International journal for parasitology最新文献_第4页

Genomic and metagenomic analyses of the domestic mite Tyrophagus putrescentiae identify it as a widespread environmental contaminant and a host of a basal, mite-specific Wolbachia lineage (supergroup Q) 对家养螨 Tyrophagus putrescentiae 的基因组和元基因组分析表明，它是一种广泛存在的环境污染物，也是螨虫特异性沃尔巴奇菌系（超群 Q）的宿主。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-11-01 DOI: 10.1016/j.ijpara.2024.07.001

Pavel B. Klimov , Jan Hubert , Tomas Erban , M. Alejandra Perotti , Henk R. Braig , Alex Flynt , Qixin He , Yubao Cui

Tyrophagus putrescentiae (mould mite) is a global, microscopic trophic generalist that commonly occurs in various human-created habitats, causing allergies and damaging stored food. Its ubiquity and extraordinary ability to penetrate research samples or cultures through air currents or by active walking through tights spaces (such as treads of screw caps) may lead to sample contamination and introduction of its DNA to research materials in the laboratory. This prompts a thorough investigation into potential sequence contamination in public genomic databases. The trophic success of T. putrescentiae is primarily attributed to the symbiotic bacteria housed in specialized internal mite structures, facilitating adaptation to varied nutritional niches. However, recent work suggests that horizontal transfer of bacterial/fungal genes related to nutritional functionality may also contribute to the mite’s trophic versatility. This aspect requires independent confirmation. Additionally, T. putrescentiae harbors an uncharacterized and genetically divergent bacterium, Wolbachia, displaying blocking and microbiome-modifying effects. The phylogenomic position and supergroup assignment of this bacterium are unknown. Here, we sequenced and assembled the T. putrescentiae genome, analyzed its microbiome, and performed detailed phylogenomic analyses of the mite-specific Wolbachia. We show that T. putrescentiae DNA is a substantial source of contamination of research samples. Its DNA may inadvertently be co-extracted with the DNA of the target organism, eventually leading to sequence contamination in public databases. We identified a diversity of bacterial species associated with T. putrescentiae, including those capable of rapidly developing antibiotic resistance, such as Escherichia coli. Despite the presence of diverse bacterial communities in T. putrescentiae, we did not detect any recent horizontal gene transfers in this mite species and/or in astigmatid (domestic) mites in general. Our phylogenomic analysis of Wolbachia recovered a basal, mite-specific lineage (supergroup Q) represented by two Wolbachia spp. from the mould mite and a gall-inducing plant mite. Fluorescence in situ hybridization confirmed the presence of Wolbachia inside the mould mite. The discovery of an early derivative Wolbachia lineage (supergroup Q) in two phylogenetically unrelated and ecologically dissimilar mites suggests that this endosymbiotic bacterial lineage formed a long-term association with mites. This finding provides a unique insight into the early evolution and host associations of Wolbachia. Further discoveries of Wolbachia diversity in acariform mites are anticipated.

霉螨（Tyrophagus putrescentiae）是一种全球性的微小营养性通病，通常出现在人类创造的各种栖息地中，会引起过敏并破坏储存的食物。它无处不在，而且具有超强的穿透能力，可以通过气流或在狭小空间（如螺旋盖的踏板）中主动行走而穿透研究样本或培养物，可能会导致样本污染，并将其 DNA 带入实验室的研究材料中。这促使我们对公共基因组数据库中潜在的序列污染进行彻底调查。T.putrescentiae在营养方面的成功主要归功于寄生在螨虫内部特化结构中的共生细菌，这有助于适应各种营养环境。不过，最近的研究表明，与营养功能相关的细菌/真菌基因的水平转移也可能有助于螨虫的多营养性。这方面还需要独立证实。此外，T. putrescentiae 还携带一种尚未定性且基因不同的细菌，即 Wolbachia，它具有阻断和改变微生物群的作用。这种细菌的系统发生组位置和超群分配尚不清楚。在这里，我们对T. putrescentiae基因组进行了测序和组装，分析了其微生物组，并对螨特异性狼杆菌进行了详细的系统发生组分析。我们发现，T. putrescentiae DNA 是研究样本的一个重要污染源。它的 DNA 可能会无意中与目标生物的 DNA 共同提取，最终导致公共数据库中的序列污染。我们发现了与腐生菌相关的多种细菌，包括那些能够迅速产生抗生素耐药性的细菌，如大肠杆菌。尽管T. putrescentiae中存在多种细菌群落，但我们并未在该螨类和/或一般的星螨中发现任何近期的水平基因转移。我们的沃尔巴克氏体系统发生组分析发现，霉螨和引胆植物螨中的两个沃尔巴克氏体属代表了一个基础的、螨虫特异的品系（超群 Q）。荧光原位杂交证实了霉螨体内存在沃尔巴克氏体。在两种在系统发育上不相关、生态上不同的螨类体内发现了沃尔巴奇菌的早期衍生物（超群 Q），这表明这种内共生细菌菌系与螨类形成了长期的联系。这一发现为了解沃尔巴克氏菌的早期进化和宿主关联提供了独特的视角。我们期待着在螨类中进一步发现沃尔巴克氏菌的多样性。

