Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin.
Membrane Integrity Contributes to Resistance of Cryptococcus neoformans to the Cell Wall Inhibitor Caspofungin.
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真菌病原体新型隐球菌每年在全球引起多达278，000例感染，每年导致多达180，000人死亡，主要影响免疫功能低下的人群。C的治疗选择新生儿感染非常有限。卡泊芬净是棘白菌素类抗真菌药中的一员，一般耐受性良好，但临床上对念珠菌无效。新人类我们试图通过筛选在KN 99 α背景下制备的卡泊芬净敏感性基因缺失突变体的可用文库，确定可靶向使细胞对棘白菌素更敏感的生物学过程。我们采用了白色念珠菌真菌生物膜检测法，以了解白色念珠菌的生长特性。在一式三份平板测定中系统地筛选了4，030个个体基因缺失突变体。我们确定了25株卡泊芬净敏感性。我们随后进行了剂量依赖性试验，确认25个中的17个敏感，其中5个在琼脂平板试验中也敏感。我们对其中四个基因进行了新的缺失突变株：CFT 1，编码铁转运蛋白; ERG 4，编码固醇去饱和酶; MYO 1，编码肌球蛋白重链;和YSP 2，编码固醇转运蛋白。所有这些都对膜应力更敏感，并且在较高温度下对卡泊芬净的敏感性显着增加。令人惊讶的是，没有一个显示出任何明显的细胞壁缺陷，如卡泊芬净敏感菌株所预期的。我们的显微镜分析表明，膜完整性的损失有助于卡泊芬净的敏感性，无论是通过允许更多的卡泊芬净进入或留在细胞中，或通过改变酶靶点的位置或方向，使其更容易受到抑制。重要性新生隐球菌对细胞壁抑制剂卡泊芬净的内在耐药性限制了治疗隐球菌感染的可用疗法。我们筛选了4,000多个基因缺失菌株的集合，以改变卡泊芬净的敏感性，以确定可以靶向使细胞对卡泊芬净更敏感的生物过程。我们鉴定了多个影响卡泊芬净敏感性的基因，发现它们与细胞膜通透性的改变有关，而不是与预期的细胞壁缺陷有关。这表明靶向这些基因或影响膜通透性的其他基因是开发治疗这种全球真菌病原体的新疗法的可行途径。
The fungal pathogen Cryptococcus neoformans causes up to 278 000 infections each year globally, resulting in up to 180,000 deaths annually, mostly impacting immunocompromised people. Therapeutic options for C. neoformans infections are very limited. Caspofungin, a member of the echinocandin class of antifungals, is generally well tolerated but clinically ineffective against C. neoformans. We sought to identify biological processes that can be targeted to render the cell more susceptible to echinocandins by screening the available libraries of gene deletion mutants made in the KN99α background for caspofungin sensitivity. We adapted a Candida albicans fungal biofilm assay for the growth characteristics of C. neoformans and systematically screened 4,030 individual gene deletion mutants in triplicate plate assays. We identified 25 strains that showed caspofungin sensitivity. We followed up with a dose dependence assay, and 17 of the 25 were confirmed sensitive, 5 of which were also sensitive in an agar plate assay. We made new deletion mutant strains for four of these genes: CFT1, encoding an iron transporter; ERG4, encoding a sterol desaturase; MYO1, encoding a myosin heavy chain; and YSP2, encoding a sterol transporter. All were more sensitive to membrane stress and showed significantly increased sensitivity to caspofungin at higher temperatures. Surprisingly, none showed any obvious cell wall defects such as would be expected for caspofungin-sensitive strains. Our microscopy analyses suggested that loss of membrane integrity contributed to the caspofungin sensitivity, either by allowing more caspofungin to enter or remain in the cell or by altering the location or orientation of the enzyme target to render it more susceptible to inhibition. IMPORTANCE The intrinsic resistance of Cryptococcus neoformans to the cell wall inhibitor caspofungin limits the available therapies for treating cryptococcal infections. We screened a collection of more than 4,000 gene deletion strains for altered caspofungin sensitivity to identify biological processes that could be targeted to render the cell more susceptible to caspofungin. We identified multiple genes with an effect on caspofungin susceptibility and found that they were associated with altered membrane permeability rather than the expected cell wall defects. This suggests that targeting these genes or other genes affecting membrane permeability is a viable path for developing novel therapies for treating this global fungal pathogen.
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影响因子：
4.9
作者：
Meletiadis, Joseph;Pournaras, Spyros;Walsh, Thomas J.
通讯作者：
Walsh, Thomas J.
影响因子：
2.9
作者：
McDade, HC;Cox, GM
通讯作者：
Cox, GM
影响因子：
4.6
作者：
Cao, Chengjun;Xue, Chaoyang
通讯作者：
Xue, Chaoyang
DOI：
10.1016/j.ijfoodmicro.2010.05.020
发表时间：
2010-07-15
影响因子：
5.4
作者：
Landolfo, Sara;Zara, Giacomo;Mannazzu, Ilaria
通讯作者：
Mannazzu, Ilaria
DOI：
10.1083/jcb.201502033
发表时间：
2015-05-25
期刊：
The Journal of cell biology
影响因子：
--
作者：
Murley A;Sarsam RD;Toulmay A;Yamada J;Prinz WA;Nunnari J
通讯作者：
Nunnari J