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《厦门免费医学论文发表-调节非洲锥虫的血管外分布》期刊简介

厦门免费医学论文发表-调节非洲锥虫的血管外分布

抽象

在哺乳动物宿主中，组织驻留的布氏锥虫寄生虫的生物学尚未完全了解，尤其是其血管外定植所涉及的机制。锥虫鞭毛是寄生虫发育多个方面必不可少的细胞器。称为 FLAgellar 成员 8 （FLAM8）的鞭毛蛋白充当参与信号传导的环 AMP 反应蛋白 3 （CARP3）池的对接平台。FLAM8 表现出阶段特异性分布，表明寄生虫在哺乳动物和载体阶段具有特定功能。对哺乳动物形式的敲除锥虫和敲除锥虫的分析表明，FLAM8 在体外对于存活、生长、运动和残肢分化不是必需的。实验性感染的功能研究表明，FLAM8剥夺的锥虫可以在血液循环中建立和维持感染，并分化为昆虫传播形式。然而，定量生物发光成像和基因表达分析显示，FLAM8-null 寄生虫在血管外隔室中表现出显着的播散受损，通过添加 FLAM8 的单个抢救拷贝可以恢复。体外跨内皮迁移试验显示缺乏FLAM8的锥虫存在显著缺陷。FLAM8 是第一个被证明调节 T 的鞭毛成分。布鲁氏菌在宿主组织中的分布，可能通过传感功能，有助于维持哺乳动物解剖生态位中血管外寄生虫种群，尤其是在皮肤中。

作者摘要

布氏锥虫寄生虫引起被忽视的热带病，称为人类和动物非洲锥虫病。这些寄生虫通过受感染的采采蝇叮咬传播，沉积在哺乳动物宿主的皮肤中，占据脉管系统和血管外组织。目前，组织驻留寄生虫的生物学尚不清楚，介导血管外定植的寄生虫因子尚不清楚。使用定量体内生物发光成像和宿主感染组织和血液中的离体基因表达定量，我们发现鞭毛寄生虫蛋白 FLAM8 调节哺乳动物宿主锥虫的血管外播散。已知 FLAM8 可作为鞭毛中信号复合物的对接平台，但我们观察到它不会影响寄生虫分化为可传播阶段。然而，我们发现 FLAM8 的缺失导致鞭毛腺苷酸环化酶信号转导复合物的关键成分丢失，并减少寄生虫通过内皮细胞单层的迁移，这表明 FLAM8 对于血管内和血管外隔室之间的寄生虫交换很重要。这项工作确定了参与宿主-寄生虫相互作用的关键锥虫鞭毛成分，包括寄生虫嗜性和血管外播散的调节。

数字

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引文： Alvarez EC、Ngoune JMT、Sharma P、Cooper A、Camara A、Travaillé C 等人（2023） FLAgellum Member 8 调节非洲锥虫的血管外分布。PLoS 病理学 19（12）： E1011220 中。 https://doi.org/10.1371/journal.ppat.1011220

编辑器： Kent L. Hill，加州大学洛杉矶分校，美国

收到： 18年2023月6日;接受： 2023年21月2023日;发表： <>月 <>， <>

数据可用性：所有相关数据都在论文及其支持信息文件中。

资金：这项工作得到了巴斯德研究所的支持。这项工作得到了法国政府 Investissement d'Avenir 计划、Laboratoires d'Excellence ANR-10-LABX-62-IBEID（为 ECA 和 JMTN 提供资金）和 ANR-11-LABX-0024-ParaFrap（为 PS 提供资金）和法国国家科学研究局通过项目 ANR-14-CE14-0019-01 EnTrypa、ANR-18-CE15-0012 TrypaDerm 和 ANR-19-CE15-0004-02 AdipoTryp 授予 BR 的支持。BR 和 AlC 由巴斯德研究所资助，AiC 由几内亚巴斯德研究所资助。AML 和 AnC 由惠康 AML 高级奖学金（209511/Z/17/Z）资助。资助者在研究设计、数据收集和分析、发表决定或手稿准备方面没有任何作用。

利益争夺：作者宣称不存在任何利益冲突。

介绍

布氏锥虫是一种细胞外寄生虫，负责非洲锥虫病，包括人类昏睡病和牛的纳加纳。非洲锥虫是以血液为食的采采蝇（舌蝇属）叮咬传播的血液和组织原生生物。在哺乳动物宿主中，寄生虫面临着不同的微环境，包括多种宿主免疫反应的致命挑战和碳源的可变可用性。这需要由特定基因表达程序激活驱动的重大形态和代谢适应，这对生命周期进展至关重要[1\u3]。最近，血管外趋向性对T的重要性。布鲁氏菌在动物模型中被重新发现：除大脑外，寄生虫还占据大多数哺乳动物组织，尤其是皮肤和脂肪组织[4\u7]。然而，锥虫外渗和隔离在特定解剖生态位和潜在分子过程中的作用尚不清楚。

锥虫鞭毛是一种重要的细胞器，锚定在细胞体表面，存在于其发育的各个阶段[8]。它对寄生虫活力[9]、细胞分裂和形态发生[10]、采采蝇唾液腺附着[11]和运动[12]至关重要。在昆虫宿主中，鞭毛仍处于细胞的最前沿，可能参与宿主-寄生虫相互作用所需的感觉和信号功能[13,14]。在哺乳动物宿主中，鞭毛运动被证明对血流感染的建立和维持至关重要[15]。然而，锥虫鞭毛对哺乳动物宿主血管外寄生虫趋向性和时空传播的贡献仍有待探索。

对从寄生虫昆虫阶段纯化的完整鞭毛进行蛋白质组学分析，鉴定出一组具有独特模式和动力学的鞭毛膜和基质蛋白[16]。其中，一种称为FLAgellar Member 3（FLAM075）的大蛋白（8,8个氨基酸）仅以昆虫前环形式存在于鞭毛的远端[16\u18]。有趣的是，FLAM8以哺乳动物的形式沿着鞭毛的整个长度重新分布，包括在残肢传播阶段[19]，这可能意味着这种蛋白质具有阶段特异性功能。因此，我们假设 FLAM8 可能参与宿主-寄生虫相互作用或发育形态发生。在这里，我们研究了 FLAM8 在体外哺乳动物寄生虫中存活、增殖、运动和分化的作用，以及小鼠感染过程中血管内和血管外寄生虫负荷的体内动力学。有趣的是，通过生物发光成像和基因表达分析监测和量化的小鼠实验感染表明，FLAM8参与血管外隔室中的寄生虫传播。此外，体外迁移研究检测到 FLAM8 剥夺寄生虫的外渗能力受损，这可能是由于鞭毛腺苷酸环化酶信号转导复合物成分的丢失所致。

结果

FLAM8型RNAi沉默不影响寄生虫存活

FLAM8在不同锥虫阶段的鞭毛中的差异分布[19]提出了其在寄生虫生命周期中的特定功能的问题。为了研究 FLAM8 在哺乳动物宿主中的潜在作用，首先对血流形式的寄生虫进行了工程改造，用于在表达 mNeonGreen 标记的 FLAM8 的单态菌株中诱导 RNAi 敲低 FLAM8。为了通过全身成像方法监测它们在小鼠中的行为，这些FLAM8：：mNG FLAM8 核糖核酸（RNAi）突变体随后被转化为过表达嵌合三重报告蛋白[20]。在体外用四环素诱导RNAi72小时后，FLAM8在mRNA水平上降低了60%（图1A），并且在免疫荧光水平上无法检测到（图1B）。在诱导RNAi后6天内监测寄生虫生长，未观察到对增殖的影响（图1C），这表明FLAM8对于哺乳动物形式的寄生虫在细胞培养条件下的存活可能不是必需的。

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图 1. FLAM8：：mNG FLAM8 的表征核糖核酸（RNAi）哺乳动物宿主的体外和体内功能研究菌株。