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引用次数: 0

Chromosome-contiguous genome for the Haecon-5 strain of Haemonchus contortus reveals marked genetic variability and enables the discovery of essential gene candidates Haecon-5血吸虫菌株的染色体连续基因组揭示了明显的遗传变异，有助于发现重要的候选基因。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-11-01 DOI: 10.1016/j.ijpara.2024.08.003

Yuanting Zheng , Neil D. Young , Tulio L. Campos , Pasi K. Korhonen , Tao Wang , Sunita B. Sumanam , Aya C. Taki , Joseph J. Byrne , Bill C.H. Chang , Jiangning Song , Robin B. Gasser

Millions of livestock animals worldwide are infected with the haematophagous barber’s pole worm, Haemonchus contortus, the aetiological agent of haemonchosis. Despite the major significance of this parasite worldwide and its widespread resistance to current treatments, the lack of a high-quality genome for the well-defined strain of this parasite from Australia, called Haecon-5, has constrained research in a number of areas including host-parasite interactions, drug discovery and population genetics. To enable research in these areas, we report here a chromosome-contiguous genome (∼280 Mb) for Haecon-5 with high-quality models for 19,234 protein-coding genes. Comparative genomic analyses show significant genomic similarity (synteny) with a UK strain of H. contortus, called MHco3(ISE).N1 (abbreviated as “ISE”), but we also discover marked differences in genomic structure/gene arrangements, distribution of nucleotide variability (single nucleotide polymorphisms (SNPs) and indels) and orthology between Haecon-5 and ISE. We used the genome and extensive transcriptomic resources for Haecon-5 to predict a subset of essential single-copy genes employing a “cross-species” machine learning (ML) approach using a range of features from nucleotide/protein sequences, protein orthology, subcellular localisation, single-cell RNA-seq and/or histone methylation data available for the model organisms Caenorhabditis elegans and Drosophila melanogaster. From a set of 1,464 conserved single copy genes, transcribed in key life-cycle stages of H. contortus, we identified 232 genes whose homologs have critical functions in C. elegans and/or D. melanogaster, and prioritised 10 of them for further characterisation; nine of the 10 genes likely play roles in neurophysiological processes, germline, hypodermis and/or respiration, and one is an unknown (orphan) gene for which no detailed functional information exists. Future studies of these genes/gene products are warranted to elucidate their roles in parasite biology, host-parasite interplay and/or disease. Clearly, the present Haecon-5 reference genome and associated resources now underpin a broad range of fundamental investigations of H. contortus and could assist in accelerating the discovery of novel intervention targets and drug candidates to combat haemonchosis.

全世界有数以百万计的牲畜感染了噬血性理发杆蠕虫（Haemonchus contortus），这是血吸虫病的病原体。尽管这种寄生虫在世界范围内具有重要意义，而且对目前的治疗方法普遍存在抗药性，但由于缺乏澳大利亚寄生虫明确菌株（Haecon-5）的高质量基因组，制约了宿主与寄生虫相互作用、药物发现和种群遗传学等多个领域的研究。为了促进这些领域的研究，我们在此报告了 Haecon-5 的染色体连续基因组（∼280 Mb），其中包含 19,234 个蛋白质编码基因的高质量模型。比较基因组分析表明，Haecon-5 与英国的一个 H. contortus 株系 MHco3(ISE).N1（缩写为 "ISE"）在基因组结构/基因排列、核苷酸变异性（单核苷酸多态性（SNP）和嵌合体）分布和选系方面存在显著的相似性（同源），但我们也发现 Haecon-5 与 ISE 在基因组结构/基因排列、核苷酸变异性（单核苷酸多态性（SNP）和嵌合体）分布和选系方面存在显著的差异。我们利用 Haecon-5 的基因组和广泛的转录组资源，采用 "跨物种 "机器学习（ML）方法，利用模式生物秀丽隐杆线虫（Caenorhabditis elegans）和黑腹果蝇（Drosophila melanogaster）的核苷酸/蛋白质序列、蛋白质同源、亚细胞定位、单细胞 RNA-seq 和/或组蛋白甲基化数据等一系列特征，预测了基本单拷贝基因的子集。在一组 1464 个保守的单拷贝基因中，我们确定了 232 个在秀丽隐杆线虫和/或黑腹果蝇中具有关键功能的同源基因，并优先对其中 10 个基因进行了进一步鉴定；这 10 个基因中的 9 个可能在神经生理过程、生殖细胞、下胚层和/或呼吸中发挥作用，还有一个是未知（孤儿）基因，没有详细的功能信息。今后有必要对这些基因/基因产物进行研究，以阐明它们在寄生虫生物学、宿主-寄生虫相互作用和/或疾病中的作用。很明显，目前的 Haecon-5 参考基因组及相关资源是对霍乱弧菌进行广泛基础研究的基础，并有助于加快发现新的干预目标和候选药物，以防治霍乱弧菌病。

