Bakir Gorkem, Dahms Tanya E S, Martin-Yken Helene, Bechtel Hans A, Gough Kathleen M
Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada.
Department of Chemistry and Biochemistry, University of Regina, Regina, Saskatchewan, Canada.
Appl Spectrosc. 2024 Apr;78(4):355-364. doi: 10.1177/00037028231213658. Epub 2024 Feb 20.
The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, β-1,3- and β-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. , a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: Δ and Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of β-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 310 μm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ∼25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the β-1,6-glucan content is decreased in Δ, while all glucan content is decreased in the Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior.
细胞壁完整性（CWI）信号通路调节酵母细胞壁生物合成、细胞分裂以及对外界应激的反应。细胞壁由几丁质、β-1,3-和β-1,6-葡聚糖以及甘露糖蛋白的致密网络组成，非常薄，<100 nm。细胞壁组成的改变可能会激活CWI通路。酿酒酵母作为一种模式酵母，被用于研究单个细胞壁成分在改变酵母细胞壁结构和生物物理特性中的作用。近场傅里叶变换红外光谱（nano-FT-IR）首次用于在背景（野生型）菌株以及来自酵母基因敲除文库的两个缺失突变体：Δ和Δ中对细胞壁成分进行直接的光谱化学鉴定。杀伤毒素抗性6（Kre6）是β-1,6-葡聚糖生物合成所需的整合膜蛋白，而Knr4是一种参与控制细胞壁生物合成，特别是几丁质生物合成和沉积的细胞信号蛋白。通过远场（FF）-FT-IR透射光谱以及具有310μm波长依赖性空间分辨率的衰减全反射（ATR）光谱显微镜获得了互补光谱数据。细胞的FF-FT-IR光谱和分离的细胞壁成分光谱表明，细胞体成分在透射光谱中占主导，并且在ATR光谱中仍然明显。相比之下，空间分辨率约为25 nm的nano-FT-IR可用于表征酵母细胞壁的化学结构。我们的结果表明，Δ中β-1,6-葡聚糖含量降低，而Δ细胞壁中所有葡聚糖含量均降低。后者可能比野生型更薄，因为通过nano-FT-IR不仅可以检测到甘露聚糖和几丁质，还可以检测到脂质膜和蛋白质，这表明是细胞内部成分。
