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Divergence and convergence in epiphytic and endophytic phyllosphere bacterial communities of rice landraces. 水稻陆稻附生和内生叶球细菌群落的分化与趋同。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/msphere.00765-24
Pratibha Sanjenbam, Deepa Agashe
<p><p>Phyllosphere-associated microbes can significantly alter host plant fitness, with distinct functions provided by bacteria inhabiting the epiphytic (external surface) vs endophytic niches (internal leaf tissue). Hence, it is important to understand the assembly and stability of these phyllosphere communities, especially in field conditions. Broadly, epiphytic communities should encounter more environmental fluctuations and frequent immigration, whereas endophytic microbiota should face stronger host selection. As a result, we expect greater variability in epiphytic than endophytic communities. We analyzed the structure and stability of leaf phyllosphere microbiota of four traditionally cultivated rice landraces and one commercial variety from northeast India grown in the field for 3 consecutive years, supplemented with opportunistic sampling of eight other landraces. Epiphytic and endophytic bacterial communities shared dominant core genera such as <i>Methylobacterium</i> and <i>Sphingomonas</i>. Consistent with an overall strong environmental effect, both communities varied more across sampling years than across host landraces. Seeds sampled from a focal landrace did not support vertical transmission of phyllosphere bacteria, suggesting that both types of communities are assembled anew each generation. Despite these points of convergence, epiphytic communities had distinct composition and significantly higher microbial load and were more rich, diverse, modular, and unstable than endophytic communities. Finally, focused sampling of one landrace across developmental stages showed that the divergence between the two types of communities arose primarily at the flowering stage. Thus, our results show both convergent and divergent patterns of community assembly and composition in distinct phyllosphere niches in rice, identifying key bacterial genera and host developmental stages that may aid agricultural interventions to increase rice yield.IMPORTANCEPhyllosphere (leaf-associated) microbes significantly impact plant fitness, making it crucial to understand how these communities are assembled and maintained. While many studies have analyzed epiphytic (surface) phyllosphere communities, we have a relatively poor understanding of endophytic communities which colonize the very distinct niche formed inside leaf tissues. We found that across several rice landraces, both communities are largely colonized by the same core genera, indicating divergence at the species level across the two leaf niches and highlighting the need to understand the mechanisms underlying this divergence. Surprisingly, both epiphytic and endophytic communities were only weakly shaped by the host landrace, with a much greater role for environmental factors that likely vary over time. Thus, microbiome-based agricultural interventions for increasing productivity could perhaps be generalized across rice varieties but would need to account for the temporal instability of the microbiot
叶球相关微生物可显著改变寄主植物的适应性,栖息在附生(外部表面)和内生(内部叶片组织)壁龛中的细菌具有不同的功能。因此,了解这些叶球群落的组成和稳定性非常重要,尤其是在田间条件下。一般来说,附生群落会遇到更多的环境波动和频繁的移民,而内生微生物群落则会面临更强的宿主选择。因此,我们预计附生生物群落的变异性要大于内生生物群落。我们分析了印度东北部连续 3 年在田间种植的 4 个传统栽培水稻陆稻品种和 1 个商业品种的叶肉微生物群的结构和稳定性,并对其他 8 个陆稻品种进行了机会性采样。附生细菌群落和内生细菌群落共享优势核心菌属,如 Methylobacterium 和 Sphingomonas。这两种群落在不同采样年份的差异比不同寄主品种的差异更大,这与整体环境的强烈影响是一致的。从一个重点陆生品系采样的种子不支持叶球细菌的垂直传播,这表明这两种群落每一代都是重新组合的。尽管存在这些趋同点,但附生群落具有独特的组成,微生物负载量明显高于内生群落,并且更加丰富、多样、模块化和不稳定。最后,对一个陆生品种不同发育阶段的集中取样表明,两种群落之间的差异主要出现在开花阶段。因此,我们的研究结果显示了水稻不同叶球壁龛中群落组装和组成的趋同和分化模式,确定了关键的细菌属和宿主发育阶段,这可能有助于农业干预措施以提高水稻产量。虽然许多研究分析了附生(表面)叶球群落,但我们对定植于叶片组织内部形成的非常独特的生态位的内生群落的了解相对较少。我们发现,在几个水稻品种中,这两种群落主要由相同的核心种属定殖,这表明两种叶片壁龛在物种水平上存在差异,并强调了了解这种差异背后机制的必要性。令人惊讶的是,附生和内生生物群落受宿主品种的影响都很微弱,环境因素的作用要大得多,而环境因素很可能随时间而变化。因此,基于微生物群的农业干预措施或许可以在不同水稻品种间推广,但需要考虑微生物群的时间不稳定性。因此,我们的研究结果凸显了像我们这样的数据集--跨品种、跨年份的广泛采样--对于了解叶球微生物群及其在田间应用的重要性。