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引用次数: 0

Micro RNA profiles of host extracellular vesicles are modulated by Ascaris suum infection but parasite extracellular vesicle miRNAs are systemically undetectable using in-depth miRNA sequencing 宿主细胞外囊泡的微RNA谱受蛔虫感染的影响，但寄生虫细胞外囊泡的miRNA在深度miRNA测序中是系统检测不到的。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-11-01 DOI: 10.1016/j.ijpara.2024.08.001

Bradley Whitehead , Litten Sørensen Rossen , Mads Zippor , Anders T. Boysen , Vineesh Indira Chandran , Per Skallerup , Stig M. Thamsborg , Peter Nejsum

The intestinal helminth Ascaris lumbricoides infects over 800 million people. Infections are often chronic and immunity is not sterilizing due to host-immune modulation, therefore reinfection is common after antihelmintic treatment. We have previously demonstrated a role for Ascaris spp. extracellular vesicles (EVs) in host immune modulation but whether EVs are recognized by the adaptive immune system and are present systemically in the host remains unknown. Therefore, we employed a well-established trickle infection model in pigs to mimic natural Ascaris infection in humans. EVs were isolated from adult Ascaris suum followed by immunoblotting of EV and EV-depleted secretory fractions using plasma from infected and uninfected pigs. Next, EVs were isolated from pig plasma at day 56 post first infection and subjected to deep small RNAseq analysis. RNAs were aligned to A. suum and Sus scrofa miRNA complements to detect A. suum EVs and elucidate the host EV micro RNA (miRNA) response to infection, respectively. Infection generates robust antibody responses against A. suum EVs that is distinct from EV-depleted fractions. However, A. suum miRNAs were not detectable in EVs from the peripheral blood. Notably, host plasma-derived EV miRNA profiles showed significant changes between infected and uninfected pigs, indicating that Ascaris infection drives systemic changes in host EV composition.

肠道蠕虫蛔虫感染了 8 亿多人。感染通常是慢性的，由于宿主免疫调节作用，免疫不能杀菌，因此抗蠕虫药治疗后再感染很常见。我们之前已经证明了蛔虫胞外囊泡（EVs）在宿主免疫调节中的作用，但EVs是否能被适应性免疫系统识别并系统地存在于宿主体内仍是未知数。因此，我们采用了一种成熟的猪涓流感染模型来模拟人类的自然蛔虫感染。从成年蛔虫体内分离出 EVs，然后用感染猪和未感染猪的血浆对 EV 和 EV 贫化分泌物部分进行免疫印迹。然后，从首次感染后第 56 天的猪血浆中分离出 EVs，并对其进行深度小 RNAseq 分析。将 RNA 与 A. suum 和 Sus scrofa miRNA 互补序列进行比对，分别检测 A. suum EVs 和阐明宿主 EV 微 RNA（miRNA）对感染的反应。感染会产生针对鼠EVs的强抗体反应，这种反应与EV去除了的部分不同。然而，在外周血的EVs中检测不到鼠疫miRNA。值得注意的是，宿主血浆来源的 EV miRNA 图谱在感染猪和未感染猪之间发生了显著变化，这表明蛔虫感染驱动了宿主 EV 组成的系统性变化。

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引用次数: 0

DNA N-glycosylases Ogg1 and EndoIII as components of base excision repair in Plasmodium falciparum organelles DNA N-糖基化酶 Ogg1 和 EndoIII 是恶性疟原虫细胞器中碱基切除修复的组成部分。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-11-01 DOI: 10.1016/j.ijpara.2024.06.005

Anupama Tiwari , Neetu Verma , Himadri Shukla , Shivani Mishra , Kit Kennedy , Tribeni Chatterjee , Jitendra Kuldeep , Shahid Parwez , MI Siddiqi , Stuart A. Ralph , Satish Mishra , Saman Habib

The integrity of genomes of the two crucial organelles of the malaria parasite — an apicoplast and mitochondrion in each cell − must be maintained by DNA repair mediated by proteins targeted to these compartments. We explored the localisation and function of Plasmodium falciparum base excision repair (BER) DNA N-glycosylase homologs PfEndoIII and PfOgg1. These N-glycosylases would putatively recognise DNA lesions prior to the action of apurinic/apyrimidinic (AP)-endonucleases. Both Ape1 and Apn1 endonucleases have earlier been shown to function solely in the parasite mitochondrion. Immunofluorescence localisation showed that PfEndoIII was exclusively mitochondrial. PfOgg1 was not seen clearly in mitochondria when expressed as a PfOgg1_leader-GFP fusion, although chromatin immunoprecipitation assays showed that it could interact with both mitochondrial and apicoplast DNA. Recombinant PfEndoIII functioned as a DNA N-glycosylase as well as an AP-lyase on thymine glycol (Tg) lesions. We further studied the importance of Ogg1 in the malaria life cycle using reverse genetic approaches in Plasmodium berghei. Targeted disruption of PbOgg1 resulted in loss of 8-oxo-G specific DNA glycosylase/lyase activity. PbOgg1 knockout did not affect blood, mosquito or liver stage development but caused reduced blood stage infection after inoculation of sporozoites in mice. A significant reduction in erythrocyte infectivity by PbOgg1 knockout hepatic merozoites was also observed, thus showing that PbOgg1 ensures smooth transition from liver to blood stage infection. Our results strengthen the view that the Plasmodium mitochondrial genome is an important site for DNA repair by the BER pathway.

