Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization.
Cellular barcoding of protozoan pathogens reveals the within-host population dynamics of Toxoplasma gondii host colonization.
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DOI：
10.1016/j.crmeth.2022.100274
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发表时间：
2022-08-22
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细胞条形码技术是了解微生物发病机制的有力工具。然而，条形码策略尚未广泛应用于原生动物寄生虫，与病毒和细菌病原体相比，原生动物寄生虫具有独特的基因组结构和毒力策略。在这里，我们提出了一种基于 CRISPR 的原生动物条形码方法，并成功应用于弓形虫和布氏锥虫。使用带条形码的弓形虫文库，我们评估了小鼠从急性感染到慢性感染的群体结构变化。与预期相反，近交 CBA/J 和远交瑞士小鼠腹膜内感染后一个月，大多数条形码都出现在大脑中。尽管寄生虫包囊数量和条形码多样性随着时间的推移而下降，但代表接种物一小部分的条形码可能会在感染后三个月成为大脑中的主要群体。这些数据建立了原生动物的细胞条形码方法，并证明血脑屏障并不是弓形虫定植的主要瓶颈。弓形虫和布氏锥虫可以进行细胞条形码 一种简单的方法从单次转染中生成复杂的条形码群体 弓形虫的复杂条形码群体在体外具有遗传稳定性 细胞条形码揭示了弓形虫的混杂宿主脑定植 弓形虫对宿主的慢性感染与大脑定植和寄生虫生命周期阶段分化有关，但这如何影响弓形虫在宿主体内的种群动态尚不清楚。细胞条形码已被用来提供有关病毒和细菌的这些见解，但这些方法尚未广泛适用于弓形虫等真核寄生虫。这些知识可以揭示选择瓶颈，促进我们对宿主内病原体相互作用的这一方面如何影响发病机制的理解。病原体的细胞条形码使得能够定量研究宿主生物体的定殖。温科特等人。建立了一种基于 CRISPR 的多功能方法来对弓形虫和布氏锥虫进行条形码，并展示了他们的发现：弓形虫在小鼠宿主大脑中的定殖是一个令人惊讶的允许过程。
Cellular barcoding techniques are powerful tools to understand microbial pathogenesis. However, barcoding strategies have not been broadly applied to protozoan parasites, which have unique genomic structures and virulence strategies compared with viral and bacterial pathogens. Here, we present a CRISPR-based method to barcode protozoa, which we successfully apply to Toxoplasma gondii and Trypanosoma brucei. Using libraries of barcoded T. gondii, we evaluate shifts in the population structure from acute to chronic infection of mice. Contrary to expectation, most barcodes were present in the brain one month post-intraperitoneal infection in both inbred CBA/J and outbred Swiss mice. Although parasite cyst number and barcode diversity declined over time, barcodes representing a minor fraction of the inoculum could become a dominant population in the brain by three months post-infection. These data establish a cellular barcoding approach for protozoa and evidence that the blood-brain barrier is not a major bottleneck to colonization by T. gondii. Toxoplasma gondii and Trypanosoma brucei can be cellularly barcoded A simple method generates complex barcoded populations from single transfections Complex barcoded populations of T. gondii are genetically stable in vitro Cellular barcoding reveals promiscuous host-brain colonization by T. gondii Toxoplasma gondii’s chronic infection of the host is associated with brain colonization and parasite life cycle stage differentiation, but how this affects T. gondii’s within-host population dynamics is unclear. Cellular barcoding has been used to provide these insights for viruses and bacteria, but these methods have not been widely adapted to eukaryotic parasites such as T. gondii. Such knowledge can reveal selection bottlenecks, advancing our understanding of how this aspect of the within-host-pathogen interaction influences pathogenesis. Cellular barcoding of pathogens enable colonization of the host organism to be quantitatively investigated. Wincott et al. establish a versatile CRISPR-based method to barcode Toxoplasma gondii and Trypanosoma brucei and present their discovery that colonization of the murine host brain by T. gondii is a surprisingly permissive process.