• 【用羟胺武装荧光探针探索新型隐球菌胶囊的结构和组装。】 复制标题 收藏 收藏
    DOI:10.1074/jbc.RA119.012251 复制DOI
    作者列表:Crawford CJ,Cordero RJB,Guazzelli L,Wear MP,Bowen A,Oscarson S,Casadevall A
    BACKGROUND & AIMS: :Chemical biology is an emerging field that enables the study and manipulation of biological systems with probes whose reactivities provide structural insights. The opportunistic fungal pathogen Cryptococcus neoformans possesses a polysaccharide capsule that is a major virulence factor, but is challenging to study. We report here the synthesis of a hydroxylamine-armed fluorescent probe that reacts with reducing glycans and its application to study the architecture of the C. neoformans capsule under a variety of conditions. The probe signal localized intracellularly and at the cell wall-membrane interface, implying the presence of reducing-end glycans at this location where the capsule is attached to the cell body. In contrast, no fluorescence signal was detected in the capsule body. We observed vesicle-like structures containing the reducing-end probe, both intra- and extracellularly, consistent with the importance of vesicles in capsular assembly. Disrupting the capsule with DMSO, ultrasound, or mechanical shear stress resulted in capsule alterations that affected the binding of the probe, as reducing ends were exposed and cell membrane integrity was compromised. Unlike the polysaccharides in the assembled capsule, isolated exopolysaccharides contained reducing ends. The reactivity of the hydroxylamine-armed fluorescent probe suggests a model for capsule assembly whereby reducing ends localize to the cell wall surface, supporting previous findings suggesting that this is an initiation point for capsular assembly. We propose that chemical biology is a promising approach for studying the C. neoformans capsule and its associated polysaccharides to unravel their roles in fungal virulence.
    背景与目标: : 化学生物学是一个新兴领域,可以通过探针研究和操纵生物系统,探针的反应性提供了结构见解。机会性真菌病原体新型隐球菌具有多糖胶囊,是主要的毒力因子,但研究具有挑战性。我们在这里报告了与还原性聚糖反应的羟胺武装荧光探针的合成及其在各种条件下研究新孢子虫胶囊结构的应用。探针信号位于细胞内和细胞壁-膜界面处,这意味着在胶囊附着于细胞体的位置存在还原端聚糖。相反,在胶囊体内未检测到荧光信号。我们观察到在细胞内和细胞外均包含还原端探针的囊泡状结构,这与囊泡在荚膜组装中的重要性一致。用DMSO,超声或机械剪切应力破坏胶囊会导致胶囊改变,从而影响探针的结合,因为减少端暴露了,细胞膜的完整性受到损害。与组装胶囊中的多糖不同,分离的胞外多糖含有还原端。羟胺武装荧光探针的反应性表明了胶囊组装的模型,其中还原端定位于细胞壁表面,支持先前的发现,表明这是胶囊组装的起点。我们认为化学生物学是研究新生梭菌胶囊及其相关多糖以阐明其在真菌毒力中的作用的一种有前途的方法。
  • 【隐球菌新生脑膜炎相关免疫重建炎症综合征的临床和影像学特征。】 复制标题 收藏 收藏
    DOI:10.1038/s41598-020-67031-4 复制DOI
    作者列表:Wu G,Guo X,Wang Y,Hu Z
    BACKGROUND & AIMS: :Cryptococcal meningitis is the most common intracranial infectious fungal disease. After a period of antifungal treatment, as the number of cells in the cerebrospinal fluid decreases, the biochemical indexes improve and the number of cryptococcus reduces, the patient's condition suddenly worsen. Most of the symptoms are severe headache, raised intracranial pressure, together with impaired clinical nerve function. These presentations are often mistaken for a failure of antifungal treatment. In fact it's an encephalitis syndrome which is unrecognized by most clinicians: Immune reconstitution inflammatory syndrome (IRIS). To increase awareness we retrospectively analyzed clinical data of 100 cases of cryptococcal neoformans meningitis, among which 26 patients develop CM-IRIS. All patients have been divided into three groups: Group 1, patients who were not treated with glucocorticoid and didn't experienced IRIS; Group 2, patients who were not treated with glucocorticoid although developed CM-IRIS; Group 3, patients started treatment with glucocorticoid for two weeks with new onset CM-IRIS. Compared with the group treated with glucocorticoid, treatment without glucocorticoid was subjected to a higher risk of incident IRIS. The difference was statistically significant (P < 0.05). Imaging findings demonstrated diseased area of the white matter area, and it looked like commonly in the supratentorial region. Moreover, if it appears in the infratentorial region then must be combined with supratentorial region.