疟原虫的两个关键细胞器--每个细胞中的顶体和线粒体--的基因组的完整性必须通过这些细胞器的靶向蛋白介导的 DNA 修复来维持。我们研究了恶性疟原虫碱基切除修复（BER）DNA N-糖基化酶同源物 PfEndoIII 和 PfOgg1 的定位和功能。这些 N-糖基化酶可能会在嘌呤/近嘧啶（AP）内切酶发挥作用之前识别 DNA 病变。Ape1 和 Apn1 内切酶早先都被证明只在寄生虫线粒体中发挥作用。免疫荧光定位显示，PfEndoIII 只存在于线粒体中。PfOgg1 以 PfOgg1leader-GFP 融合体的形式表达时，线粒体中看不到明显的 PfOgg1，但染色质免疫沉淀测定显示，PfOgg1 可与线粒体和 apicoplast DNA 相互作用。重组 PfEndoIII 可作为 DNA N-糖基化酶以及胸腺嘧啶乙二醇（Tg）病变的 AP-裂解酶发挥作用。我们利用反向遗传方法进一步研究了 Ogg1 在伯格氏疟原虫疟疾生命周期中的重要性。PbOgg1的靶向破坏导致8-氧代-G特异性DNA糖基化酶/裂解酶活性的丧失。PbOgg1 基因敲除不会影响血液、蚊子或肝脏阶段的发育，但会降低小鼠接种孢子虫后血液阶段的感染率。此外，还观察到 PbOgg1 基因敲除肝脏裂殖子对红细胞的感染率明显降低，从而表明 PbOgg1 确保了从肝脏感染到血液感染阶段的顺利过渡。我们的研究结果进一步证实，疟原虫线粒体基因组是通过 BER 途径进行 DNA 修复的重要场所。

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引用次数: 0

The concentration McMaster method for diagnosis of patent Ascaris and Trichuris infections in humans 用于诊断人类蛔虫和毛滴虫感染的麦克马斯特浓缩法。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-11-01 DOI: 10.1016/j.ijpara.2024.08.004

Sozan Kader , Christine E. Helmersen , Stig M. Thamsborg , Lise T. Erikstrup , Peter Nejsum

In tropical and subtropical regions, soil-transmitted helminth (STH) infections such as Ascaris lumbricoides, Trichuris trichiura, and hookworms have a significant impact on public health. Globally, A. lumbricoides infects approximately 0.8 billion people, while T. trichiura infects around 500 million. This study involved a comparison of three diagnostic methods, Kato-Katz and two flotation methods (concentration McMaster, and simple McMaster), for the detection of Ascaris and Trichuris in human faeces. We conducted a comparison of the number of eggs in faeces (or faecal egg counts (FECs)) obtained with these methods using freshly collected samples that were positive for T. trichiura and spiked with a known quantity of Ascaris sp. eggs. Additionally, for the concentration McMaster method we assessed FECs after storing the samples at 5 °C for up to 21 days. The concentration McMaster method demonstrated superiority over the simple McMaster method in terms of higher detection levels for both helminths, while the Kato-Katz method yielded FEC values very close to the ’true’ spiking values. Importantly, the concentration McMaster method was considerably easier to read compared with the Kato-Katz method, and it allowed for sample storage at 5 °C for up to 14 days without affecting FEC results. Consequently, we conclude that the concentration McMaster method is an effective and user-friendly alternative for diagnosis of Ascaris and Trichuris infections in humans. Furthermore, it offers the advantage of sample storage before analysis, enhancing flexibility in the workflow.

在热带和亚热带地区，蛔虫、毛滴虫和钩虫等土壤传播蠕虫（STH）感染对公共卫生产生了重大影响。全球约有 8 亿人感染了蛔虫，约有 5 亿人感染了毛滴虫。本研究比较了三种诊断方法（卡托-卡茨法和两种浮选法（浓缩麦克马斯特法和简单麦克马斯特法）），以检测人类粪便中的蛔虫和毛滴虫。我们使用新采集的对毛滴虫呈阳性的样本，并在样本中添加已知数量的蛔虫卵，对使用这些方法获得的粪便中的虫卵数量（或粪便虫卵计数（FEC））进行了比较。此外，对于浓度麦克马斯特法，我们评估了在 5°C 下保存样本长达 21 天后的 FECs。浓度麦克马斯特法比简单麦克马斯特法更优越，对两种蠕虫的检测水平更高，而卡托-卡茨法得出的 FEC 值非常接近 "真实 "加标值。重要的是，与卡托-卡茨法相比，浓缩麦克马斯特法更容易读取，而且它允许样品在 5°C 下保存长达 14 天，而不会影响 FEC 结果。因此，我们得出结论，浓度麦克马斯特法是诊断人类蛔虫和毛滴虫感染的一种有效且方便使用的替代方法。此外，它还具有在分析前储存样本的优点，提高了工作流程的灵活性。