A）通过比较 ΔΔC 通过 RT-PCR 评估 FLAM8 mRNA 的表达T对照、非诱导和诱导 FLAM8：：mNG FLAM8 的方法核糖核酸（RNAi）寄生虫（72小时）。B）非诱导（上图）和诱导（下图）FLAM8：：mNG FLAM8 的免疫荧光图片核糖核酸（RNAi）BSF 在 72 小时内。用抗mNG抗体（绿色）和DAPI染色DNA含量（蓝色）对甲醇固定锥虫进行染色。比例尺表示 5 μm。C）对照组、非诱导组和诱导组 FLAM8：：mNG FLAM8 的生长曲线核糖核酸（RNAi）BSF寄生虫。所有细胞系均接受 1 μg 四环素治疗 6 天。对照寄生虫是李斯特427“单标记物”BSF寄生虫，不携带用于RNAi沉默的pZJM-FLAM8质粒。结果代表三个独立实验的平均值（±标准差，SD）。D）向 4 只 BALB/c 小鼠组注射 IP 对照、非诱导或诱导 FLAM8核糖核酸（RNAi）BSF锥虫。1只注射PBS的BALB/c动物作为阴性对照。生物发光辐射信号的代表性归一化体内图像（单位为光子/秒/cm2/ 球面）从感染对照、非诱导和诱导 FLAM8：：mNG FLAM8 的 BALB/c 小鼠发出核糖核酸（RNAi）感染后 4 天寄生虫（未感染的技术对照小鼠的生物发光呈阴性，未显示）。通过在感染前 8 小时在含糖饮用水中加入多西环素来维持 FLAM48 的 RNAi 沉默，直到实验结束。E）在感染过程中（5 天）感染的 BALB/c 小鼠血液（血管内，IV）中的寄生虫数量，使用细胞计数器从尾部出血中计数。F）与E中相同的感染小鼠的血管外隔室（血管外，EV）中的寄生虫数量）。G）对照、非诱导和诱导 FLAM8：：mNG FLAM8 的传播核糖核酸（RNAi）寄生虫，在整个动物身体上测量（单位：cm2）在整个感染过程中通过整个生物发光表面。结果表示均值±标准差（SD）。

https://doi.org/10.1371/journal.ppat.1011220.g001

然后，在体内攻击之前，在IVIS光谱成像仪中评估发射的生物发光与寄生虫数量之间的线性相关性（S1图）。为了深入了解 FLAM8 在哺乳动物宿主中的功能，雄性 BALB/c 小鼠组通过腹膜内途径感染 105亲本、非诱导和诱导细胞系的寄生虫（图1D）。体内研究通过在感染前 8 小时在含糖饮用水中加入多西环素来维持小鼠 FLAM48 的 RNAi 沉默，直到实验结束。每天通过以下方式监测感染过程：i）量化寄生虫血症，ii）使用IVIS光谱成像仪获取整个生物体中寄生虫发出的生物发光信号。通过从生物体中的寄生虫总数（总生物发光）中减去血管系统中已知的锥虫数量（寄生虫血症 x 血容量，根据体重）来推断给定时间点血管外隔室中的寄生虫数量。在感染的建立和随后的血管内寄生虫数量变化（图1E，IV）和占据血管外组织的寄生虫数量（图1F，EV）方面均未检测到差异。在感染过程中，在三个独立的感染小鼠组中，每组都观察到血管内和血管外寄生虫的相似种群概况（图1D-1F）。在血管外隔室中，在任何组的整个动物体内的寄生虫传播方面均未检测到显着差异（图1G）。

FLAM8敲除不影响锥虫在体外的生长

考虑到 i） FLAM8 RNAi 沉默效率仅部分（诱导 40 小时后剩余 8% FLAM72 mRNA），ii）多西环素诱导的 FLAM8 抑制在体内的功效可能更低，并且 iii）用于第一种策略的亲本菌株是单态的（即无法分化为采采蝇适应的树桩阶段），我们推断多形性菌株中的基因敲除方法更适合评估 FLAM8 在哺乳动物宿主感染期间的潜在作用。因此，通过同源重组在多形性锥虫中产生 ΔFLAM8 敲除细胞系。通过全基因组测序和PCR（S8图）验证了一个FLAM8等位基因的完全替代和第二个等位基因的部分替代（以允许使用比长FLAM2编码序列更短的原位救援序列）。使用靶向第二个等位基因中未被替换的 FLAM8 区域的抗 FLAM8 抗体 [19] 进行定量免疫荧光分析，进一步评估 FLAM8 蛋白表达是否完整或截短（图 2A）。RT-qPCR（S1表）在mRNA水平上也证实了这一点。将生成的锥虫细胞系进一步转化以表达嵌合三重报告基因构建体[20]，并分析了所有菌株的体外和体内生物发光信号与寄生虫总数之间的线性相关性（S3图）。在 Δ FLAM8 敲除寄生虫中，将一个 FLAM8 等位基因的救援拷贝重新添加到其内源性位点中，通过免疫荧光分析评估，FLAM8 分布沿大部分鞭毛长度恢复（图 2A）。通过量化所有寄生虫菌株中沿整个鞭毛长度的FLAM8信号的总荧光强度证实了这一点（图2B）：在敲除锥虫中检测到背景荧光信号，而救援细胞系呈现中等水平的FLAM8表达。在培养条件下未观察到对寄生虫生长的影响（图2C）。接下来，我们根据轴突标记物 mAb8 获得的信号测量，研究了 FLAM25 的丢失是否会影响鞭毛的总长度：BSF 系之间没有观察到差异（图 2D）。此外，在8.0%甲基纤维素液体培养基（图5E和2F）和2.1%甲基纤维素粘性培养基（图1G和2H）中，FLAM2的缺失不影响寄生虫在体外的速度或线性。这些结果表明，多形性锥虫在体外耐受FLAM8的丢失，这与我们之前在RNAi沉默后的观察结果一致。

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图 2. ΔFLAM8 无效突变体的体外表征。

A）用抗 FLAM8（绿色）和 mAb8（品红色中的轴突）抗体标记的亲本、ΔFLAM25 敲除和拯救多形性 BSF 寄生虫的免疫荧光图片，DNA 含量的 DAPI 染色（蓝色）。比例尺显示 5 μm。B）在甲醇固定的亲本、ΔFLAM8 敲除和拯救寄生虫中沿整个鞭毛长度的总荧光强度的定量。C）一个亲本、三个 ΔFLAM8 亚克隆和 8 个拯救多形性 BSF 锥虫细胞系连续 6 天的生长曲线。D）基于亲本、三个 ΔFLAM25 亚克隆和救援寄生虫中的轴突标记 mAb8 谱测量鞭毛长度。未发现统计学差异。E-H）运动跟踪分析显示了BSF细胞系在含有0.5%（E-F）或1.1%（G-H）甲基纤维素的基质依赖性培养基中的平均速度（E和G）和线性（F和H）。未观察到统计学差异。考虑用于定量（N）的寄生虫数量分别在（B）和（D-H）的图表下方和上方表示。结果代表三个独立实验的平均值±标准差（SD）。统计检验包括单因素方差分析和Tukey的多重比较的临时后检验。

https://doi.org/10.1371/journal.ppat.1011220.g002

FLAM8敲除影响哺乳动物宿主锥虫分布

然后，通过用多形性亲本菌株、三个不同的 Δ FLAM8 敲除亚克隆（由独立重组事件产生）或一个携带 FLAM8 拯救拷贝的 ΔFLAM8 菌株感染 BALB/c 小鼠，在哺乳动物宿主中进行功能研究（图 3 和 S4）。通过量化寄生虫血症和从整个动物发出的生物发光信号，每天监测感染，持续 4 周（图 3A）。无效突变寄生虫能够在血液和血管外隔室中建立感染。在脉管系统中，与感染亲本菌株的小鼠相比，感染了三个 ΔFLAM8 亚克隆的小鼠在整个实验感染过程中的寄生虫总量略高，除了在第 5 天和第 12 天之间，在寄生虫血症的第一个峰值之后观察到较低且可变的寄生虫血症（图 3B 和 3D).另一方面，对血管外寄生虫的定量分析显示了不同的情况。与血管内隔室不同，在第 5 天至第 12 天之间以及从感染后第 19 天到第 27 天实验结束期间观察到的血管外锥虫数量显着减少，证明与亲本对照相比，ΔFLAM8 寄生虫的血管外定植受损（图 3C 和 3E）。需要注意的是，所有多形性菌株的分布曲线都与在感染单形性寄生虫的小鼠中观察到的分布曲线不同：观察到大量锥虫占据血管外隔室，达到5-8x109寄生虫，而在血液中发现的最大数量从未超过 7x107锥虫（分别为图3C和3B）。

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图 3. 哺乳动物宿主体内 ΔFLAM8 无效突变体的功能研究。