    背景与目标: : 隐球菌性脑膜炎是最常见的颅内感染性真菌病。经过一段时间的抗真菌治疗后,随着脑脊液细胞数量的减少,生化指标的改善和隐球菌数量的减少,患者的病情突然恶化。大多数症状是严重的头痛,颅内压升高,以及临床神经功能受损。这些表现通常被误认为抗真菌治疗失败。实际上,这是一种脑炎综合征,大多数临床医生都无法识别: 免疫重建炎症综合征 (IRIS)。为了提高认识,我们回顾性分析了100例新型隐球菌脑膜炎的临床资料,其中26例发生二1212-虹膜。将所有患者分为三组: 第1组,未接受糖皮质激素治疗且未出现虹膜的患者; 第2组,尽管出现二1212-虹膜,但未接受糖皮质激素治疗的患者; 第3组,开始使用糖皮质激素治疗两周并新发二1212-虹膜的患者。与使用糖皮质激素的组相比,不使用糖皮质激素的治疗发生虹膜的风险更高。差异有统计学意义 (p  <  0.05)。影像学发现显示白质区域的病变区域,看起来像在幕上区域一样。此外,如果它出现在幕下区域,则必须与幕上区域结合。
  • 【一种通过真菌金属蛋白酶的新型天然产物抑制剂防止新型隐球菌穿过血脑屏障的抗毒方法。】 复制标题 收藏 收藏
    影响因子 :
    发表时间:2020-07-21
    来源期刊:mBio
    DOI:10.1128/mBio.01249-20 复制DOI
    作者列表:Aaron PA,Vu K,Gelli A
    BACKGROUND & AIMS: :Cryptococcus neoformans (Cn) is the leading cause of fungal meningitis, a deadly disease with limited therapeutic options. Dissemination to the central nervous system hinges on the ability of Cn to breach the blood-brain barrier (BBB) and is considered an attribute of Cn virulence. Targeting virulence instead of growth for antifungal drug development has not been fully exploited despite the benefits of this approach. Mpr1 is a secreted fungal metalloprotease not required for fungal growth, but rather, it functions as a virulence factor by facilitating Cn migration across the BBB. This central role for Mpr1, its extracellular location, and lack of expression in mammalian cells make Mpr1 a high-value target for an antivirulence approach aimed at developing therapeutics for cryptococcal meningitis. To test this notion, we devised a large-scale screen to identify compounds that prohibited Cn from crossing the BBB by selectively blocking Mpr1 proteolytic activity, without inhibiting the growth of Cn A phytochemical natural product-derived library was screened to identify new molecular scaffolds of prototypes unique to a Cn microecosystem. Of the 240 pure natural products examined, 3 lead compounds, abietic acid, diosgenin, and lupinine inhibited Mpr1 proteolytic activity with 50% inhibitory concentration (IC50) values of <10 μM, displayed little to no mammalian cell toxicity, and did not affect Cn growth. Notably, the lead compounds blocked Cn from crossing the BBB, without damaging the barrier integrity, suggesting the bioactive molecules had no off-target effects. We propose that these new drug scaffolds are promising candidates for the development of antivirulence therapy against cryptococcal meningitis.IMPORTANCE Fungal infections like cryptococcal meningitis are difficult to resolve because of the limited therapies available. The small arsenal of antifungal drugs reflect the difficulty in finding available targets in fungi because like mammalian cells, fungi are eukaryotes. The limited efficacy, toxicity, and rising resistance of antifungals contribute to the high morbidity and mortality of fungal infections and further underscore the dire but unmet need for new antifungal drugs. The traditional approach in antifungal drug development has been to target fungal growth, but an attractive alternative is to target mechanisms of pathogenesis. An important attribute of Cryptococcus neoformans (Cn) pathogenesis is its ability to enter the central nervous system. Here, we describe a large-scale screen that identified three natural products that prevented Cn from crossing the blood-brain barrier by inhibiting the virulence factor Mpr1 without affecting the growth of Cn We propose that compounds identified here could be further developed as antivirulence therapy that would be administered preemptively or serve as a prophylactic in patients at high risk for developing cryptococcal meningitis.