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引用次数: 0

Potential effects of host competence and schooling behavior on parasite transmission in a host-pathogen system: a test of the dilution effect 宿主能力和学校行为对宿主-病原体系统中寄生虫传播的潜在影响：稀释效应测试。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-11-01 DOI: 10.1016/j.ijpara.2024.08.002

Shun Zhou , Xiao Jin , Ming Duan , Hong Zou , Ming Li , David J. Marcogliese , Guitang Wang , Wenxiang Li

High species diversity in a community may reduce the risk of infectious disease, termed the dilution effect. However, the generality of the dilution effect in different disease systems remains controversial as both host competence and behaviors of hosts may play roles in dilution or amplification of disease. Using the goldfish (Carassius auratus)-monogenean ectoparasite (Gyrodactylus kobayashii) system, effects of host competence and schooling behavior on parasite transmission were investigated while holding focal host density constant. Following competency tests of 12 fish species as potential hosts for the parasite, infection by G. kobayashii was determined on fins of goldfish mixed with each of three different species based on their level of host competence, including Prussian carp, Carassius gibelio (low competence), grass carp, Ctenopharyngodon idellus (non-competent), swordtail, Xiphophorus helleri (non-competent), and the four species combined. Compared with mean abundance (85.8 ± 25.1) on goldfish in the control group, the mean abundance on goldfish decreased significantly when paired with 10 Prussian carp (30.0 ± 16.5), but did not differ significantly when paired with 10 swordtail (70.0 ± 22.2), 10 grass carp (116.1 ± 33.2), or the multi-species of three Prussian carp, four grass carp and three swordtail (75.9 ± 30.8) during the 11-day experiment. The parasite was also found on the Prussian carp in the Prussian carp group and the multi-species group at a mean abundance of 7.1 and 10.9, respectively. Video recording showed that the school of goldfish mixed well with the Prussian carp, while they maintained separation from the grass carp and swordtail when mixed together. The distance between goldfish increased, and swimming speed and contact time decreased with the additional of other fish species for all groups. The results suggested that the presence of a low-competence host in sufficient numbers was a necessary condition for a dilution effect due to encounter reduction, and the dilution effect may also be enhanced by changes in schooling behavior of goldfish in the presence of low competence hosts. However, the presence of non-competent hosts did not result in any dilution effect owing to the specialist nature of the parasites and the lack of mixing with schools of goldfish.

群落中物种的高度多样性可能会降低传染病的风险，这被称为稀释效应。然而，稀释效应在不同疾病系统中的普遍性仍然存在争议，因为宿主的能力和行为都可能在稀释或放大疾病方面发挥作用。研究人员利用金鱼（Carassius auratus）-单基因外寄生虫（Gyrodactylus kobayashii）系统，在保持焦点寄生虫密度不变的情况下，研究了寄生虫传播中宿主能力和游动行为的影响。在对作为寄生虫潜在宿主的 12 种鱼类进行能力测试之后，根据其宿主能力水平（包括普氏鲫（低能力）、草鱼（无能力）、剑尾鱼（无能力）以及四种鱼类的总和），分别测定了与三种不同鱼类混合的金鱼鳍上的小林天牛感染情况。在为期 11 天的实验中，与对照组金鱼身上的平均虫量（85.8 ± 25.1）相比，金鱼身上的平均虫量在与 10 条普氏鲤配对时显著下降（30.0 ± 16.5），但在与 10 条剑尾（70.0 ± 22.2）、10 条草鱼（116.1 ± 33.2）或 3 条普氏鲤、4 条草鱼和 3 条剑尾的多鱼种（75.9 ± 30.8）配对时没有显著差异。在普氏鲤组和多鱼种组的普氏鲤身上也发现了寄生虫，平均数量分别为 7.1 条和 10.9 条。视频记录显示，金鱼群与普氏鲤混养得很好，而与草鱼和剑尾鱼混养时则保持了一定的距离。各组金鱼之间的距离随着其他鱼种的增加而增加，游泳速度和接触时间则随着其他鱼种的增加而减少。结果表明，足够数量的低能力宿主的存在是因相遇减少而产生稀释效应的必要条件，稀释效应也可能因金鱼在低能力宿主存在时的游动行为变化而增强。然而，由于寄生虫的专一性和不与金鱼群混合，非能力寄主的存在不会导致任何稀释效应。

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引用次数: 0

Acanthamoeba castellanii trophozoites need oxygen for normal functioning and lipids are their preferred substrate, offering new possibilities for treatment. 卡斯特拉氏棘阿米巴滋养体需要氧气才能正常运作，而脂质是它们的首选底物，这为治疗提供了新的可能性。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-10-26 DOI: 10.1016/j.ijpara.2024.10.004