向 3 只 BALB/c 小鼠组注射 IP 中 8 个亲本、<> 个 ΔFLAM<> 无效亚克隆或 <> 个挽救菌株。<>只注射PBS的BALB/c动物作为阴性对照。A）生物发光辐射强度的归一化体内图像（以光子/秒/cm为单位）2/ 球面）在感染亲本、三个 ΔFLAM8 亚克隆或拯救寄生虫的 BALB/c 小鼠感染后 8 天发射（未感染的对照小鼠 C- 生物发光呈阴性）。B）在 4 周内使用细胞计数器从尾部出血中每天计数感染小鼠（血管内，IV）血液中的寄生虫总数。统计学上的显著差异（p<0.01）用一个、两个或三个星号（*、**、***）分别表示亲本菌株与一个、两个或三个ΔFLAM8亚克隆之间的差异。C）同一小鼠的血管外（EV）锥虫总数。亲本菌株和 ΔFLAM8 亚克隆之间的统计学显着差异如 B）所示。D-E）同一小鼠中血管内（IV 如 B）和血管外（EV 如 C）寄生虫种群总数的变化与在同一时间点感染亲本锥虫的小鼠获得的值的倍数差异。F）亲本、三个 ΔFLAM8 亚克隆和拯救寄生虫菌株的传播，在整个动物体内测量（以厘米为单位）2）在整个感染过程中通过生物发光信号的总表面。如上所述，亲本菌株和 ΔFLAM8 亚克隆之间的统计学显着差异。结果表示均值±标准差（SD）。统计检验包括双向方差分析和Tukey的临时后检验，用于B-F中的多重比较。详细的个人数据在S4图中提供。

https://doi.org/10.1371/journal.ppat.1011220.g003

此外，对在整个动物体内扩散的寄生虫的定量表明，FLAM8 的耗竭导致 ΔFLAM8 无效突变寄生虫在血管外隔室的传播显着受损（感染后第 5 至 14、19 和 27 天）。这主要在携带FLAM8挽救拷贝的锥虫中恢复（图3F）。

FLAM8 敲除损害血管外锥虫播散

我们推断，在感兴趣的小区域进行生物发光检测的血管内与血管外寄生虫种群估计的准确性可能会受到限制。因此，重复相同的实验，时间分辨率较低，但在血管外寄生虫种群和寄生虫传播方面具有可比的趋势（图4A-4C和S5）。在第二次实验挑战结束时（第 24 天），在器官解剖之前通过盐水灌注将大多数锥虫从血管系统中去除。然后收集单个器官，通过RT-qPCR定量不同寄生虫基因的表达。使用微管蛋白RT-qPCR标准曲线计算每个样品中每mg组织中的寄生虫总数。为了更好地比较不同菌株之间每个隔室中寄生虫种群的变化，将寄生虫的Delta数量计算为给定小鼠每个组织样本中的寄生虫数量与同一小鼠血液样本中的寄生虫数量之间的差异。

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图 4. FLAM8 的缺失可减少血管外锥虫播散。

在第二次实验感染中，将 3 只 BALB/c 动物组注射 IP 与亲本、三个 ΔFLAM8 无效亚克隆或挽救菌株，而 24 只注射 PBS 的 BALB/c 动物用作阴性对照。感染后第8天，对动物实施安乐死，采集肠道、肾脏、肝脏、肺、皮肤、脾脏和睾丸，量化各器官中的寄生虫比例。A）感染期间血管内寄生虫（IV）总数。B）同一小鼠血管外（EV）锥虫的总数。C）亲本、三个 ΔFLAM<> 亚克隆和拯救寄生虫菌株的传播，在整个动物体内测量（以厘米为单位）2）在整个感染过程中通过生物发光信号的总表面。统计学上的显著差异（p<0.01）用一个或三个星号（*， ***）表示，分别表示亲本菌株与一个或三个 ΔFLAM8 亚克隆之间的差异。详细的个人数据在S5图D）中提供，每个解剖器官和菌株的寄生虫数量Delta。使用微管蛋白RT-qPCR标准曲线计算每个样品中每mg组织中的寄生虫总数。为了更好地比较不同菌株之间每个隔室中寄生虫种群的变化，将寄生虫的Delta数量计算为给定小鼠每个组织样本中的寄生虫数量与同一小鼠血液样本中的寄生虫数量之间的差异。E）每个菌株的寄生虫 Delta 数量计算为 D 中所示的单个器官值的平均值。根据单因素方差分析和 Dunnett 比较检验的统计差异（p<0.0001）。详细的个人数据在S1表和S6图中提供。

https://doi.org/10.1371/journal.ppat.1011220.g004

首先，在感染 Δ FLAM8 寄生虫的动物中证实了 FLAM8 转录本的缺失，而在感染了亲本和救援菌株的小鼠中检测到 FLAM8 mRNA（S1 表）。然后使用标准化微管蛋白表达来比较器官和菌株之间的寄生虫密度（图 4D、4E 和 S6）。在所有小鼠中，在皮肤中观察到的血管外寄生虫数量最多。在所有器官中，与亲本和救援寄生虫感染的小鼠相比，感染ΔFLAM8寄生虫的小鼠的血管外寄生虫数量显着减少（图4D和S6）。当考虑器官与血液中寄生虫数量之间的平均差异时，血管外FLAM8剥夺寄生虫密度的统计学显着降低更加明显（图4E）。救援细胞系概括了所有器官中的 EV 寄生虫谱，平均 Delta 寄生虫数量与亲本系的寄生虫数量没有统计学差异。

我们推断，FLAM8剥夺寄生虫的血管外播散减少可能是由于：（1）较低的增殖率，（2）运动缺陷，（3）较高的分化率为非增殖性残端形式，和/或（4）外渗缺陷。前两个假设可以立即被抛弃，因为菌株之间没有观察到差异，无论是在体外（图2C）和体内（图3B和4A）的细胞生长，还是在体外的细胞运动（图2E-2H）。然后，对最后两个假设进行了连续的检验。

FLAM8 的缺失不影响寄生虫分化

血管外 ΔFLAM8 寄生虫群的全身性减少可能是由于寄生虫分化为非增殖性传播形式的不平衡所致。因此，我们通过免疫荧光或RT-qPCR评估了FLAM8的缺失是否会影响血液和器官中增殖细长体分化为可传播的残端形式。在培养的寄生虫中，FLAM8 的缺失不会显着改变分化（图 5A 和 5B），并且新分化的 FLAM8 剥夺的残肢寄生虫能够在体外进一步分化和维持为前环锥虫，亲本和救援菌株也是如此（图 5C 和 5D）。在体内，在实验结束时，通过对血液和解剖器官的RT-qPCR证实了增殖性细长寄生虫与非增殖性残肢寄生虫的自然分化（图5E，第二次体内挑战的第24天）。对于每个样品，微管蛋白表达用于标准化 PAD1 mRNA 水平，以比较器官和菌株之间表达 PAD1 mRNA 的寄生虫在总寄生虫种群中的平均水平（图 5E），较高的 Delta CqPAD1-CqTub 与器官中较少的 PAD1 转录本相关。在至少一个个体的每个器官中检测到PAD1转录本，表明所有测试的器官都代表了寄生虫分化为可传播形式的合适环境（S1表）。在感染亲本和救援菌株的小鼠中，在血液和皮肤中检测到最高水平的PAD1转录本，表明直接参与寄生虫传播的器官的积累或分化率增加（S1表）。在感染亲本和挽救菌株的小鼠的肠道、肝脏和脾脏中未检测到PAD1转录本，而在感染ΔFLAM1菌株的小鼠的这三个器官中检测到PAD8转录本。然而，当按菌株考虑整个生物体中的平均Delta CqPAD1-CqTub时，表达PAD1 mRNA的寄生虫的相对比例在组间没有显着差异，证实了FLAM8不参与寄生虫分化（图5E）。此外，在寄生虫血症的第一个峰值取样的血涂片的抗PAD1免疫荧光染色在视觉上证实了所有感染动物组中存在分化的残端寄生虫（图5F，第一次体内攻击的第5天）。

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图 5. 寄生虫分化在 FLAM8 耗尽的锥虫中不受影响。

A）用核苷酸 8'-AMP 类似物体外处理后，从亲本（左图）、三个 ΔFLAM5 亚克隆（中图）和挽救（右图）寄生虫菌株的增殖细长者体外分化后残肢寄生虫的代表性免疫荧光图像。用抗 PAD-1 抗体（红色）标记甲醇固定寄生虫，并用 DAPI 染色检测 DNA 含量（蓝色）。比例尺显示 5 μm。B）体外分化后所有多形性细胞系中残肢锥虫比例的定量。考虑定量的寄生虫数量（n）显示在图表上方。未发现显著差异（单因素方差分析和Tukey比较检验）。结果代表三个独立实验的平均值±标准差（SD）。C）新鲜体外分化的原环细胞的选定免疫荧光图像。锥虫用抗 FLAM8（绿色）和 mAb25（品红色轴突）双重标记，DAPI 染色显示蓝色的 DNA 含量。比例尺显示 5 μm。D）分化后，将亲本、三个 ΔFLAM8 亚克隆和挽救寄生虫菌株的前环形式（PCF）均匀稀释，并评估其体外生长。仅在比较亲本和 KO 0 PCF 锥虫（单因素方差分析和 Tukey 比较检验）时观察到统计学差异（p<01.2）。结果代表了四个独立实验的平均值±标准差（SD）。E）在第二次体内攻击的第 1 天，通过 RT-qPCR 定量的血液和解剖器官上 PAD-24 表达水平的平均相对比例。微管蛋白表达用于使所有亲本、ΔFLAM1 和抢救感染小鼠样本中的 PAD-8 mRNA 水平标准化。F）在寄生虫血症的第一个高峰期（第一次实验性体内感染）期间，从亲本、一个选定的 ΔFLAM8 亚克隆和从小鼠血液中分离的拯救菌株的自然分化的残肢锥虫的代表性免疫荧光图像。用抗 PAD-1 抗体（红色）标记寄生虫，并用 DAPI 染色检测 DNA 含量（蓝色）。比例尺显示 5 μm。

