbomo H,Stack D,Mody CH
    BACKGROUND & AIMS: :Cryptococcus neoformans is a fungal pathogen that causes fatal meningitis and pneumonia. During host defense to Cryptococcus, NK cells directly recognize and kill C. neoformans using cytolytic degranulation analogous to killing of tumor cells. This fungal killing requires independent activation of Src family kinase (SFK) and Rac1-mediated pathways. Recognition of C. neoformans requires the natural cytotoxicity receptor, NKp30; however, it is not known whether NKp30 activates both signal transduction pathways or whether a second receptor is involved in activation of one of the pathways. We used primary human NK cells and a human NK cell line and found that NKp30 activates SFK → PI3K but not Rac1 cytotoxic signaling, which led to a search for the receptor leading to Rac1 activation. We found that NK cells require integrin-linked kinase (ILK) to activate Rac1 for effective fungal killing. This observation led to our identification of β1 integrin as an essential anticryptococcal receptor. These findings demonstrate that multiple receptors, including β1 integrins and NKp30 and their proximal signaling pathways, are required for recognition of Cryptococcus, which activates a central cytolytic antimicrobial pathway leading to fungal killing.
    背景与目标: : 新型隐球菌是一种真菌病原体,会导致致命的脑膜炎和肺炎。在宿主对隐球菌的防御过程中,NK细胞使用类似于杀死肿瘤细胞的溶细胞脱颗粒法直接识别并杀死新生梭菌。这种真菌杀死需要独立激活Src家族激酶 (SFK) 和Rac1-mediated途径。新生梭菌的识别需要天然细胞毒性受体NKp30; 但是,尚不清楚NKp30是否激活了这两种信号转导途径,或者第二种受体是否参与了其中一条途径的激活。我们使用原代人类NK细胞和人类NK细胞系,发现NKp30激活SFK → PI3K,但不激活Rac1细胞毒性信号,这导致寻找导致Rac1激活的受体。我们发现NK细胞需要整合素连接激酶 (ILK) 来激活Rac1以有效杀死真菌。这一观察结果导致我们将 β1整联蛋白鉴定为必不可少的抗链球菌受体。这些发现表明,识别隐球菌需要多种受体,包括 β1整联蛋白和NKp30及其近端信号通路,隐球菌激活了导致真菌杀死的中央溶细胞抗菌途径。
  • 【体外传代过程中DNA聚合酶POL3的自发突变会导致隐球菌物种的高突变表型。】 复制标题 收藏 收藏
    DOI:10.1016/j.dnarep.2019.102751 复制DOI
    作者列表:Boyce KJ,Cao C,Xue C,Idnurm A
    BACKGROUND & AIMS: :Passaging of microbes in vitro can lead to the selection of microevolved derivatives with differing properties to their original parent strains. One well characterised instance is the phenotypic differences observed between the series of strains derived from the type strain of the human pathogenic fungus Cryptococcus neoformans. A second case was reported in the close relative Cryptococcus deneoformans, in which a well-studied isolate ATCC 24067 (52D) altered its phenotypic characteristics after in vitro passaging in different laboratories. One of these derivatives, ATCC 24067A, has decreased virulence and also exhibits a hypermutator phenotype, in which the mutation rate is increased compared to wild type. In this study, the molecular basis behind the changes in the lineage of ATCC 24067 was determined by next-generation sequencing of the parent and passaged strain genomes. This analysis resulted in the identification of a point mutation that causes a D270G amino acid substitution within the exonuclease proofreading domain of the DNA polymerase delta subunit encoded by POL3. Complementation with POL3 confirmed that this mutation is responsible for the hypermutator phenotype of this strain. Regeneration of the mutation in C. neoformans, to eliminate the additional mutations present in the ATCC 24067A genetic background, demonstrated that the hypermutator phenotype of the pol3D270G mutant causes rapid microevolution in vitro but does not result in decreased virulence. These findings indicate that mutator strains can emerge in these pathogenic fungi without conferring a fitness cost, but the subsequent rapid accumulation of mutations can be deleterious.
    背景与目标: : 微生物在体外传代可以导致选择与其原始亲本菌株具有不同性质的微进化衍生物。一个特征良好的实例是在源自人类病原真菌新型隐球菌的类型菌株的一系列菌株之间观察到的表型差异。在近亲隐球菌中报告了第二例,其中经过充分研究的分离株ATCC 24067 (52D) 在不同实验室中体外传代后改变了其表型特征。这些衍生物之一ATCC 24067A具有降低的毒力,并且还表现出超突变表型,与野生型相比,突变率增加。在这项研究中,通过亲本和传代菌株基因组的下一代测序确定了ATCC 24067谱系变化背后的分子基础。此分析导致鉴定出点突变,该点突变导致由pol3编码的DNA聚合酶 δ 亚基的核酸外切酶校对结构域内的D270G氨基酸取代。与POL3的互补证实,该突变是该菌株的超突变表型的原因。新生梭菌突变的再生,以消除ATCC 24067A遗传背景中存在的其他突变,表明pol3D270G突变体的超突变表型在体外引起快速的微进化,但不会导致毒力降低。这些发现表明,突变菌株可以在这些致病真菌中出现而不增加适应性成本,但是随后快速积累的突变可能是有害的。

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