Maarten J Sarink, Anna Z Mykytyn, Aïsha Jedidi, Martin Houweling, Jos F Brouwers, George Ruijter, Annelies Verbon, Jaap J van Hellemond, Aloysius G M Tielens

Acanthamoebae, pathogenic free-living amoebae, can cause Granulomatous Amoebic Encephalitis (GAE) and keratitis, and for both types of infection, no adequate treatment options are available. As the metabolism of pathogens is an attractive treatment target, we set out to examine the energy metabolism of Acanthamoeba castellanii and studied the aerobic and anaerobic capacities of the trophozoites. Under anaerobic conditions, or in the presence of inhibitors of the electron-transport chain, A. castellanii trophozoites became rounded, moved sluggishly and stopped multiplying. This demonstrates that oxygen and the respiratory chain are essential for movement and replication. Furthermore, the simultaneous activities of both terminal oxidases, cytochrome c oxidase and the plant-like alternative oxidase, are essential for normal functioning and replication. The inhibition of normal function caused by the inactivity of the respiratory chain was reversible. Once respiration was made possible again, the rounded, rather inactive amoebae formed acanthopodia within 4 h and resumed moving, feeding and multiplying. Experiments with radiolabelled nutrients revealed a preference for lipids over glucose and amino acids as food. Subsequent experiments showed that adding lipids to a standard culture medium of trophozoites strongly increased the growth rate. Acanthamoeba castellanii trophozoites have a strictly aerobic energy metabolism and β-oxidation of fatty acids, the Krebs cycle, and an aerobic electron-transport chain coupled to the ATP synthase, producing most of the used ATP. The preference for lipids can be exploited, as we show that three known inhibitors of lipid oxidation strongly inhibited the growth of A. castellanii. In particular, thioridazine and perhexiline showed potent effects in low micromolar concentrations. Therefore, this study revealed a new drug target with possibly new options to treat Acanthamoeba infections.

棘阿米巴虫是一种致病性自由生活阿米巴虫，可引起肉芽肿阿米巴脑炎（GAE）和角膜炎，对于这两种类型的感染，目前还没有适当的治疗方案。由于病原体的新陈代谢是一个有吸引力的治疗目标，我们开始研究卡斯特拉氏阿米巴原虫的能量代谢，并研究滋养体的需氧和厌氧能力。在厌氧条件下，或在存在电子传递链抑制剂的情况下，卡斯特拉氏棘阿米巴滋养体变得浑圆，运动迟缓并停止繁殖。这表明氧气和呼吸链对运动和复制至关重要。此外，细胞色素 c 氧化酶和植物样替代氧化酶这两种末端氧化酶的同时活动对正常功能和复制至关重要。呼吸链不活跃对正常功能的抑制是可逆的。一旦呼吸作用得以恢复，这些圆形的、相当不活跃的变形虫就会在 4 小时内形成棘足，并恢复运动、进食和繁殖。用放射性标记的营养物质进行的实验表明，阿米巴虫更喜欢脂类而不是葡萄糖和氨基酸作为食物。随后的实验表明，在滋养体的标准培养基中添加脂质可大大提高滋养体的生长速度。卡氏棘阿米巴滋养体具有严格的有氧能量代谢和脂肪酸的β-氧化、克雷布斯循环，以及与 ATP 合成酶耦合的有氧电子传递链，可产生大部分用过的 ATP。我们发现，三种已知的脂质氧化抑制剂都能强烈抑制卡斯特氏菌的生长。特别是，硫利达嗪和过氧苯胺在低微摩尔浓度下显示出了强效作用。因此，这项研究揭示了一个新的药物靶点，可能是治疗棘阿米巴感染的新选择。

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引用次数: 0

Optimized plasmid loading of human erythrocytes for Plasmodium falciparum DNA transfections 用于恶性疟原虫 DNA 转染的人红细胞质粒装载优化。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-10-01 DOI: 10.1016/j.ijpara.2024.04.011

In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods. As these methods also require expensive instrumentation and consumables that are not broadly available, we explored a simpler method based on plasmid loading through hypotonic lysis and resealing of erythrocytes. We used parasite expression of a sensitive NanoLuc reporter for rapid evaluation and optimization of each step. Hypotonic buffer composition, resealing buffer volume and composition, and subsequent incubation affected plasmid retention and successful transfection. While ATP was critical for erythrocyte resealing, addition of Ca⁺⁺ or glutathione did not improve transfection efficiency, with increasing Ca⁺⁺ concentrations proving detrimental to outcomes. Compared with either the standard electroporation method or a previously reported hypotonic loading protocol, the optimized method yields greater plasmid loading and higher expression of the NanoLuc reporter 48 h after transfection. It also produced significantly faster outgrowth of parasites in transfections utilizing either episomal expression or CRISPR-Cas9 mediated integration. This new method produces higher P. falciparum transfection efficiency, reduces resource requirements and should accelerate molecular studies of malaria drug and vaccine targets.