    背景与目标: : 新型隐球菌 (Cn) 是真菌性脑膜炎的主要原因,真菌性脑膜炎是一种致命的疾病,治疗选择有限。传播到中枢神经系统取决于Cn突破血脑屏障 (BBB) 的能力,被认为是Cn毒力的属性。尽管这种方法的好处,但尚未充分利用针对抗真菌药物开发的毒力而不是生长。Mpr1是真菌生长不需要的分泌性真菌金属蛋白酶,而是通过促进Cn在BBB上的迁移而充当毒力因子。Mpr1的核心作用,其细胞外位置以及在哺乳动物细胞中缺乏表达,使Mpr1成为旨在开发隐球菌性脑膜炎治疗药物的抗病毒方法的高价值靶标。为了验证这一概念,我们设计了一种大规模筛选,通过选择性阻断Mpr1蛋白水解活性来鉴定禁止Cn穿越BBB的化合物,而不抑制Cn的生长。筛选了植物化学天然产物衍生的文库,以鉴定Cn微生态系统特有的新分子支架。在所检查的240种纯天然产物中,3种铅化合物,松香酸,diosgenin和lupine抑制Mpr1蛋白水解活性,50% 抑制浓度 (IC50) 值为   <10μm,几乎没有哺乳动物细胞毒性,并且不影响Cn生长。值得注意的是,铅化合物阻止Cn穿过BBB,而不损害屏障的完整性,表明生物活性分子没有脱靶作用。我们建议这些新的药物支架是开发抗隐球菌性脑膜炎的抗病毒治疗的有希望的候选者。重要的是真菌感染,如隐球菌性脑膜炎很难解决,因为可用的治疗有限。抗真菌药物的小武库反映了在真菌中寻找可用靶标的困难,因为像哺乳动物细胞一样,真菌是真核生物。抗真菌药物的有限功效,毒性和不断上升的耐药性导致了真菌感染的高发病率和死亡率,并进一步强调了对新抗真菌药物的迫切但未得到满足的需求。抗真菌药物开发的传统方法是靶向真菌生长,但有吸引力的替代方法是靶向发病机制。新型隐球菌 (Cn) 发病的一个重要属性是其进入中枢神经系统的能力。在这里,我们描述了一个大规模的筛选,该筛选确定了三种天然产物,它们通过抑制毒力因子Mpr1而不影响Cn的生长而阻止Cn穿过血脑屏障。发生隐球菌性脑膜炎的高风险。
  • 【新隐球菌物种复合体哥伦比亚分离株的体外交配。】 复制标题 收藏 收藏
    DOI:/S0120-41572007000800017 复制DOI
    作者列表:Escandón P,Ngamskulrungroj P,Meyer W,Castañeda E
    BACKGROUND & AIMS: INTRODUCTION:Within the Cryptococcus neoformans species complex, two species and five serotypes are recognized: C. neoformans (var. grubii, serotype A; var. neoformans, serotype D and a hybrid, serotype AD) and C. gattii (serotypes B and C). Mating types a and alpha are designated by a single locus, with the mating type alpha being most prevalent in serotype A and D strains. OBJECTIVE:To evaluate the ability of Colombian isolates of the C. neoformans species complex to mate in vitro with tester strains of the opposite mating type. MATERIALS AND METHODS:Fifty three clinical isolates were included in this study, 33 C. neoformans var. grubii serotype A, 4 C. neoformans var. neoformans serotype D, all mating type alpha, and 16 C. gattii, 13 serotype B (mating type a) and 3 serotype C (mating type alpha), were mixed on V8 juice agar, using a modified method, with the appropriate tester strains to determine the mating types in vitro. RESULTS:Mating studies revealed that 9 of 33 (27.3%) serotype A isolates and 6 of 13 (46.2%) serotype B isolates were able to mate. Clamp connections and basidia with basidiospores were observed microscopically, indicating that the mating process had occurred. All mating competent serotype A strains were mating type alpha and the serotype B mating competent strains were mating type a. CONCLUSION:This is the first report of the determination of the mating ability of Colombian Cryptococcus neoformans isolates to mate in vitro with appropriate tester strains, which is of great importance to study the propagation of the fungus around the globe.
    背景与目标:
  • 【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。

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