N-acetylglucosamine (GlcNAc) Triggers a Rapid, Temperature-Responsive Morphogenetic Program in Thermally Dimorphic Fungi

Thermally dimorphic human fungal pathogens undergo a reversible program of cellular differentiation in response to their environment that is essential for infectivity and pathogenicity. In the soil, these organisms grow as highly polarized, multicellular hyphal filaments that produce infectious particles. When inhaled by a mammalian host, these cells switch to a unicellular yeast form that causes disease even in healthy hosts. Temperature is considered to be the primary environmental cue that promotes reversible cellular differentiation; however, a shift to a lower temperature in vitro induces filamentous growth in an inefficient and asynchronous manner. In a search for other signals that regulate morphogenesis, we considered the monosaccharide N-acetylglucosamine (GlcNAc), which is a major component of microbial cell walls and is ubiquitous in the environment. GlcNAc was a potent and specific inducer of the yeast-to-filament transition in two thermally dimorphic fungi, Histoplasma capsulatum and Blastomyces dermatitidis. Micromolar concentrations of GlcNAc induced a robust morphological transition of H. capsulatum after temperature shift, indicating that fungal cells sense GlcNAc to promote filamentation. The synchronous morphologic transition stimulated by low temperature and GlcNAc allowed us to examine the temporal regulation of the transcriptome during morphogenesis to reveal candidate genes involved in establishing the filamentous growth program. Through this analysis, we identified two genes encoding GlcNAc transporters, NGT1 and NGT2, that were necessary for H. capsulatum cells to robustly filament in response to GlcNAc. Unexpectedly, NGT1 and NGT2 were important for efficient H. capsulatum yeast-to-filament conversion in standard glucose medium, suggesting that Ngt1 and Ngt2 monitor endogenous levels of GlcNAc to control multicellular filamentous growth in response to temperature. Overall, our work indicates that GlcNAc functions as a highly conserved cue of morphogenesis in fungi, which further enhances the significance of this ubiquitous sugar in cellular signaling in eukaryotes. For each time-course sample, cDNA was coupled to Cy5 and a reference cDNA pool was made by combining RNA from t = 0 and all late time course samples, which was coupled to Cy3. For end point microarray experiments (i.e., established yeast samples compared to established filamentous samples), G217B yeast cDNA was coupled to Cy5 and filament cDNA was coupled to Cy3.

热二态性人类致病真菌可响应环境触发可逆的细胞分化程序，该过程对其侵染性与致病性不可或缺。在土壤中，此类真菌以高度极化的多核菌丝体形式生长，并产生感染性粒子；当被哺乳动物宿主吸入后，这些细胞会转换为单细胞酵母型，即便在健康宿主中也可引发疾病。温度被认为是介导可逆细胞分化的核心环境信号，但体外低温诱导菌丝生长的过程往往低效且不同步。
在筛选其他调控形态发生的信号分子时，我们关注到单糖N-乙酰葡糖胺（N-acetylglucosamine，GlcNAc）：它是微生物细胞壁的主要组成成分，且在环境中广泛分布。实验证实，GlcNAc是两种热二态性真菌——荚膜组织胞浆菌（Histoplasma capsulatum）与皮炎芽生菌（Blastomyces dermatitidis）——酵母向菌丝形态转换的强效且特异性诱导剂。微摩尔浓度的GlcNAc可在温度转换后显著诱导荚膜组织胞浆菌的形态转换，提示真菌细胞可通过感知GlcNAc信号促进菌丝形成。
低温与GlcNAc协同诱导的同步形态转换体系，使我们能够解析形态发生过程中转录组的时序调控规律，进而挖掘参与构建菌丝生长程序的候选基因。通过该分析，我们鉴定出两个编码GlcNAc转运蛋白的基因NGT1与NGT2，二者对于荚膜组织胞浆菌响应GlcNAc高效形成菌丝是必需的。出乎意料的是，在标准葡萄糖培养基中，NGT1与NGT2同样对荚膜组织胞浆菌高效的酵母-菌丝转化过程至关重要，这表明Ngt1与Ngt2可通过感知内源性GlcNAc水平，响应温度信号调控多核菌丝生长。
综上，本研究证实GlcNAc是真菌中高度保守的形态发生信号，这进一步提升了这种广泛存在的糖类在真核细胞信号传导中的生物学意义。
对于每个时间进程样本，互补DNA（complementary DNA，cDNA）与Cy5荧光基团偶联；将t=0时刻与所有晚期时间进程样本的RNA混合构建参考cDNA库，该参考库与Cy3荧光基团偶联。在终点微阵列实验（即比较成熟酵母样本与成熟菌丝样本）中，G217B株酵母cDNA与Cy5偶联，菌丝cDNA与Cy3偶联。