Organoid-based in vitro systems to model Cryptosporidium parvum infection in 2D and 3D.
Organoid-based in vitro systems to model Cryptosporidium parvum infection in 2D and 3D.
复制标题
基于类器官的体外系统，用于模拟 2D 和 3D 隐孢子虫感染。
DOI：
10.1101/2023.09.29.560165
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Huston,ChristopherD
中科院分区：
文献类型：
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作者：
Korwin-Mihavics,BethanyR;Dews,EmmettA;diGenova,BrunoMartorelli;Huston,ChristopherD
隐孢子虫的无性生殖周期和性分化研究取得了很大进展。然而，受精的过程，这是必要的传染性卵囊的传播，并没有得到很好的理解。典型的基于癌细胞的培养仅允许对隐孢子虫的无性周期和性分化进行强有力的探索。为了促进对C. parvumwe开发了一种基于类器官的培养系统，支持隐孢子虫的整个生命周期和一种新的受精报告基因。类器官来源的单层（ODMs）支持受精和卵囊生产，并维持感染长达3周。ODM卵囊在体内具有感染性。通过两株C. parvumand与一个新的受精开关报告。受精开关报告基因利用DiCre系统，其中cre片段在有性阶段启动子的控制下表达，导致受精后荧光蛋白表达从mCherry到mNeonGreen的雷帕霉素诱导型开关，其在空间和时间上受到控制。这导致mCherry阳性寄生虫在第一代和后代中表达mNeonGreen.In受精开关报告基因的体内验证证明了受精开关报告基因的精度和效率，并证实了只有在雷帕霉素处理后mCherry基因序列的切除。第二代寄生虫的开始也在ODM中显示，而在HCT 8中很少。在ODM中使用这种报告基因可以帮助研究隐孢子虫在生理学相关的体外系统中的性分化后的生命周期。重要的是类器官衍生的单层细胞提供了一个机会来阐明隐孢子虫生物学以前无法获得的方面。该系统克服了以往基于类器官的隐孢子虫培养方法的缺点。它比以前描述的系统更快，更简单，使用确定的培养基，以提高再现性和一致性，使实时观察，支持寄生虫受精和卵囊生产，并提供了一个生理相关的组织培养系统，以促进隐孢子虫细胞生物学的研究。ODM系统可以促进宿主-病原体相互作用、隐孢子虫-宿主特异性或肠上皮对隐孢子虫感染刺激的先天性或细胞免疫应答的研究。受精开关报告基因可用于检测可能干扰隐孢子虫有性生殖的因素或药物。基于类器官的细胞培养物与受精开关报告基因相结合可以增加我们对隐孢子虫有性生殖的理解，从而为开发有性生殖抑制剂或疫苗提供重要信息，从而缩短疾病持续时间并防止传播。
Many advances have been made recently in our understanding ofCryptosporidium’s asexual cycle and sexual differentiation. However, the process of fertilization, which is required for transmission of infectious oocysts, is not well understood. Typical cancer cell-based culture only allows robust exploration of asexual cycle and sexual differentiation ofCryptosporidium. To facilitate exploration of sexual reproduction inC. parvumwe developed an organoid-based culture system that supportsCryptosporidium’sfull life cycle and a novel fertilization reporter. Organoid derived monolayers (ODMs) supported fertilization and oocyst production and maintained the infection for up to 3 weeks. ODM derived oocysts were infectiousin vivo. Fertilization was confirmed by successfully mating two strains ofC. parvumand with a novel fertilization switch reporter. The fertilization switch reporter utilizes a DiCre system in which cre fragments are expressed under the control of sexual stage promoters resulting in a rapamycin-inducible switch in fluorescent protein expression from mCherry to mNeonGreen after fertilization that is spatially and temporally controlled. This results in mCherry positive parasites in the first generation and offspring that express mNeonGreen.In vivovalidation of the fertilization switch reporter demonstrated the precision and efficiency of the fertilization switch reporter and confirmed excision of the mCherry gene sequence only after rapamycin treatment. The start of a second generation of parasites was also shown in the ODMs and rarely in HCT8s. Use of this reporter in ODMs can help investigate theCryptosporidiumlifecycle post sexual differentiation in a physiologically relevantin vitrosystem.ImportanceOrganoid derived monolayers provide an opportunity to elucidate previously inaccessible aspects ofCryptosporidium’s biology. This system overcomes the disadvantages of previous organoid-based methods forCryptosporidiumculture. It is faster and simpler than previously described systems, uses defined media to increase reproducibility and consistency, enables real-time observation, supports parasite fertilization and oocyst production, and provides a physiologically relevant tissue culture system to facilitate studies ofCryptosporidiumcell biology. The ODM system could facilitate the study of host-pathogen interactions,Cryptosporidium-host specificity, or innate or cellular immune responses toCryptosporidiuminfection stimulated in the intestinal epithelium. The fertilization switch reporter could be used to test factors or drugs that may have potential to interfere withCryptosporidium’ssexual reproduction. Organoid-based cell cultures in combination with the fertilization switch reporter could increase our understanding of sexual reproduction inCryptosporidium,leading to vital information for the development of sexual reproduction inhibitors or vaccines that could shorten disease duration and prevent transmission.