Veterinary Clinical Microbiology, Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.
Central Facility for Microscopy, Helmholtz Centre for Infection Research, Braunschweig, Germany.
Front Cell Infect Microbiol. 2017 Nov 6;7:465. doi: 10.3389/fcimb.2017.00465. eCollection 2017.
subsp. () is an opportunistic pathogen of several species including humans. is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the pathogenesis and investigated if was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two strains (1-4a and S31A1) both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM) was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular , and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing at some stages fused with lysosomes to form a phagolysosome. The results indicate that an intracellular phase may be one way survives in the host, and could in part explain how can cause recurrent/persistent infections. Future studies should reveal the ability of to internalize and survive in primary equine endometrial cells and during conditions.
亚种（）是几种包括人类在内的物种的机会性病原体。在健康马的黏膜上发现，但可引起急性和慢性子宫内膜炎。最近发现能够在子宫内膜中长期存在。因此，我们假设细胞内阶段可能是发病机制的一部分，并研究是否能够侵入和在上皮细胞内生存。HEp-2 和 HeLa 细胞系与两种（1-4a 和 S31A1）来自患有子宫内膜炎的母马子宫的菌株共培养。在 23 小时感染试验的不同时间点固定细胞，并使用场发射扫描电子显微镜（FESEM）来表征粘附和入侵机制。FESEM 图像显示两种细菌菌株都有三种形态不同的入侵方式。主要进入点是上皮细胞膜上的大内陷。当细胞与上皮细胞接近时，观察到类似于菌毛的细菌附属物，表明附着和入侵是活跃的过程。通过免疫荧光染色技术和荧光显微镜确定粘附和细胞内，以及与溶酶体相关的。在青霉素保护试验中确定了细胞内细菌的定量。两种研究的菌株都能够侵入上皮细胞，尽管程度不同。免疫荧光数据显示，与 S31A1 菌株相比，菌株 1-4a 的粘附和入侵率明显更高。能够在细胞内生存，但在细胞培养系统中随着时间的推移存活率下降。含有的吞噬体样隔室在某些阶段与溶酶体融合形成吞噬溶酶体。结果表明，细胞内阶段可能是在宿主中存活的一种方式，并在一定程度上解释了如何引起复发性/持续性感染。未来的研究应该揭示在原发性马子宫内膜细胞和条件下内部化和生存的能力。
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