Molecular Engineering Lab, Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Singapore, 61 Biopolis Drive, Proteos, 138673, Singapore.
Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543, Singapore.
Biochimie. 2024 Jan;216:181-193. doi: 10.1016/j.biochi.2023.09.023. Epub 2023 Sep 24.
Malassezia globosa is abundant and prevalent on sebaceous areas of the human skin. Genome annotation reveals that M. globosa possesses a repertoire of secreted hydrolytic enzymes relevant for lipid and protein metabolism. However, the functional significance of these enzymes is uncertain and presence of these genes in the genome does not always translate to expression at the cutaneous surface. In this study we utilized targeted RNA sequencing from samples isolated directly from the skin to quantify gene expression of M. globosa secreted proteases, lipases, phospholipases and sphingomyelinases. Our findings indicate that the expression of these enzymes is dynamically regulated by the environment in which the fungus resides, as different growth phases of the planktonic culture of M. globosa show distinct expression levels. Furthermore, we observed significant differences in the expression of these enzymes in culture compared to healthy sebaceous skin sites. By examining the in situ gene expression of M. globosa's secreted hydrolases, we identified a predicted aspartyl protease, MGL_3331, which is highly expressed on both healthy and disease-affected dermatological sites. However, molecular modeling and biochemical studies revealed that this protein has a non-canonical active site motif and lacks measurable proteolytic activity. This pseudoprotease MGL_3331 elicits a heightened IgE-reactivity in blood plasma isolated from patients with atopic dermatitis compared to healthy individuals and invokes a pro-inflammatory response in peripheral blood mononuclear cells. Overall, our study highlights the importance of studying fungal proteins expressed in physiologically relevant environments and underscores the notion that secreted inactive enzymes may have important functions in influencing host immunity.
马拉色菌属在人体皮肤的皮脂区域丰富且普遍存在。基因组注释表明，M. globosa 拥有一系列与脂质和蛋白质代谢相关的分泌水解酶。然而，这些酶的功能意义尚不确定，并且这些基因在基因组中的存在并不总是意味着它们在皮肤表面表达。在这项研究中，我们利用直接从皮肤分离的样本进行靶向 RNA 测序，定量测定 M. globosa 分泌的蛋白酶、脂肪酶、磷脂酶和神经鞘磷脂酶的基因表达。我们的研究结果表明，这些酶的表达受真菌所处环境的动态调节，因为 M. globosa 浮游培养的不同生长阶段显示出不同的表达水平。此外，我们观察到这些酶在培养物中的表达与健康皮脂腺皮肤部位有显著差异。通过检查 M. globosa 分泌水解酶的原位基因表达，我们鉴定出一种预测的天冬氨酸蛋白酶 MGL_3331，它在健康和受疾病影响的皮肤科部位均高度表达。然而，分子建模和生化研究表明，该蛋白具有非典型的活性位点基序，并且缺乏可测量的蛋白水解活性。与健康个体相比，这种假蛋白酶 MGL_3331 可引起特应性皮炎患者血浆中的 IgE 反应性增强，并在周围血单核细胞中引发促炎反应。总的来说，我们的研究强调了在生理相关环境中研究真菌蛋白的重要性，并强调了分泌无活性酶可能在影响宿主免疫方面具有重要功能的观点。
