Department of Oral Biology, University at Buffalo, Buffalo, New York, USA.
PLoS One. 2012;7(11):e46020. doi: 10.1371/journal.pone.0046020. Epub 2012 Nov 6.
Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence.
对外界刺激的感知和产生适当的反应对于病原体宿主定植至关重要。在致病性真菌中，丝裂原激活蛋白激酶 (MAPK) 途径调节二态性、生物膜/基质形成和毒力。信号黏蛋白以富含糖基的细胞外结构域、跨膜结构域和小细胞质结构域为特征，已知其可以调节各种信号通路。在白色念珠菌中，黏蛋白 Msb2 调节 Cek1 MAPK 途径。我们在此表明 Msb2 定位于酵母细胞壁上，并且在菌丝表面进一步富集。msb2Δ/Δ 菌株形成正常的菌丝体，但生物膜缺陷。msb2Δ/Δ 突变体中不存在 Cek1（但不是 Mkc1）磷酸化。在暴露于高温和碳源限制的细胞中，细胞外结构域的 Msb2 脱落，同时伴随着出芽和 Cek1 磷酸化。在细胞壁和渗透胁迫存在的情况下，在浮游细胞或固体表面上生长时，Msb2 的脱落会以不同的方式发生。我们进一步表明，Msb2 脱落和 Cek1 磷酸化被添加 Pepstatin A (PA) 抑制，PA 是一种天冬氨酸蛋白酶 (Saps) 的选择性抑制剂。分析 Sap 蛋白酶突变体的组合确定了一个 Sap8Δ/Δ 突变体，该突变体具有降低的 MAPK 信号传导以及生物膜形成缺陷，从而表明 Sap8 可能是 Msb2 加工的主要调节剂。我们进一步表明，缺失 Msb2（msb2Δ/Δ）或 Sap8（sap8Δ/Δ）都会导致白色念珠菌表面β-葡聚糖暴露增加，并且 msb2Δ/Δ 在口腔念珠菌病的小鼠模型中显示出减弱的毒力。因此，Sap 介导的 Msb2 蛋白水解切割是响应包括诱导出芽的环境线索激活 Cek1 MAPK 途径所必需的。在 Saps 水平上抑制 Msb2 加工可能提供一种减弱 MAPK 信号传导和降低白色念珠菌毒力的方法。
