Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America.
PLoS One. 2011 Jan 21;6(1):e15572. doi: 10.1371/journal.pone.0015572.
Cryptococcus neoformans (Cn) is a significant human pathogen that, despite current treatments, continues to have a high morbidity rate especially in sub-Saharan Africa. The need for more tolerable and specific therapies has been clearly shown. In the search for novel drug targets, the gene for glucosylceramide synthase (GCS1) was deleted in Cn, resulting in a strain (Δgcs1) that does not produce glucosylceramide (GlcCer) and is avirulent in mouse models of infection. To understand the biology behind the connection between virulence and GlcCer, the production and localization of GlcCer must be characterized in conditions that are prohibitive to the growth of Δgcs1 (neutral pH and high CO(2)). These prohibitive conditions are physiologically similar to those found in the extracellular spaces of the lung during infection. Here, using immunofluorescence, we have shown that GlcCer localization to the cell surface is significantly increased during growth in these conditions and during infection. We further seek to exploit this localization by treatment with Cerezyme (Cz), a recombinant enzyme that metabolizes GlcCer, as a potential treatment for Cn. Cz treatment was found to reduce the amount of GlcCer in vitro, in cultures, and in Cn cells inhabiting the mouse lung. Treatment with Cz induced a membrane integrity defect in wild type Cn cells similar to Δgcs1. Cz treatment also reduced the in vitro growth of Cn in a dose and condition dependent manner. Finally, Cz treatment was shown to have a protective effect on survival in mice infected with Cn. Taken together, these studies have established the legitimacy of targeting the GlcCer and other related sphingolipid systems in the development of novel therapeutics.
新型隐球菌（Cn）是一种重要的人类病原体，尽管目前有治疗方法，但它的发病率仍然很高，尤其是在撒哈拉以南的非洲地区。显然，人们需要更耐受和更具针对性的治疗方法。在寻找新的药物靶点时，人们删除了新型隐球菌中的葡糖基神经酰胺合酶（GCS1）基因，从而产生了一种不能产生葡糖基神经酰胺（GlcCer）且在感染小鼠模型中无毒性的菌株（Δgcs1）。为了了解毒力与 GlcCer 之间的联系背后的生物学机制，必须在不利于 Δgcs1 生长的条件下（中性 pH 值和高 CO2）对 GlcCer 的产生和定位进行特征描述。这些不利条件在生理上与感染期间肺部细胞外空间中发现的条件相似。在这里，我们通过免疫荧光法表明，在这些条件下和感染期间，GlcCer 向细胞表面的定位显著增加。我们进一步寻求通过用 Cerezyme（Cz）处理来利用这种定位，Cz 是一种可代谢 GlcCer 的重组酶，可作为新型隐球菌的潜在治疗方法。发现 Cz 处理可减少体外、培养物中和栖息在小鼠肺部的新型隐球菌细胞中的 GlcCer 量。Cz 处理在野生型新型隐球菌细胞中诱导了类似于 Δgcs1 的膜完整性缺陷。Cz 处理还以剂量和条件依赖的方式降低了新型隐球菌在体外的生长。最后，Cz 处理显示对感染新型隐球菌的小鼠的生存具有保护作用。总之，这些研究确立了靶向 GlcCer 和其他相关鞘脂系统以开发新型治疗方法的合理性。