恶性疟原虫基因的体外改造是疟疾基础研究和转化研究的基石。通过 DNA 转染，这些修饰可能会改变蛋白质序列或丰度。此类实验对于确定关键寄生虫表型的分子机制以及验证药物和疫苗靶点至关重要。尽管转染很重要，但成功转染仍然很困难，是恶性疟原虫研究中一个资源密集型的限制性步骤。在此，我们报告了质粒在红细胞中的低效负载限制了常用电穿孔方法的转染效果。由于这些方法还需要昂贵的仪器和耗材，而这些仪器和耗材并不能广泛使用，因此我们探索了一种更简单的方法，即通过低渗裂解红细胞并重新封口来装载质粒。我们利用寄生虫表达敏感的 NanoLuc 报告器来快速评估和优化每个步骤。低渗缓冲液的成分、再封闭缓冲液的容量和成分以及随后的培养都会影响质粒的保留和成功转染。虽然 ATP 对红细胞再封闭至关重要，但添加 Ca++ 或谷胱甘肽并不能提高转染效率，Ca++ 浓度的增加对转染结果不利。与标准电穿孔方法或之前报道的低渗装载方案相比，优化方法能在转染 48 小时后产生更多的质粒装载和更高的 NanoLuc 报告基因表达。在利用外显子表达或 CRISPR-Cas9 介导的整合进行转染时，它还能明显加快寄生虫的生长速度。这种新方法可提高恶性疟原虫的转染效率，减少资源需求，并可加速疟疾药物和疫苗靶标的分子研究。

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引用次数: 0

Tick salivary proteome and lipidome with low glycan content correlate with allergic type reactions in the zebrafish model 低糖含量的蜱唾液蛋白质组和脂质组与斑马鱼模型中的过敏型反应相关。

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-10-01 DOI: 10.1016/j.ijpara.2024.07.002

Rita Vaz-Rodrigues , Lorena Mazuecos , Margarita Villar , Marinela Contreras , Almudena González-García , Paolo Bonini , Ruth C Scimeca , Albert Mulenga , José de la Fuente

Ticks, as hematophagous ectoparasites, can manipulate host immune and metabolic processes, causing tick-borne allergies such as α-Gal syndrome (AGS). Glycolipids with bound galactose-alpha-1–3-galactose (α-Gal) are potential allergenic molecules associated with AGS. Nevertheless, proteins and lipids lacking α-Gal modifications may contribute to tick salivary allergies and be linked to AGS. In this study, we characterized the effect of deglycosylated tick salivary proteins without lipids on treated zebrafish fed with dog food formulated with mammalian (beef, lamb, pork) meat by quantitative proteomics analysis of intestinal samples. The characterization and functional annotations of tick salivary lipids with low representation of glycolipids was conducted using a lipidomics approach. Results showed a significant effect of treatment with saliva and saliva deglycosylated protein fraction on zebrafish abnormal or no feeding (p < 0.005). Treatment with this fraction affected multiple metabolic pathways, defense responses to pathogens and protein metabolism, which correlated with abnormal or no feeding. Lipidomics analysis identified 23 lipid classes with low representation of glycolipids (0.70% of identified lipids). The lipid class with highest representation was phosphatidylcholine (PC; 26.66%) and for glycolipids it corresponded to diacylglycerol (DG; 0.48%). Qualitative analysis of PC antibodies revealed that individuals bitten by ticks were more likely to produce PC-IgG antibodies (p < 0.001). DG levels were significantly higher in tick salivary glands (p < 0.05) compared with tick saliva and salivary fractions. The α-Gal content was higher in tick saliva than in deglycosylated saliva and lipid fractions. These results support a possible role for tick salivary proteins and lipids without α-Gal modifications in AGS.

蜱虫作为食血的体外寄生虫，可以操纵宿主的免疫和代谢过程，导致蜱传过敏症，如α-Gal综合征（AGS）。与半乳糖-α-1-3-半乳糖（α-Gal）结合的糖脂是与 AGS 相关的潜在过敏原分子。然而，缺乏α-Gal修饰的蛋白质和脂质也可能导致蜱唾液过敏，并与AGS有关。在这项研究中，我们通过对肠道样本进行定量蛋白质组学分析，确定了不含脂质的脱糖蜱唾液蛋白对用哺乳动物（牛肉、羊肉、猪肉）肉配制的狗粮喂养的斑马鱼的影响。利用脂质组学方法对糖脂含量较低的蜱唾液脂质进行了表征和功能注释。结果表明，用唾液和唾液脱糖蛋白部分处理斑马鱼异常或不摄食有明显影响（p < 0.005）。用唾液和唾液脱糖蛋白处理斑马鱼会影响多种代谢途径、对病原体的防御反应和蛋白质代谢，这与斑马鱼摄食异常或不摄食有关。脂质组学分析确定了 23 类脂质，其中糖脂的代表性较低（占所确定脂质的 0.70%）。代表性最高的脂类是磷脂酰胆碱（PC；26.66%），糖脂类则是二酰甘油（DG；0.48%）。PC 抗体的定性分析显示，被蜱虫叮咬的个体更容易产生 PC-IgG 抗体（p < 0.001）。与蜱唾液和唾液组分相比，蜱唾液腺中的 DG 含量明显更高（p < 0.05）。蜱唾液中的α-Gal含量高于脱糖唾液和脂质部分。这些结果支持了蜱唾液蛋白质和脂质在 AGS 中可能起的作用。

