Walwyn Odaelys, Skariah Sini, Lynch Brian, Kim Nathaniel, Ueda Yukari, Vohora Neal, Choe Josh, Mordue Dana G
Department of Microbiology and Immunology, New York Medical College.
J Vis Exp. 2015 Mar 12(97):52556. doi: 10.3791/52556.
Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in naïve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.
弓形虫是弓形虫病的病原体，是一种专性细胞内原生动物病原体。该寄生虫几乎可在任何温血脊椎动物细胞类型内侵入并复制。在寄生虫侵入宿主细胞期间，寄生虫会形成一个源自宿主细胞膜的寄生泡（PV），该寄生泡独立于吞噬作用产生，寄生虫在其中进行复制。虽然依赖干扰素的固有免疫和细胞介导免疫对于最终控制感染很重要，但包括中性粒细胞、单核细胞和树突状细胞在内的固有免疫细胞在感染早期也可作为寄生虫全身播散的载体。本文描述了一种方法，该方法利用宿主固有免疫反应，在此为巨噬细胞，进行正向遗传学筛选，以鉴定在被激活的巨噬细胞中适应性存在缺陷但在未激活的巨噬细胞中侵入和复制正常的寄生虫突变体。因此，该筛选分离出在抵抗巨噬细胞激活作用能力方面存在特定缺陷的寄生虫突变体。本文描述了感染巨噬细胞被激活后突变寄生虫的两种广泛表型：寄生虫停滞与寄生虫降解，通常发生在无定形空泡中。然后对寄生虫突变体进行分析，以鉴定对抵抗细胞自主免疫诱导介质特别重要的负责寄生虫基因。本文提出了一种正向遗传学筛选的通用方法，理论上该方法可进行修改，以靶向对抵抗特定抗菌介质重要的寄生虫基因。本文还描述了一种评估寄生虫突变体易感性的特定巨噬细胞抗菌介质的方法。感染巨噬细胞的激活还可促进寄生虫从速殖子向缓殖子阶段的分化，从而维持慢性感染。因此，本文提出了评估已鉴定寄生虫基因对建立慢性感染重要性的方法。