https://doi.org/10.1371/journal.ppat.1011220.g005

FLAM8剥夺锥虫的寄生虫外渗受损

为了检验最后一个假设，我们询问了FLAM8的缺失是否会影响寄生虫穿过血管壁进入血管外组织的方式。我们首先使用人脐静脉内皮细胞（HUVEC）在具有 3 μM 孔的聚酯转孔插入物上生长汇合，分离两个腔室以模拟体外血管内皮细胞（图 6A）。一旦 HUVEC 达到汇合点，106将亲本、FLAM8 剥夺或挽救锥虫加入上室并孵育 24 小时。通过流式细胞术计算通过内皮单层迁移到下腔室的锥虫数量和残留在transwell系统上室中的非迁移锥虫数量，以确定迁移百分比。有趣的是，与亲本对照组相比，所有FLAM8敲除亚克隆的迁移率均显著降低（KO25、6和70的寄生虫交叉率分别为7.33%、8.1%和2.3%）（图6B）。仅表达一个 FLAM8 等位基因的挽救锥虫表现出中间迁移表型，其中 59.2% 的寄生虫通过内皮单层迁移（图 6B），低于亲本锥虫，但显着高于 KO1 和 KO3 无效突变体（图 6B）。这表明缺乏 FLAM8 的寄生虫严重受损，无法穿过内皮细胞的汇合层，这在救援寄生虫中部分恢复。

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图 6. 缺乏FLAM8的寄生虫在体外通过内皮细胞迁移时受损。

A）跨内皮迁移测定的示意图，显示包含上下隔室、内皮 HUVEC 细胞单层和细长锥虫的跨孔系统（Boyden 室）接种在室顶部 24 小时。B）在此时期之后，对上腔室和下腔内的寄生虫进行计数，并进一步使用值来计算通过内皮单层迁移到下腔室的亲本、三个 ΔFLAM8 亚克隆和挽救寄生虫的比例。使用 R 中的广义线性模型函数，以高斯家族函数和迁移比例为因变量，比较 FLAM8 突变细胞系和亲本参考系（100% 的跨内皮迁移）之间的锥虫迁移。p<0.001 的概率值被认为是显著的（*）。误差线显示 SD。 C）来自亲本、ΔFLAM8 亚克隆和挽救菌株的培养细长锥虫的代表性免疫荧光图像。甲醇固定的寄生虫用抗CARP3抗体（绿色强度标准化）和DAPI染色的DNA含量（蓝色）标记。比例尺显示 5 μm。D）来自甲醇固定亲本、ΔFLAM3 亚克隆和拯救锥虫的归一化图像中 CARP8 荧光信号的定量，表示为沿整个鞭毛的荧光 CARP3 信号强度除以单个鞭毛长度之间的比率。考虑定量的寄生虫数量（n）显示在图表下方。根据单因素方差分析和 Dunnett 比较检验的统计差异（p<0.0001）。结果表示两个独立实验的平均值±标准差（SD）。

https://doi.org/10.1371/journal.ppat.1011220.g006

我们知道，在哺乳动物形式中，FLAM8最近被发现是鞭毛复合物的一部分，包括环AMP反应蛋白3（cyclic AMP response protein 3， CARP21）[8]，我们推断这种跨内皮交叉损伤可能是由传感和/或信号传导缺陷引起的。为了评估 FLAM3 的缺失如何影响 CARP3 的定位和/或丰度，在甲醇固定后通过定量免疫荧光研究了所有菌株中 CARP6 的分布（图 6C 和 3D）。正如预期的那样，在亲本哺乳动物形式中沿整个鞭毛长度检测到 CARP8，但在 ΔFLAM6 寄生虫中未检测到，其中荧光信号较弱且仅限于鞭毛的远端部分（图 6C 和 3D）。这表明存在至少两个不同的 CARP8 池，其中一个独立于 FLAM8，因为它定位于鞭毛的前部。在 Δ FLAM8 寄生虫中存在 FLAM3 救援拷贝，恢复了沿整个鞭毛的 CARP6 检测，但数量低于亲本细胞（图 6C 和 <>D）。

总的来说，这些数据表明 FLAM8 可能参与调节锥虫外渗的细胞通路，从而参与哺乳动物宿主血管外室的锥虫播散。

讨论

FLAM8在哺乳动物感染阶段从采采蝇中肠前环寄生虫的最远端到整个鞭毛长度的差异定位[19]促使我们推测FLAM8可以在每个宿主中发挥独特而特定的作用。在这里，我们首次提出了鞭毛蛋白与锥虫在哺乳动物宿主脉管系统外传播的效率的联系，特别是在皮肤中。通过生物发光成像和基因表达分析监测的实验动物感染的定量分析表明，FLAM8的缺失会损害寄生虫在感染期间在宿主血管外室的外渗和播散，这主要是通过整合FLAM8基因的单个拯救拷贝在内源性位点中恢复的。

1. 个体间和个体内部的差异

个体间异质性在实验性体内感染中很常见。然而，感染了 3 个 ΔFLAM3 亚克隆的 8 组小鼠（共 18 只小鼠）的总体寄生虫种群动态在两个主要实验中遵循非常可比的趋势（图 3 和 4）。两组数据都表明，ΔFLAM8寄生虫可以在血管外隔室定植，但在感染过程中的大部分时间里，其密度仍低于父母和救援人群的密度。在第二次实验结束时，RT-qPCR分析证实了这一点（图4D和4E）。

在个体水平上，宿主每个隔室（不同的血管亚隔室、器官和组织）中的整体寄生虫分布在感染过程中以不同的动态方式进化。此外，在每个隔室内，寄生虫分布不均匀。例如，这反映在皮肤中寄生虫的可变传播上。通过全生物体生物发光成像和特定器官的RT-qPCR进行寄生虫定量相结合，还表明每个器官中血管外寄生虫种群的大小对血管外总种群有不同影响，皮肤是所有测试器官中最重要的解剖生态位。

在相同多形性菌株的克隆之间观察到不同的毒力是很常见的，因此测试不同的寄生虫克隆非常重要。在这里，所有8个ΔFLAM2亚克隆的迁移效率都明显低于其亲本菌株，尽管与其他两个亚克隆相比，8亚克隆的迁移效率更高。此外，由于 FLAM<> 基因的单个等位基因的恢复，加回锥虫在进行的所有实验中仅呈现“中间”挽救表型。

2. FLAM8和锥虫传播

在血液中，增殖的细长寄生虫和采采蝇适应的残肢形式之间的平衡对群体感应机制有反应，该机制涉及寡肽的产生及其通过特定转运蛋白的接收[22]。FLAM8 的缺失并没有改变寄生虫在两个观察到的时间点（寄生虫血症的第一个高峰和感染后 3 周）分化为可传播的残端形式的能力。尽管在整个感染过程中没有评估残肢比例，但这一证据表明 FLAM8 在这一过程中并未发挥不可或缺的作用。此外，FLAM8剥夺的残端细胞在体外分化成前环状形式的能力表明，它们可以在被采采蝇摄入后进一步追求其周期性发育。

血管外锥虫占据多个器官的间质空间，包括中枢神经系统、睾丸、脂肪组织和皮肤[4–7,23–25]。皮肤锥虫在寄生虫传播中的相关性已通过异种诊断实验得到证实，即使在没有可检测到的寄生虫血症的情况下，在感染性叮咬后的早期[26]或感染后期[4]。最近，在确诊和疑似昏睡病病例的皮肤中证实了血管外锥虫的存在[27]。在这里，我们表明所有测试的器官都代表了寄生虫分化成可传播形式的合适环境，但在血液和皮肤中检测到的PAD1转录本量最高，这表明分化率增加，或在直接参与寄生虫传播的器官中积累分化的残肢形式。总的来说，FLAM8-null寄生虫在血管外隔室的扩散受损将在数学上降低寄生蝇被采采蝇摄入的可能性。这对于皮肤来说尤其重要，因为皮肤的寄生虫密度最高，并且由于其大小而具有最高的整体寄生虫负荷，在与昆虫媒介的直接接触处。