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引用次数: 0

Detection of Toxoplasma gondii in wild bivalves from the Kerguelen and Galapagos archipelagos: influence of proximity to cat populations, exposure to marine currents and kelp density 在凯尔盖朗群岛和加拉帕戈斯群岛的野生双壳贝中检测到弓形虫：与猫群的距离、暴露于海流和海藻密度的影响

IF 3.7 2区医学 Q1 PARASITOLOGY

International journal for parasitology

Pub Date : 2024-10-01 DOI: 10.1016/j.ijpara.2024.06.001

Oocysts of the protozoan Toxoplasma gondii are found in felid feces and can be washed into coastal waters, where they persist for months, attaching to algae and accumulating in invertebrates. We used wild bivalves to assess contamination of coastal waters of the Kerguelen and Galapagos archipelagos by this zoonotic parasite. Additionally, we leveraged the contrasting situations of these archipelagos to identify some potential drivers of contamination. In the Galapagos, with a cat density reaching 142 per km², 15.38% of the sampled oysters (Saccostrea palmula) tested positive for T. gondii by quantitative real-time PCR (qPCR) (n = 260), and positive samples were found in all eight sampling sites. In Kerguelen, with 1-3 cats per km², 40.83% of 120 tested mussels (Mytilus edulis platensis) were positive, and positive samples were found in four out of the five sampling sites. These findings provide evidence of T. gondii contamination in the coastal waters of these archipelagos. Furthermore, T. gondii-positive bivalves were found on islands located 20 km away (Galapagos) and 5 km away (Kerguelen) from the nearest cat population, indicating that T. gondii oocysts can disperse through waterborne mechanisms over several kilometers from their initial deposition site. In the Galapagos, where runoff is infrequent and all sites are exposed to currents, the prevalence of qPCR-positive bivalves did not show significant variations between sites (p = 0.107). In Kerguelen where runoff is frequent and site exposure variable, the prevalence varied significantly (p < 0.001). The detection of T. gondii in Kerguelen mussels was significantly correlated with the site exposure to currents (odds ratio (OR) 60.2, p < 0.001) and the on-site density of giant kelp forests (OR 2.624, p < 0.001). This suggests that bivalves can be contaminated not only by oocysts transported by currents but also by consuming marine aggregates containing oocysts that tend to form in kelp forests.

原生动物弓形虫的卵囊存在于猫科动物的粪便中，会被冲入沿岸水域，并在那里存留数月，附着在藻类上，并在无脊椎动物体内积累。我们利用野生双壳贝类来评估这种人畜共患病寄生虫对凯尔盖朗群岛和加拉帕戈斯群岛沿岸水域的污染情况。此外，我们还利用这两个群岛截然不同的情况，确定了一些潜在的污染驱动因素。在加拉帕戈斯群岛，猫的密度达到每平方公里 142 只，通过实时定量 PCR（qPCR）检测，15.38% 的采样牡蛎（Saccostrea palmula）的淋病双球菌检测呈阳性（n = 260），在所有八个采样点都发现了阳性样本。在凯尔盖朗（Kerguelen），每平方公里有 1-3 只猫，在 120 个检测的贻贝（Mytilus edulis platensis）中，有 40.83% 呈阳性，在五个采样点中的四个采样点都发现了阳性样本。这些发现提供了这些群岛沿海水域受到淋病双球菌污染的证据。此外，在距离最近的猫群 20 千米（加拉帕戈斯群岛）和 5 千米（凯尔盖朗岛）以外的岛屿上也发现了 T. gondii 阳性双壳贝，这表明 T. gondii 卵囊可以通过水传播机制从最初的沉积地点扩散到几千米以外的地方。在加拉帕戈斯群岛，径流并不频繁，而且所有地点都暴露在水流中，qPCR 阳性双壳贝的流行率在不同地点之间并无显著差异（p = 0.107）。在凯尔盖朗岛，由于径流频繁，各地点的水流情况各不相同，因此双壳类动物的发病率差异很大（p = 0.001）。在凯尔盖朗贻贝中检测到的淋病双球菌与现场暴露于水流的情况（几率比（OR）60.2，p <0.001）和现场巨型海藻林的密度（OR 2.624，p <0.001）有明显的相关性。这表明，双壳类动物不仅会受到由水流传播的卵囊的污染，也会因食用海藻林中往往会形成的含有卵囊的海洋聚集体而受到污染。

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引用次数: 0