School of Veterinary Science, Hopkirk Research Institute, Massey University, Palmerston North, New Zealand.
Flowjoanna Tāpui Ltd, Palmerston North, New Zealand.
Front Cell Infect Microbiol. 2023 May 22;13:1178576. doi: 10.3389/fcimb.2023.1178576. eCollection 2023.
Cryptosporidiosis is a worldwide diarrheal disease caused by the protozoan . The primary symptom is diarrhea, but patients may exhibit different symptoms based on the species of the parasite they are infected with. Furthermore, some genotypes within species are more transmissible and apparently virulent than others. The mechanisms underpinning these differences are not understood, and an effective system for culture would help advance our understanding of these differences. Using COLO-680N cells, we employed flow cytometry and microscopy along with the -specific antibody Sporo-Glo™ to characterize infected cells 48 h following an infection with or . The -infected cells showed higher levels of signal using Sporo-Glo™ than -infected cells, which was likely because Sporo-Glo™ was generated against . We found a subset of cells from infected cultures that expressed a novel, dose-dependent auto-fluorescent signal that was detectable across a range of wavelengths. The population of cells that expressed this signal increased proportionately to the multiplicity of infection. The spectral cytometry results confirmed that the signature of this subset of host cells closely matched that of oocysts present in the infectious ecosystem, pointing to a parasitic origin. Present in both and cultures, we named this Sig M, and due to its distinct profile in cells from both infections, it could be a better marker for assessing infection in COLO-680N cells than Sporo-Glo™. We also noted Sig M's impact on Sporo-Glo™ detection as Sporo-Glo™ uses fluoroscein-isothiocynate, which is detected where Sig M also fluoresces. Lastly, we used NanoString nCounter analysis to investigate the transcriptomic landscape for the two species, assessing the gene expression of 144 host and parasite genes. Despite the host gene expression being at high levels, the levels of putative intracellular gene expression were low, with no significant difference from controls, which could be, in part, explained by the abundance of uninfected cells present as determined by both Sporo-Glo™ and Sig M analyses. This study shows for the first time that a natural auto-fluorescent signal, Sig M, linked to infection can be detected in infected host cells without any fluorescent labeling strategies and that the COLO-680N cell line and spectral cytometry could be useful tools to advance the understanding of infectivity.
隐孢子虫病是一种由原生动物引起的世界性腹泻病。主要症状是腹泻，但患者可能会因感染的寄生虫种类不同而出现不同的症状。此外，一些种内基因型比其他基因型更具传染性和明显的毒力。这些差异的潜在机制尚不清楚，一个有效的培养系统将有助于我们理解这些差异。本研究使用 COLO-680N 细胞，通过流式细胞术和显微镜观察，以及针对的特异性抗体 Sporo-GloTM，分析感染后 48 小时内感染 或 的感染细胞。与感染的细胞相比，感染的细胞使用 Sporo-GloTM 显示出更高水平的信号，这可能是因为 Sporo-GloTM 是针对 生成的。我们从感染培养物中发现了一组细胞，它们表达一种新的、剂量依赖性的自发荧光信号，该信号可在一系列波长下检测到。表达这种信号的细胞群体与感染倍数成比例增加。光谱细胞术结果证实，该宿主细胞亚群的特征与感染性生态系统中存在的卵囊非常吻合，表明其具有寄生起源。在 和 培养物中均存在，我们将其命名为 Sig M，由于其在两种感染细胞中的特征明显不同，因此它可能是评估 COLO-680N 细胞中 感染的比 Sporo-GloTM 更好的标志物。我们还注意到 Sig M 对 Sporo-GloTM 检测的影响，因为 Sporo-GloTM 使用异硫氰酸荧光素，而 Sig M 也在该位置发出荧光。最后，我们使用 NanoString nCounter 分析来研究两种 物种的转录组图谱，评估 144 种宿主和寄生虫基因的表达。尽管宿主基因表达水平较高，但假定的细胞内 基因表达水平较低，与对照无显著差异，这在一定程度上可以解释为通过 Sporo-GloTM 和 Sig M 分析确定的未感染细胞的丰度。本研究首次表明，可以在未感染的宿主细胞中检测到与 感染相关的天然自发荧光信号 Sig M，而无需任何荧光标记策略，并且 COLO-680N 细胞系和光谱细胞术可能是深入了解 感染性的有用工具。