3. 关于FLAM8可能的细胞功能

增殖的细长锥虫具有高度的流动性[28]，这种运动性被证明会影响体内的毒力。例如，鞭毛动力蛋白轻链1（LC1）敲除突变体缺乏推进动力，导致锥虫无法在血液中建立感染[15]。在这里，多形性哺乳动物形式的FLAM8耗竭不会改变液体培养基中的寄生虫生长和细胞运动，也不会改变体内寄生虫微环境粘度更接近寄生虫微环境的含基质培养基。然而，定量分析表明，与父母对照组相比，FLAM8-null锥虫在血管外组织中的数量较少。假设寄生虫在组织和间质空间中的运动可能不同，其生物物理特性与血液中的生物物理特性不同[28,29]，则不能排除ΔFLAM8敲除寄生虫的运动性可能在血管外隔室中以某种方式改变。需要在细胞水平上进行活体成像以进行运动分析以证实这一假设。

从历史上看，大多数关于 T.实验性感染中的布鲁氏病毒力几乎将血液循环视为锥虫寄生的唯一宿主隔室，而血管外寄生虫生态位和两个隔室之间的潜在交换长期以来一直被低估。De Niz及其同事最近发现粘附分子是组织趋向性的关键参与者[30]。他们表明，储层的建立发生在血管通透性受损之前，这表明外渗是一种活跃的机制，并且取决于锥虫与内皮表面粘附分子（如E-选择素、P-选择素或ICAM2）的相互作用[30]。在这里，我们发现FLAM8的缺失导致了寄生虫外渗的强烈损害。FLAM8无效锥虫不能在血管外隔室上正确传播这一事实可能在某种程度上可能意味着寄生虫感知其微环境或与宿主内皮细胞相互作用的方式存在缺陷，导致其血管外趋性改变。

The insect forms′ coordinated social motility in vitro is linked to cAMP signaling at the flagellar tip [31, 32], i. e. where FLAM8 localizes [19]. The architecture of an adenylate cyclase complex in the tip nanodomain was recently shown to be essential for social motility and salivary gland colonization [21]. In this complex, CARP3 interacts with the catalytic domain of adenylate cyclases and regulates abundance of multiple adenylate cyclase isoforms. We recently demonstrated that the CARP3 tip localization depends on the presence of FLAM8 acting as a scaffold protein [21]. Thus, trypanosome migration and transmission in the tsetse vector specifically depend on adenylate cyclase complex-mediated signaling in the tip nanodomain including FLAM8 [21]. Interestingly, we have recently shown that CARP3 and FLAM8 are both redistributed along the entire length of the flagellum during their differentiation to the mammalian stage, and that they further remain associated in flagellar complexes in mammalian forms [21]. Here, this was confirmed by the significant decrease of the pool of CARP3 detected in the proximal flagellum region of FLAM8-deprived parasites. Altogether, these data could reflect a possible specific function of some CARP3-containing signaling complexes depending on FLAM8 for their sub-flagellar localization. Environmental sensing and / or signaling, possibly through direct contact with host cell receptors may play a role in extravasation. We hypothesize that the absence of FLAM8 would destabilize or delocalize these signaling complexes, impairing parasite sensing, signaling and / or adhesion functions, hence preventing the parasite to efficiently cross vascular endothelia.

To our knowledge, FLAM8 is the first flagellar component affecting parasite extravasation and their subsequent dissemination in the extravascular host tissues in vivo. Further investigations on the FLAM8 interactions with other possible partners in the flagellum would help to unravel the roles of this fascinating and essential organelle, especially regarding the modulation of trypanosome tropism, extravasation and spreading in their mammalian hosts, and its implications in parasite virulence and transmission.

Materials and methods

Ethics statement

This study was conducted in strict accordance with the recommendations from the Guide for the Care and Use of Laboratory Animals of the European Union (European Directive 2010/63/UE) and the French Government. The protocol was approved by the “Comité d’éthique en expérimentation animale de l’Institut Pasteur” CETEA 89 (Permit number: 2012–0043 and 2016–0017) and undertaken in compliance with the Institut Pasteur Biosafety Committee (protocol CHSCT 12.131).

Strains, culture and in vitro differentiation

The AnTat 1.1E Paris pleomorphic clone of Trypanosoma brucei brucei was derived from a strain originally isolated from a bushbuck in Uganda in 1966 [33]. The monomorphic T. brucei strain Lister 427 [34] was also used. All bloodstream forms (BSF) were cultivated in HMI-11 medium supplemented with 10% (v/v) fetal bovine serum (FBS) [35] at 37°C in 5% CO2. For in vitro slender to stumpy BSF differentiation, we used 8-pCPT-2′-O-Me-5′-AMP, a nucleotide analog of 5’-AMP from BIOLOG Life Science Institute (Germany). Briefly, 2x106 cultured pleomorphic AnTat 1.1E slender forms were incubated with 8-pCPT-2′-O-Me-5′-AMP (5 μM) for 48 h [36]. For specific experiments, in vitro differentiation of BSF into procyclic forms was performed by transferring freshly differentiated short stumpy forms into SDM-79 medium supplemented with 10% (v/v) FBS, 6 mM cis-aconitate and 20 mM glycerol at 27°C [37].

Monomorphic BSF “Single Marker” (SM) trypanosomes are derivatives of the Lister 427 strain, antigenic type MITat 1.2, clone 221a [38], and express the T7 RNA polymerase and tetracycline repressor. FLAM8RNAi cells express complementary single-stranded RNA corresponding to a fragment of the FLAM8 gene from two tetracycline-inducible T7 promoters facing each other in the pZJM vector [39] integrated in the rDNA locus [40]. Addition of tetracycline (1 μg/mL) to the medium induces expression of sense and anti-sense RNA strands that can anneal to form double-stranded RNA (dsRNA) and trigger RNAi. For in vivo RNAi studies in mice, doxycycline hyclate (Sigma Aldrich) was added in sugared drinking water (0.2 g/L doxycycline hyclate combined with 50 g/L sucrose).

Generation of FLAM8 RNAi mutants

For the generation of the FLAM8RNAi cell lines, a 380 bp (nucleotides 6665–7044) fragment of FLAM8 (Tb927.2.5760), flanked by 5’ HindIII and 3’ XhoI restriction sites to facilitate subsequent cloning, was selected using the RNAit2 algorithm (https://dag.compbio.dundee.ac.uk/RNAit/) to ensure that the targeted sequence was distinct from any other genes to avoid any cross-RNAi effects [41]. This FLAM8 DNA fragment was synthesized by GeneCust Europe (Dudelange, Luxembourg) and inserted into the HindIII-XhoI digested pZJM vector [39].

The pZJM-FLAM8 plasmid was linearized with NotI prior to transfection using nucleofector technology (Lonza, Italy) as described previously [42]. The cell line was further engineered for endogenous tagging of FLAM8 with an mNeonGreen (mNG) at its C-terminal end by using the p3329 plasmid [43], carrying a FLAM8 gene fragment corresponding to FLAM8 ORF nucleotides 8892–9391. Prior to nucleofection, NruI linearization of p3329-FLAM8-mNG plasmid was performed.

For in vivo experiments in mice, FLAM8RNAi parasites were finally modified by integrating a plasmid encoding for the chimeric triple reporter which combines the red-shifted firefly luciferase PpyREH9 and the tdTomato red fluorescent protein fused with a TY1 tag [20]. Transformants were selected with the appropriate antibiotic concentrations: phleomycin (1 μg/mL), blasticidin (5 μg/mL), G418 (2 μg/mL), and puromycin (0.1 μg/mL). Clonal populations were obtained by limiting dilution. Cell culture growth was monitored with an automatic Muse cell analyzer (Merck Millipore, Paris).

Generation of FLAM8 KO mutants

For generating the FLAM8 knockout and rescue cell lines, all insert templates were synthesized by GeneCust Europe (Dudelange, Luxembourg). For breaking the first allele, the 300 first nucleotides of the FLAM8 gene flanking sequences were added on each side of a HYG resistance cassette (S2 Fig). For a complete disruption of the FLAM8 locus, a second selectable marker (PAC) was flanked with the FLAM8-flanking sequence at 5’ and by 300 nucleotides of the FLAM8 ORF (nucleotides 501–800) at 3’. For generating an add-back rescue cell line, due to the large size of the FLAM8 ORF (9,228 nucleotides), the PAC selection marker was replaced by a BLE marker cassette flanked by the 300 first nucleotides of the FLAM8 5’ untranslated region (UTR) and by the nucleotides 1 to 500 of the FLAM8 ORF for reinsertion into the endogenous locus of the knockout clone 1. PCR amplifications of the DNA fragments bearing the FLAM8 flanking sequences and the appropriate resistance markers were used for nucleofection and generation of all cell lines. The primers used are listed below: 5’- CATGACTTTACGTGTTTGGGCAC-3’ (FW, located 82 bp upstream the flanking 5’UTR sequence); 5’-CTTGCTTGTTTCTGTTTCGCAAC-3’ (RV, 130 bp downstream the flanking 3’UTR sequence, used to replace one WT allele by HYG resistance cassette); 5’- GCACACTAAAACTCATTGAAAGCC-3’ (RV, 926 bp downstream the ATG codon of FLAM8, used for second WT allele replacement by PAC cassette and rescue line generation). All knockout and rescue cell lines were further transfected to express the chimeric triple reporter protein PpyRE9H/TY1/tdTomato for multimodal in vivo imaging approaches as described elsewhere [20]. Selection-marker recovery was confirmed by screening individual clones in multi-well plates. Transformants were selected with the appropriate antibiotic concentrations: phleomycin (1 μg/mL), blasticidin (5 μg/mL), puromycin (0.1 μg/mL) and hygromycin (2.5 μg/mL). Clonal populations were obtained by limiting dilution and cell culture growth was monitored with an automatic Muse cell analyzer (Merck Millipore, Paris).

