背景与目的
梅毒螺旋体（Treponema pallidum, Tp）作为高度依赖宿主的病原体，对其侵袭机制的研究长期受限于缺乏理想的体外培养模型，而传统的家兔感染梅毒螺旋体的动物模型因物种差异难以准确反映人类感染特征。类器官技术作为近些年新兴的前沿技术高速发展，通过利用人类多能干细胞（human pluripotent stem cell，hPSC）自组织潜能进行诱导分化形成的三维组织可以高度重现人体多种器官的关键结构和功能，已经成为了研究感染性疾病的重要工具。因此，本研究基于皮肤类器官（skin organoids, SO）技术构建梅毒螺旋体体外三维感染模型，以模拟梅毒螺旋体早期感染人类皮肤的动态过程，并利用液相色谱-串联质谱（1iquid chromatography tandem mass spectrometry, LC-MS/MS）与数据非依赖采集（data independent acquisition, DIA）技术对宿主蛋白表达谱的时间动态变化进行系统解析，以探索梅毒螺旋体在早期感染过程中的侵袭机制。

方法
本研究基于人类诱导多能干细胞（human-induced pluripotent stem cell, hiPSCs）技术构建皮肤类器官，并将分化成熟的皮肤类器官与纯化的梅毒螺旋体菌株进行共培养，通过采用定量PCR（polymerase chain reaction, PCR）、免疫组化、免疫荧光及透射电镜技术对梅毒螺旋体在皮肤类器官中的侵袭和增殖情况进行观察和多重验证。然后，我们分别在Tp感染皮肤类器官后的第4、8、16、24天进行取样，将感染后不同时间点的皮肤类器官设置为实验组，未感染的皮肤类器官设为对照组，利用LC-MS/MS联合DIA技术获取不同组别皮肤类器官的蛋白质组学特征图谱，并筛选出实验组和对照组间的差异表达蛋白。然后通过GO/KEGG功能富集分析、PPI蛋白互作网络分析及STEM时间序列蛋白表达谱分析等生物信息学技术对梅毒螺旋体诱导的宿主蛋白表达变化及关键调控网络进行系统解析。

结果
本研究成功构建了梅毒螺旋体感染皮肤类器官的三维体外培养模型，分化成熟的类器官具备完整皮肤层次及毛囊、皮脂腺等附属器结构，其分子特征与人类皮肤高度一致。将梅毒螺旋体接种至成熟的皮肤类器官后，定量PCR证实其在皮肤类器官中呈现显著增殖，免疫组化及免疫荧光显示了其主要定植于真表皮交界区及毛囊外根鞘的分布模式，透射电镜则进一步证实了梅毒螺旋体周质间隙、薄层细胞壁等超微结构特征。基于DIA蛋白组学技术对梅毒螺旋体感染组与未感染组的皮肤类器官蛋白表达进行分析，共筛选出了158种差异表达蛋白，其中上调蛋白36种，下调蛋白122种。对差异蛋白及不同时间点宿主蛋白表达谱进行功能富集分析、PPI蛋白互作网络分析及STEM时间序列聚类分析，结果显示梅毒螺旋体在早期感染中主要通过以下三个途径促进自身增殖及侵袭：1）诱导角质细胞分化异常及细胞外基质重塑破坏皮肤屏障功能；2）激活氧化应激及代谢重编程形成利于自身增殖微环境；3）干扰剪接体功能及调控核糖体翻译促进自身侵袭及免疫逃逸。

结论
1. 本研究成功构建了首个可高度模拟梅毒螺旋体感染人类皮肤动态过程的体外三维培养模型，为研究其与宿主的互作网络提供了重要平台。
2. 利用LC-MS/MS联合DIA技术绘制了梅毒螺旋体感染皮肤类器官后的蛋白质组学特征图谱，揭示了其在早期感染中诱导的宿主蛋白表达时间动态变化及其调控网络。
3. 通过蛋白组学分析系统解析了梅毒螺旋体在早期感染中通过协同调控细胞外基质重塑、氧化应激、代谢重编程及宿主基因表达系统实现自身增殖、侵袭与免疫逃逸的核心机制。

