Poh Si En, Koh Winston L C, Lim Shi Yu Derek, Wang Etienne C E, Yew Yik Weng, Common John E A, Oon Hazel H, Li Hao
Molecular Engineering Lab, Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research, Singapore, Singapore.
National Skin Centre, National Healthcare Group, Singapore, Singapore.
JID Innov. 2022 Apr 15;2(4):100130. doi: 10.1016/j.xjidi.2022.100130. eCollection 2022 Jul.
Atopic dermatitis (AD) is a skin inflammatory disease in which the opportunistic pathogen is prevalent and abundant. harbors several secreted virulence factors that have well-studied functions in infection models, but it is unclear whether these extracellular microbial factors are relevant in the context of AD. To address this question, we designed a culture-independent method to detect and quantify virulence factors expressed at the skin sites. We utilized RNase-H‒dependent multiplex PCR for preamplification of reverse-transcribed RNA extracted from tape strips of patients with AD sampled at skin sites with differing severity and assessed the expression of a panel of virulence factors using qPCR. We observed an increase in viable abundance on sites with increased severity of disease, and many virulence factors were expressed at the AD skin sites. Surprisingly, we did not observe any significant upregulation of the virulence factors at the lesional sites compared with those at the nonlesional control. Overall, we utilized a robust assay to directly detect and quantify viable and its associated virulence factors at the site of AD skin lesions. This method can be extended to study the expression of skin microbial genes at the sites of various dermatological conditions.
特应性皮炎(AD)是一种皮肤炎症性疾病,其中机会性病原体普遍且大量存在。该病原体具有几种在感染模型中功能已得到充分研究的分泌型毒力因子,但尚不清楚这些细胞外微生物因子在AD背景下是否相关。为了解决这个问题,我们设计了一种不依赖培养的方法来检测和定量在皮肤部位表达的毒力因子。我们利用依赖核糖核酸酶H的多重PCR对从不同严重程度的皮肤部位采集的AD患者胶带条中提取的逆转录RNA进行预扩增,并使用定量PCR评估一组毒力因子的表达。我们观察到随着疾病严重程度的增加,存活的病原体数量增加,并且许多毒力因子在AD皮肤部位表达。令人惊讶的是,与非皮损对照部位相比,我们在皮损部位未观察到毒力因子有任何显著上调。总体而言,我们利用一种可靠的检测方法直接检测和定量AD皮肤病变部位存活的病原体及其相关毒力因子。该方法可扩展用于研究各种皮肤病状况部位皮肤微生物基因的表达。