Knockout and rescue cell lines were validated by whole-genome sequencing (BGI, Hong Kong). Briefly, genomic DNA from parental and mutant cell lines were sequenced by the HiSeq4000 sequence system (Illumina), generating about 10 million 100-bp reads and compared to that of the T. brucei brucei AnTat 1.1E Paris reference strain. In addition, some validation of the construct integrations in mutants were performed by PCR analysis according to standard protocols (S2 Fig and Table 1).

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Table 1. Oligonucleotides used for PCR validation of the ΔFLAM8 knockout and rescue cell lines.

https://doi.org/10.1371/journal.ppat.1011220.t001

Motility analyses

In silico 2D tracking was performed as previously described [44]. For each BSF strain, 10 to 20 movies were recorded for 20 seconds (50 ms of exposure). Trypanosomes at 1x106 parasites/mL were maintained in matrix-dependent HMI-11 medium containing 0,5% or 1.1% methylcellulose at 37°C and were observed under the 10x objective of an inverted DMI-4000B microscope (Leica) coupled to an ORCA-03G (Hamamatsu) or a PRIME 95B (Photometrics) camera. Movies were converted with the MPEG Streamclip V.1.9b3 software (Squared 5) and analyzed with the medeaLAB CASA Tracking V.5.9 software (medea AV GmbH). Results were analyzed as mean ± SD of three independent experiments.

In vitro bioluminescence quantification and analysis

To perform the parasite density / bioluminescence intensity assay, BSF parasites were counted, centrifuged, and resuspended in fresh HMI-11 medium. Then, 100 μL of this suspension containing 106 parasites were transferred into black clear-bottom 96-well plates and serial 2-fold dilutions were performed in triplicate adjusting the final volume to 200 μL with 300 μg/mL of beetle luciferin (Promega, France). Luciferase activity was quantified after 10 min of incubation with an IVIS Spectrum imager (PerkinElmer). Imaging data analysis was performed with the Living Image 4.3.1 software (PerkinElmer) by drawing regions of interest with constant size for well plate quantification. Total photon flux was calculated after removal of intensity values from WT parasites and / or parasite-free medium corresponding to the background noise. Results were analyzed as mean ± SD of three independent experiments (S1 and S3A and S3B Figs).

Mouse infection and ethical statements

Seven-week-old male BALB/c mice were purchased from Janvier Laboratory (sub-strain BALB/cAnNRj) and used as models for experimental infection and monitoring of the bioluminescence signal with the IVIS Spectrum imager (PerkinElmer). BR is authorized to perform experiments on vertebrate animals (license #A-75-2035) and is responsible for all the experiments conducted personally or under his supervision. For in vivo infections, groups of four and three animals (FLAM8 knockdown and knockout-infected mice, respectively) were injected intraperitoneally (IP) with 105 slender BSF parasites, washed in TDB (Trypanosome Dilution Buffer: 5 mM KCl, 80 mM NaCl, 1 mM MgSO4*7H2O, 20 mM Na2HPO4, 2 mM NaH2PO4, 20 mM glucose) and resuspended in 100 μl of PBS prior animal inoculation. To study the effects of the genotype factor (FLAM8 knockdown) on parasitemia and EV parasite densities (variables), the most adapted statistical analysis was a multiple comparison of the means of 5 groups by ANOVA, using two-sided tests on the 5 experimental groups. With 3 individuals per group, mixed into 3 cages (5 individuals per cage) to limit any cage effect, the experimental design allows us to obtain significative results for strong effects superior to 1.13 with 0.05% confidence and a power fixed at 0.8. This experiment was performed in two independent replicates.

In vivo bioluminescence imaging (BLI) analyses

Infection with bioluminescent parasites was monitored daily by detecting the bioluminescence signal in whole animals with the IVIS Spectrum imager (PerkinElmer). The equipment consists of a cooled charge-coupled camera mounted on a light-tight chamber with a nose cone delivery device to keep the mice anaesthetized during image acquisition with 1.5–2% isoflurane. A heated stage is comprised within the IVIS Spectrum imager to maintain optimum body temperature. D-luciferin potassium salt (Promega) stock solution was prepared in sterile PBS at 33.33 mg/mL and stored in a -20°C freezer. To produce bioluminescence, mice were inoculated by the intraperitoneal route (IP) with 150 μL of D-luciferin stock solution (250 mg/Kg body weight). After 10 minutes of incubation to allow substrate dissemination, all mice were anaesthetized in an oxygen-rich induction chamber with 1.5–2% isoflurane, and dorsal and ventral BLI images were acquired by using automatic exposure (0.5 seconds to 5 minutes) depending on signal intensity.

Images were analyzed with the Living Image software version 4.3.1 (PerkinElmer). Data were expressed in total photons/second (p/s) corresponding to the total flux of bioluminescent signal according to the selected area (ventral and dorsal regions of interest with constant size covering the total body of the mouse). The background noise was removed by subtracting the bioluminescent signal of the control mouse from the infected ones for each acquisition. For parasite dissemination analyses, a minimum value of photons/second (p/s) was set for all animals in every time point to quantify the exact dissemination area (in cm2) over the whole animal body. Parasitemia was determined daily following tail bleeds and assayed by automated fluorescent cell counting with a Muse cytometer (Merck-Millipore, detection limit at 102 parasites/mL) according to the manufacturer’s recommendations. The quantification of the total intravascular parasite population was assessed by calculating the blood volume of all animals according to their body weight and referring to daily parasitemia.

To quantify the total number of parasites by BLI (intravascular plus extravascular trypanosomes), an in vivo standard curve was performed (S3C and S3D Fig). Since the bioluminescent emission of cultured parental, KO subclones and rescue parasites was not significantly different (S3A and S3B Fig), the in vivo standard curve was obtained by injecting IP increasing numbers (103, 104, 105, 106 and 107 parasites/animal) of parental trypanosomes only (S3C and S3D Fig). After 2.5h, animals received 150 μL of D-luciferin stock solution IP (250 mg/kg body weight), 10 minutes prior image acquisition. During this time, mice were anaesthetized with 1.5–2% isoflurane, and images were acquired in the IVIS Spectrum imager by using automatic exposure settings. A region of interest with a constant size was used to correlate the number of injected parasites with the whole-body BLI signal. Non-infected controls were imaged and the total BLI values used to subtract the background signal or noise. The signals in photons/second were used to construct a standard curve to further interpolate the total number of trypanosomes present in each animal during the entire experimental infection period (S3D Fig). Subsequently, to obtain the number of extravascular parasites, the calculated total number of parasites present in the vascular system was subtracted from the total number of trypanosomes per animal body, resulting in estimating the total parasite population colonizing the extravascular compartments at a given time point.

