Department of Microbiology, Faculty of Medicine, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong 999077, China.
Department of Surgery, Faculty of Medicine, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong 999077, China.
Int J Mol Sci. 2021 Dec 28;23(1):299. doi: 10.3390/ijms23010299.
The invasion of skin tissue by is mediated by mechanisms that involve sequential breaching of the different stratified layers of the epidermis. Induction of cell death in keratinocytes is a measure of virulence and plays a crucial role in the infection progression. We established a 3D-organotypic keratinocyte-fibroblast co-culture model to evaluate whether a 3D-skin model is more effective in elucidating the differences in the induction of cell death by Methicillin-resistant (MRSA) than in comparison to 2D-HaCaT monolayers. We investigated the difference in adhesion, internalization, and the apoptotic index in HaCaT monolayers and our 3D-skin model using six strains of MRSA representing different clonal types, namely, ST8, ST30, ST59, ST22, ST45 and ST239. All the six strains exhibited internalization in HaCaT cells. Due to cell detachment, the invasion study was limited up to two and a half hours. TUNEL assay showed no significant difference in the cell death induced by the six MRSA strains in the HaCaT cells. Our 3D-skin model provided a better insight into the interactions between the MRSA strains and the human skin during the infection establishment as we could study the infection of MRSA in our skin model up to 48 h. Immunohistochemical staining together with TUNEL assay in the 3D-skin model showed co-localization of the bacteria with the apoptotic cells demonstrating the induction of apoptosis by the bacteria and revealed the variation in bacterial transmigration among the MRSA strains. The strain representing ST59 showed maximum internalization in HaCaT cells and the maximum cell death as measured by Apoptotic index in the 3D-skin model. Our results show that 3D-skin model might be more likely to imitate the physiological response of skin to MRSA infection than 2D-HaCaT monolayer keratinocyte cultures and will enhance our understanding of the difference in pathogenesis among different MRSA strains.
细菌入侵皮肤组织是通过一系列机制介导的，这些机制涉及到表皮不同分层结构的连续破坏。角质形成细胞的细胞死亡诱导是毒力的一个衡量标准，在感染进展中起着至关重要的作用。我们建立了 3D 器官型角质形成细胞-成纤维细胞共培养模型，以评估 3D 皮肤模型是否比 2D-HaCaT 单层更有效地阐明耐甲氧西林金黄色葡萄球菌 (MRSA)诱导细胞死亡的差异。我们使用代表不同克隆类型的 6 株 MRSA 菌株研究了在 HaCaT 单层和我们的 3D 皮肤模型中的粘附、内化和凋亡指数的差异，这 6 株 MRSA 菌株分别为 ST8、ST30、ST59、ST22、ST45 和 ST239。所有 6 株菌株都表现出在 HaCaT 细胞中的内化。由于细胞脱落，入侵研究仅限于两个半小时。TUNEL 检测显示，这 6 株 MRSA 菌株在 HaCaT 细胞中诱导的细胞死亡没有显著差异。我们的 3D 皮肤模型为更好地了解感染建立过程中 MRSA 菌株与人类皮肤之间的相互作用提供了更好的认识，因为我们可以在我们的皮肤模型中研究 MRSA 的感染长达 48 小时。3D 皮肤模型中的免疫组织化学染色和 TUNEL 检测显示，细菌与凋亡细胞共定位，证明了细菌诱导细胞凋亡，并揭示了不同 MRSA 菌株之间细菌迁移的差异。代表 ST59 的菌株在 HaCaT 细胞中表现出最大的内化，在 3D 皮肤模型中通过凋亡指数测量的最大细胞死亡。我们的结果表明，3D 皮肤模型可能比 2D-HaCaT 单层角质形成细胞培养更能模拟皮肤对 MRSA 感染的生理反应，并将增强我们对不同 MRSA 菌株发病机制差异的理解。