Background

As an obligate pathogen highly dependent on its host, research on the mechanisms of Treponema pallidum (Tp) has long been hindered by the lack of an ideal in vitro culture model. The traditional rabbit infection model, due to species differences, fails to accurately reflect the characteristics of human infection. Organoid technology, as an emerging frontier in biomedical research, has undergone remarkable advancements in recent years. By harnessing the self-organizing potential of human pluripotent stem cells (hPSCs) through directed differentiation, this approach enables the generation of three-dimensional (3D) tissue constructs that recapitulate the key structural and functional characteristics of diverse human organs. These biomimetic systems have emerged as transformative tools for modeling host-pathogen interactions and investigating the pathogenesis of infectious diseases.In this study, we constructed a three-dimensional in vitro infection model of Tp using skin organoid technology to simulate the dynamic process of early Tp infection in human skin. By employing liquid chromatography-tandem mass spectrometry (LC-MS/MS) and data-independent acquisition (DIA) techniques, we systematically analyzed the temporal dynamics of host protein expression to explore the invasion mechanisms of Tp during early infection.

Methods

This study used human-induced pluripotent stem cell (hiPSC) to construct skin organoids, which were then co-cultured with purified Tp strains. Quantitative PCR (qPCR), immunohistochemistry (IHC), immunofluorescence (IF), and transmission electron microscopy (TEM) were employed to observe and validate the invasion and proliferation of Tp in the skin organoids. Samples were collected at 4, 8, 16, and 24 days after Tp infection of the skin organoids. Infected skin organoids were designated as the experimental groups, while uninfected skin organoids served as the control group. LC-MS/MS combined with DIA technology was employed to acquire proteomic profiles of the different groups, and differentially expressed proteins (DEPs) between the experimental and control groups were identified. Subsequently, comprehensive bioinformatic profiling, including GO/KEGG functional enrichment, protein-protein interaction (PPI) network analysis, and STEM time-series protein expression analysis, were conducted to systematically investigate Tp-induced changes in host protein expression and key regulatory networks.

Results

In this study, we successfully established a three-dimensional ex vivo co-culture model of Tp-infected skin organoids. The fully differentiated organoids exhibited a complete skin layer structure with appendages such as hair follicles and sebaceous glands, and their molecular characteristics were highly consistent with human skin. After inoculating mature skin organoids with Tp, qPCR confirmed its significant proliferation within the organoids. IHC and IF showed that Tp primarily localized at the dermo-epidermal junction and the outer root sheath of hair follicles, while TEM further revealed ultrastructural features of Tp, including its periplasmic space and thin-layered cell wall. Proteomic analysis of infected and uninfected organoids using DIA identified 158 DEPs, of which 36 were upregulated and 122 were downregulated. Functional enrichment, PPI network and STEM analyses of the DEPs and time-series expression profiles revealed that Tp primarily facilitates its proliferation and invasion during early infection through three key mechanisms:: 1) Inducing keratinocyte differentiation abnormalities and extracellular matrix (ECM) remodeling to impair skin barrier function; 2) Activating oxidative stress and metabolic reprogramming to create a microenvironment conducive to its proliferation; 3) Disrupting spliceosome function and regulating ribosomal translation to facilitate immune evasion.

Conclusion

1. This study successfully established the first three dimensional in vitro model capable of simulating the dynamic process of Tp infection in human skin, providing a crucial platform for investigating its interactions with the host.

2. By leveraging LC-MS/MS and DIA technologies, we generated a proteomic profile of Tp-infected skin organoids, uncovering the temporal dynamics of host protein expression and its regulatory networks during early infection.

3. Proteomic analyses systematically elucidated the core mechanisms by which Tp coordinates ECM remodeling, oxidative stress, metabolic reprogramming, and host gene expression systems to achieve proliferation, invasion, and immune evasion during early infection.