Endothelial transmigration assay

Single donor cryopreserved Primary Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from Promocell and maintained as per the manufacturer’s instructions in 75 cm2 flasks at 37°C with 5% CO2 in endothelial cell growth medium (Promocell) with 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco). Cells were passaged at 80–90% confluence by dissociation with 0.04% Trypsin-0.03% EDTA (Promocell), split at 1:3–1:5 ratios, and maintained for up to six passages. Polyester transwell inserts for 24 well plates with 3 μm pore size (Corning) were coated with 10 μg/mL bovine fibronectin (Promocell) for one hour, the excess removed, and 600 μL pre-warmed endothelial cell growth medium added to the lower chamber (Fig 6A). The upper chamber was seeded with 2x104 HUVECs per insert in a volume of 100 μL endothelial cell growth medium. Media was exchanged in the upper and lower chamber every two days until monolayer confluence reached (approximately 6 days). Confluence was confirmed by FITC-dextran permeability assay and crystal violet staining of a sacrificed transwell. Briefly, 1 mg/mL FITC-70kDa dextran (Sigma-Aldrich) in endothelial cell growth medium was added to the upper chamber, incubated for 20 minutes and leakage to the lower chamber measured on a Qubit 4 Fluorometer (Invitrogen). Leakage of <1% of the FITC-dextran was observed at confluence. Monolayer integrity was confirmed by brightfield imaging of a transwell insert stained with crystal violet as per the manufacturer’s instructions (Millipore). To perform the trypanosome transmigration assays, the confluent transwells were exchanged into a new 24 well plate containing pre-warmed assay media (endothelial cell growth medium supplemented with 20% trypanosome growth media) for two hours prior to performing the assay. Cultured trypanosomes were collected by centrifugation at 900×g for 5 min and resuspended in assay media at 2x106/mL. The media in the upper chamber was replaced with 100 μL of assay media containing 2x105 trypanosomes and incubated overnight at 37°C, 5% CO2. Each cell line was tested in triplicate transwells. Unbiased quantification of trypanosome transmigration was determined after 24 hours by collecting the media from the upper and lower chambers and transferring 100 μL to 96 well plates for automated counting using a Guava Easycyte HT system with a green laser and a custom counting protocol for tdTomato fluorescent trypanosomes. The cell counts per ml were used to calculate the number of trypanosomes in each compartment and proportion of transmigration into the lower chamber. Trypanosome transmigration was compared between the FLAM8 mutant cell lines and the parental reference cell line using the Generalised Linear Model function in R with a Gaussian family function and proportion transmigration as the dependant variable. A probability value of p<0.05 was considered significant.

Ex vivo RT-qPCR of mouse tissues

Biological samples.

After four weeks of in vivo monitoring, parental-, KO- and rescue-infected mice were euthanized by an overdose of an anesthetic/analgesic mixture of ketamine and xylazine (100 mg/kg and 10 mg/kg, respectively). Terminal bleeding was then performed through the inferior vena cava, and the collected blood was directly transferred in 1 ml RNAlater (ThermoFisher Scientific), snap frozen in liquid nitrogen for long-term preservation. Next, perfusion was initiated by injecting 0.9% NaCl (pre-heated at 37°C), to completely remove the blood from the animal’s body (both organs and vasculature), thus ensuring that subsequent analyses would only contain tissue-dwelling trypanosomes. Finally, spleen, lungs, kidney, gut, testis, liver and skin tissue samples were preserved in 1 ml RNAlater, snap frozen and stored in liquid nitrogen.

RNA isolation. Total RNA from samples was isolated using the QIAzol Reagent (Qiagen) according to the manufacturer’s instructions. Frozen tissue samples were weighed and processed on ice to prevent thawing. Briefly, 30 mg of tissue were added to 700 μl of QIAzol lysis reagent, or 250 μl of blood in 550 μl of QIAzol lysis reagent, and samples were homogenized using the Precellys Evolution Homogenizer (Bertin, USA) with 2.8 mm stainless steel beads for 2 cycles of 1 min at 5000 rpm each, followed by 15 sec of resting between them. Homogenates were then incubated for 5 min at room temperature before the addition of 140 μl chloroform (0.2 volume of starting QIAzol lysis reagent), thoroughly mixed by vortexing for 15 seconds, incubated 5 min at room temperature and centrifuged at 12,000×g, 4°C, 15 min. The aqueous phase containing the RNA was subsequently mixed with 1.5 volumes of absolute ethanol and transferred into a RNeasy Mini spin column (Qiagen) following the manufacturer’s recommendations. The concentration of each RNA sample was measured by spectrophotometric analysis in a NanoDrop 2000c (ThermoFisher Scientific). Finally, RNA quality was determined by capillary electrophoresis in a 2100 Bioanalyzer (Agilent). Extracted RNA was stored at -80°C prior to RT-qPCR analyses.

DNAse treatment and validation.

Extracted RNA samples were subjected to a second DNase treatment using Invitrogen’s DNA-free kit (Life Technologies) according to manufacturer’s protocol. DNase treatment confirmation was performed by running a qPCR targeting the Tubulin locus as housekeeping gene control. The primer pair used is as follows: forward (FW), 5’-ACTGGGCAAAGGGCCACTAC-3’; reverse (RV), 5’-CTCCTTGCAGCACACATCGA-3’, with an amplicon size of 105 bp. Reactions were done in a volume of 20?μl containing: 1?μl of RNA template (17 ng), 10 μl of 2X GoTaq qPCR Master Mix Buffer, 2 μl of both FW and RV primers (at 10X) and 5 μl of Nuclease-Free Water. Amplification was accomplished in a QuantStudio 3 thermal cycler (Applied Biosystems) using the following program: 2?min at 95°C; 40 cycles at 94°C 15?sec; 55°C 1?min; and final 30?sec at 60°C. The absence of residual DNA in the DNAse-treated RNA samples was thus confirmed when no amplification was observed. RNA samples were further employed for gene expression quantification.

Primer design of target genes.

For FLAM8 amplification, primers were designed to recognize the 324-bp region suppressed during FLAM8 knockout generation: FW, 5’-GCATCGTTCGTGAGGTTGGA-3’; RV, 5’-GTTCCTCTTCGTCATCTGGTTCA-3’. The amplicon size was 88 bp. For Protein Associated to Differentiation 1 (PAD1) quantification in infected samples, primer sequences were described elsewhere [45] and are listed below: FW, 5’-TCATGGTTTCGCCATTCTCGTAACC-3’; RV, 5’-CTCAGCCACTTCTCTCCTACAACAC-3’. Amplicon size is 156 bp.

Real time RT-PCR assay.

One step RT-PCR kit (Promega) was used to amplify Tubulin, FLAM8 or PAD1 targets (Table 2). Reactions were prepared in a volume of 20?μl, containing: 1?μl of RNA template (17 ng), 10 μl of 2X GoTaq qPCR Master Mix Buffer, 0.4 μl of 50X GoScript RT Mix for 1-Step RT-qPCR, 2 μl of both FW and RV primers (10X), and 4,6 μl of Nuclease-Free Water. Reverse transcription and amplification were accomplished in one step in a QuantStudio 3 thermal cycler (Applied Biosystems) using the following incubation program: 15?min at 42°C; 10?min at 95°C; 40 cycles of 95°C during 30?sec; 55°C for Tubulin, 58°C for FLAM8 or 60°C for PAD1 during 1?min; final 72°C during 30 sec. A melt curve program was included: 15 sec at 95°C; 55°C for Tubulin, 58°C for FLAM8 or 60°C for PAD1 during 1?min; 95°C for 10 sec. Amplicons were then analyzed by gel electrophoresis. Negative and positive controls consisted of RNA extracted from uninfected mice and cultured trypanosomes, respectively.

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Table 2. Oligonucleotides used for ex vivo RT-qPCR of infected tissues.

https://doi.org/10.1371/journal.ppat.1011220.t002

RT-qPCR data analysis.

All samples were amplified in triplicates and Cq mean values were calculated. Considering that the same initial amounts of total mRNAs extracted from each organ were used as RT-qPCR templates, the total number of parasites in each sample was calculated for comparisons by using a Tubulin RT-qPCR standard curve. Nine pools of cultured parasites (p) increasing by 10-folds from 101 to 108 were extracted and tested in triplicates by Tubulin RT-qPCR to generate a standard curve. The resulting standard curve’s equation Cq = -2,87 x Log10(p) + 35,412 allowed us to calculate the total number of parasites per mg of sample according to the Cq values obtained by Tubulin RT-qPCR on each sample. For normalization purposes to better compare the variations of the parasite populations in each compartment between strains, the difference between the number of parasites in each tissue sample of a given mouse and the number of parasites in the blood sample from the same mouse was calculated and plotted as Delta number of parasites. Tubulin expression (CqTub) was also used to normalize the PAD1 mRNA levels (CqPAD1): the difference between the CqPAD1 and the CqTub values was calculated for each organ of each mouse and plotted as the Delta CqPAD1-CqTub. It allowed us to compare the relative proportions of parasites expressing PAD1 mRNAs between organs and strains, a higher Delta CqPAD1-CqTub correlating with a lower amount of PAD1 transcripts in the organ.

