ROP16's role in Toxoplasma gondii strain-specific encystment

ROP16 在弓形虫菌株特异性包囊中的作用

• 批准号: 9977930
• 负责人: Anita Koshy
• 金额: $ 22.35万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-17 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9977930
• 关键词: Acquired Immunodeficiency Syndrome; Address; Affect; Alleles; Amino Acids; Antiviral Therapy; Benign; Binding; Biological Assay; Brain; Brain Diseases; Cell Communication; Cells; Cessation of life; Chronic; Cognition; Complement; Cyst; Data; Defect; Drug Targeting; Engineering; Exposure to; Fibroblasts; Focal Neurologic Deficits; Foundations; Genes; Genetic; Genetic Transcription; Goals; Grant; HIV; Human; Immune response; In Vitro; Infection; Knock-out; Lesion; Life; Mediating; Mus; Neurons; Nuclear; Nuclear Localization Signal; Outcome; Parasites; Pathway interactions; Patients; Persons; Pharmaceutical Preparations; Phosphotransferases; Play; Population; Property; Proteins; Role; STAT3 gene; Seizures; Signal Transduction; Stat5 protein; Testing; Toxoplasma gondii; Toxoplasmosis; Transcript; Variant; Virulence; Western Blotting; Work; acquired immunodeficiency; antimicrobial; antiretroviral therapy; base; chronic infection; co-infection; cognitive function; differential expression; in vivo; latent infection; mutant; novel; toxoplasmic encephalitis; transcriptome sequencing; uptake

PROJECT SUMMARY Toxoplasma gondii is an intracellular parasite that chronically infects many hosts, including humans. Chronic infection requires T. gondii switching from a fast growing form to a slower encysted form. In humans, this life- long persistence primarily occurs in the brain where T. gondii can reactivate in the setting of acquired immune deficiencies. In AIDS patients, toxoplasmic encephalitis is the most common cause of focal brain lesions. Re- cent studies also suggest that persistent T. gondii infection may adversely affect cognition and global immune responses even in HIV+ patients on effective antiretroviral therapy. Currently, we have no drugs that target the encysted form of T. gondii. The goal of this grant is to develop new options for cyst-targeted anti-microbials by building upon our novel finding that ROP16, a parasite protein that is secreted into host cells early during inva- sion and that varies between T. gondii strains, affects encystment in a strain-specific manner. Among the ca- nonical encysting T. gondii strains– type II and III– the type III allele of ROP16 (ROP16III) phosphorylates STAT3 and 6, and possibly STAT5, while the type II allele (ROP16II) does not. In two different in vitro encyst- ment assays, we determined that a type III strain that lack ROP16 (IIIΔrop16) has a 65% decrease in encyst- ment, while a type II strain that lacks ROP16 (IIΔrop16) has a mild increase in encystment. In host cells co- infected with a wild-type type III strain, but not a type II strain, the IIIΔrop16 strain now shows normal encyst- ment. As there is no evidence that parasites re-uptake secreted effector proteins, this “trans” complementation suggests that ROP16-dependent encystment depends upon host cell manipulations. We hypothesize, there- fore, that efficient type III encystment depends upon ROP16III manipulations of host cell signaling. To test this hypothesis, we will determine which properties of ROP16III are essential for ROP16-dependent encystment by complementing the IIIΔrop16 strain with rop16III variants that lack nuclear localization, kinase activity, or STAT binding (Aim 1). To identify the host cell genes and pathways pertinent to ROP16-dependent encystment, we will use RNA-seq to perform transcriptional analysis of fibroblasts infected with the parental type III, IIIΔrop16, or the complemented strain, and exposed to encystment conditions (Aim 2). Top differentially expressed tran- scripts/genes will be validated by Q-PCR and, when possible, immunofluorescent assays or western blots. With the completion of these aims, we will have determined which functions of ROP16III are essential for type III encystment and identified the host transcripts and pathways specifically affected by ROP16III in the context of encystment. These outcomes will establish a foundation on which to build long-term, mechanistic studies to define strain-specific mechanisms for T. gondii encystment. The work proposed here represents an important first step toward developing strain-specific treatments for the persistent form of T. gondii.

项目总结 弓形虫是一种细胞内寄生虫，长期感染包括人类在内的许多宿主。慢性 感染要求弓形虫从快速生长的形态转变为较慢的包膜形态。在人类身上，这种生活- 长时间的持续主要发生在大脑中，弓形虫在获得性免疫的背景下可以重新激活 不足之处。在艾滋病患者中，弓形体脑炎是脑局灶性病变的最常见原因。重新- CENT的研究还表明，持续的弓形虫感染可能会对认知和全球免疫造成不利影响。 即使在接受有效的抗逆转录病毒治疗的HIV+患者中也是如此。目前，我们还没有针对 包囊的弓形虫。这笔赠款的目标是通过以下方式开发针对包囊的抗微生物药物的新选择 基于我们的新发现，ROP16是一种在入侵早期分泌到宿主细胞中的寄生虫蛋白。 Sion，在不同的弓形虫菌株之间不同，以菌株特有的方式影响包膜。在这些人中- 非囊化弓形虫株-II型和III型-ROP16(ROP16III)的III型等位基因磷酸化 STAT3和6，可能还有STAT5，而II型等位基因(ROP16II)没有。在两个不同的体外囊膜中- 通过检测，我们确定了一株缺乏ROP16的III型菌株(IIIΔROP16)的囊膜减少了65%。 而缺乏ROP16的II型菌株(IIΔROP16)包膜略有增加。在宿主细胞中 被野生型III型毒株感染，但不是II型毒株，IIIΔrop16毒株现在显示正常的囊膜- 门槛。由于没有证据表明寄生虫重新摄取会分泌效应蛋白，这种“反式”互补 这表明ROP16依赖的包囊化依赖于宿主细胞的操作。我们假设，在那里- 因此，有效的III型包涵体依赖于ROP16III对宿主细胞信号的操纵。为了测试这一点 假设，我们将通过以下方式确定ROP16III的哪些特性对于ROP16依赖的加密是必不可少的 用缺乏核定位、激酶活性或状态的Δ变异体补充III rop16菌株 约束性(目标1)。为了确定与ROP16依赖包囊相关的宿主细胞基因和途径，我们 将使用rna-seq对感染亲本III型Δ的成纤维细胞进行转录分析。 或补充菌株，并暴露在包囊条件下(目标2)。顶端差异表达的反式- 脚本/基因将通过Q-PCR验证，如果可能，还将通过免疫荧光分析或Western blotts进行验证。 随着这些目标的完成，我们将确定ROP16III的哪些功能是类型所必需的 III编码，并确定了在上下文中受ROP16 III特别影响的宿主转录本和途径 包裹性的。这些成果将为建立长期、机械性的研究奠定基础 确定弓形虫包囊的菌株特异性机制。这里提出的工作代表着一个重要的 为持续存在的弓形虫开发针对菌株的特异性治疗的第一步。