Immunofluorescence analysis (IFA)

Cultured parasites were washed twice in TDB and spread directly onto poly-L-lysine coated slides. For methanol fixation, slides were air-dried for 10 min, fixed in methanol at -20°C for 30 seconds and rehydrated for 20 min in PBS. For immunodetection, slides were incubated for 1 h at 37°C with the appropriate dilution of the first antibody in 0.1% BSA in PBS. After 3 consecutive 5 min washes in PBS, species and subclass-specific secondary antibodies coupled to the appropriate fluorochrome (Alexa 488, Cy3, Cy5 Jackson ImmunoResearch) were diluted 1/400 in PBS containing 0.1% BSA and were applied for 1 h at 37°C. After washing in PBS as indicated above, slides were finally stained with 4’,6-diamidino-2-phenylindole (DAPI, 1 μg/mL) for visualization of kinetoplast and nuclear DNA content and mounted under cover slips with ProLong antifade reagent (Invitrogen), as previously described [8]. Slides were observed under an epifluorescence DMI4000 microscope (Leica) with a 100x objective (NA 1.4), an EL6000 (Leica) as light excitation source and controlled by the Micro-Manager V1.4.22 software (NIH), and images were acquired using an ORCA-03G (Hamamatsu) or a PRIME 95B (Photometrics) camera. Images were analyzed with ImageJ V1.8.0 (NIH). The monoclonal antibody mAb25 (anti-mouse IgG2a, 1:10) was used as a flagellum marker as it specifically recognizes the axoneme protein TbSAXO1 [46]. FLAM8 was detected using: i) a specific rabbit serum (1:500) kindly provided by Paul McKean (University of Lancaster, UK), or ii) a monoclonal anti-mNeonGreen antibody (anti-mouse IgG2c, 1:100, ChromoTek). CARP3 was detected using a polyclonal CARP3 antiserum (1:150) [21]. Stumpy BSF were identified at the molecular level with a rabbit polyclonal anti-PAD1 antibody (kindly provided by Keith Matthews, University of Edinburgh; dilution 1:300) [47]. In the case of RNAi knockdown experiments, IFA signals were normalized using the signal obtained in non-induced controls as a reference.

Measurements, normalization, and statistical analyses

Standardization of fluorescent signals was carried out by parallel setting of raw integrated density signals in all the images to be compared in ImageJ V1.8.0 (NIH). For clarity purposes, the brightness and contrast of several pictures were adjusted after their analysis in accordance with editorial policies. Statistical analyses and plots were performed with XLSTAT 2019.2.01 (Addinsoft) in Excel 2016 (Microsoft) or Prism V9.3.1 (GraphPad). Statistical analyses include: (1) linear regression for bioluminescence / fluorescence intensity vs. parasite density and RT-qPCR standard curve, (2) two-sided ANOVA tests with Tukey or Dunnett’s ad-hoc post-tests for inter-group comparisons for growth curves, IV / EV parasite populations and parasite dissemination, and ΔCq comparisons of RT-qPCR data, all at 95% confidence.

Supporting information

Validation of the triple-reporter efficiency in monomorphic FLAM8RNAi parasite lines.

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S1 Fig. Validation of the triple-reporter efficiency in monomorphic FLAM8RNAi parasite lines.

Linear correlation between the number of parasites and the bioluminescence (in p/s) emitted by monomorphic 427 FLAM8::mNG FLAM8RNAi BSF overexpressing the triple reporter chimeric protein [20], acquired by the IVIS Spectrum imager. Parasites without the RNAi plasmid (control, C), non-treated with tetracycline (non-induced, NI) and treated with tetracycline (induced, I) are shown. Representative bioluminescent image of serial 1/2 dilutions performed in a 96-well plate (in photons / second / cm2 / steradian). RNAi induction was triggered by the addition of 1 μg tetracycline and / or doxycycline for 72 h. Results represent the mean ± standard deviation (SD) of three independent experiments.

https://doi.org/10.1371/journal.ppat.1011220.s001

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S2 Fig. Molecular validation of the ΔFLAM8 null mutant cell lines.

A) Whole-genome sequencing results showing FLAM8 wild-type allele (WT, upper panel), the partial loss of the 5’ FLAM8 ORF in ΔFLAM8 knockout trypanosomes (middle panel; the black arrow is showing the absence of reads at the FLAM8 5’ ORF in the knockout line); and the restoration of the full FLAM8 gene in rescue parasites (bottom panel; the black arrow is showing the absence of reads within the pac cassette due to the insertion of the new construct bearing the ble resistance marker), relative to the number of reads per 100-nt read length. The presence of the correct antibiotic cassettes is shown for knockout and rescue parasites (right middle and bottom panels). ΔFLAM8 knockout and rescue parasites also bear a construct for expression of a triple reporter (TR) as assessed by the detection of bsd reads. B) Schemes showing the structure of the FLAM8 locus in wild-type parasites (upper scheme) and the integration plan of the different cassettes for ΔFLAM8 knockout (middle panel, HYG- and PAC-containing schemes) and Rescue parasites (lower panel HYG- and BLE-containing schemes). C) PCR confirmation of the successful integrations of all reporter cassettes. Primer pairs used for PCRs are indicated at the bottom of each line and correspond to those drawn on the schemes in B), along with the expected band sizes of the corresponding diagnostic PCR. BSD: blasticidin; HYG: hygromycin; PAC: puromycin; BLE: phleomycin.

https://doi.org/10.1371/journal.ppat.1011220.s002

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S3 Fig. In vitro and in vivo validation of the triple-reporter efficiency in pleomorphic ΔFLAM8 mutant cell lines.

A) Representative bioluminescent image (in photons / second) of serial 1/2 dilutions performed in a 96-well plate of pleomorphic parental, ΔFLAM8 knockout subclones and rescue parasites overexpressing the triple reporter chimeric protein [20]. B) Linear correlation between the number of parasites and the emitted bioluminescence (in photons / second) acquired by the IVIS Spectrum imager. Results represent the mean ± standard deviation (SD) of three independent experiments. C) Representative ventral view images of mice infected with increasing amounts of parental trypanosomes (103, 104, 105, 106 and 107 parasites/animal) acquired with the IVIS Spectrum imager 2.5 hours after IP injection. D) In vivo standard curve showing the correlation between the number of injected parasites and the bioluminescent signal (in photons/second, R2 = 1). The standard curve was further employed to calculate the total number of parasites present in infected animals through whole-body BLI signal.

https://doi.org/10.1371/journal.ppat.1011220.s003

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S4 Fig. The absence of FLAM8 reduces extravascular trypanosome dissemination.

Detailed individual data used in Fig 3. Groups of BALB/c mice were injected IP with either one parental, three ΔFLAM8 null subclones or one rescue strains. Total number of parasites in the blood of infected mice (intravascular, IV) daily counted from tail bleeds using a cytometer over 4 weeks (left graphics). Total number of extravascular trypanosomes quantified from bioluminescence images in the same mice (middle graphs). Dissemination measured over the entire animal body (in cm2) through the total surface of bioluminescent signal (right graphs).

https://doi.org/10.1371/journal.ppat.1011220.s004

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S5 Fig. Functional investigations on the ΔFLAM8 null mutants in vivo in the mammalian host.

Detailed individual data used in Fig 4. Groups of BALB/c mice were injected IP with either one parental, three ΔFLAM8 null subclones or one rescue strains. Total number of parasites in the blood of infected mice (intravascular, IV) counted from tail bleeds using a cytometer over 3.5 weeks (left graphs). Total number of extravascular trypanosomes quantified from bioluminescence images in the same mice (middle graphs). Dissemination measured over the entire animal body (in cm2) through the total surface of bioluminescent signal (right graphics).

https://doi.org/10.1371/journal.ppat.1011220.s005

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S6 Fig. Detailed individual data used in Fig 4D.

Delta number of parasites per dissected organs and strains in each individual mouse. The total number of parasites in each sample was calculated per mg of tissues by using a Tubulin RT-qPCR standard curve. A) For normalization purposes, the difference between the number of parasites in each tissue sample of a given mouse and the number of parasites in the blood sample from the same mouse was calculated and plotted as Delta number of parasites. B) As an alternative normalization method, the ratio between the number of parasites in each tissue sample of a given mouse and the number of parasites in the blood sample from the same mouse was calculated and plotted as EV / IV parasites ratio.

https://doi.org/10.1371/journal.ppat.1011220.s006

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S1 Table. Detailed qPCR results from the second experimental infection used in Figs 4D, 4E, 5E and S6.

https://doi.org/10.1371/journal.ppat.1011220.s007

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Acknowledgments

We thank M. Bonhivers, D. Robinson, P. McKean, K. Matthews, and K. Gull for providing various plasmids and antibodies. We gratefully acknowledge the UTechS Photonic BioImaging (Imagopole), C2RT, Institut Pasteur, supported by the French National Research Agency (France BioImaging; ANR-10–INSB–04; Investments for the Future). We are grateful to P. Bastin for his strong scientific and human support. We warmly thank M. Boshart, P. Bastin, and S. Bonnefoy for their critical reading of the manuscript.

References

1.MacGregor P, Szoor B, Savill NJ, Matthews KR. Trypanosomal immune evasion, chronicity and transmission: an elegant balancing act. Nat Rev Microbiol. 2012;10(6):431–8. Epub 2012/05/01. pmid:22543519.