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引用次数: 0
Immunomodulatory activity of omadacycline in vitro and in a murine model of acute lung injury. 奥马他环素在体外和小鼠急性肺损伤模型中的免疫调节活性。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/msphere.00671-24
Madeline Sanders, Paul Beringer
<p><p>Cystic fibrosis (CF) is characterized by chronic airway obstruction, infection, and inflammation leading to progressive loss of lung function and eventual respiratory failure. Omadacycline, a tetracycline antibiotic, demonstrates <i>in vitro</i> activity against key CF pathogens, substantial lung penetration, and increasing clinical evidence for the treatment of lung infections in people with CF (PwCF). Preliminary <i>in vitro</i> data demonstrate that omadacycline exhibits anti-inflammatory activity. This study aims to determine the anti-inflammatory effects of omadacycline <i>in vitro</i> and in a murine model of lipopolysaccharide (LPS)-induced lung neutrophilia. <i>In vitro</i>, THP-1-derived macrophages were treated with omadacycline (20-100 µg/mL) 30 minutes prior to LPS stimulation. Pro-inflammatory cytokine (TNF-α, IL-1β/6), chemokine (CXCL-1/2), and MMP-9 levels were analyzed after 24 hours by ELISA. Omadacycline's effects on IL-8-induced human neutrophil chemotaxis were also investigated. <i>In vivo</i>, omadacycline (2.5-30 mg/kg), comparators dexamethasone (1 mg/kg), and azithromycin (30 mg/kg) were administered 1 hour before and 6 hours after intranasal LPS challenge, respectively. Leukocyte counts and differentials in bronchoalveolar lavage fluid (BALF), inflammatory mediator levels in BALF and lung homogenates, pulmonary edema markers, and the severity of lung injury were evaluated 24 hours or 48 hours post-challenge. Treatment with omadacycline <i>in vitro</i> resulted in significant, dose-dependent reductions in IL-6, CXCL-1, and MMP-9 expression and inhibition of IL-8-induced neutrophil chemotaxis. <i>In vivo</i>, omadacycline yielded protective and therapeutic effects by reducing the production of proinflammatory cytokines and chemokines and neutrophil infiltration into the lungs, along with modestly improving lung injury severity. These preclinical results suggest that omadacycline may provide dual anti-bacterial and anti-inflammatory activities relevant to chronic lung infection treatment in PwCF.IMPORTANCENontuberculous mycobacteria, particularly <i>Mycobacterium abscessus</i> complex (MABSC), are a major concern for people with cystic fibrosis (PwCF) due to their association with deteriorating lung function. A substantial barrier to effective treatment is the limited number of safe and effective antibiotics. Omadacycline offers a potential advancement in managing MABSC infections in cystic fibrosis due to its activity, effective penetration into pulmonary secretions, improved tolerability, and good oral bioavailability as shown in healthy volunteers. Our study is the first to explore omadacycline's effects in a model of sterile lung inflammation and acute lung injury. We found that omadacycline not only has potent anti-bacterial properties but also exhibits anti-inflammatory effects, reducing lung inflammation and injury in our preclinical models. These findings underscore omadacycline's potential as a dual-action the
囊性纤维化(CF)的特点是慢性气道阻塞、感染和炎症,导致肺功能逐渐丧失,最终出现呼吸衰竭。奥马他环素是一种四环素类抗生素,在体外对 CF 的主要病原体具有活性,具有很强的肺穿透性,而且用于治疗 CF 患者(PwCF)肺部感染的临床证据越来越多。初步体外数据显示,奥马他环素具有抗炎活性。本研究旨在确定奥马他环素在体外和脂多糖(LPS)诱导的肺中性粒细胞增多小鼠模型中的抗炎作用。在体外,源于 THP-1 的巨噬细胞在 LPS 刺激前 30 分钟接受奥马大环素(20-100 µg/mL)处理。24 小时后,用酶联免疫吸附法分析促炎细胞因子(TNF-α、IL-1β/6)、趋化因子(CXCL-1/2)和 MMP-9 的水平。此外,还研究了奥马他环素对 IL-8 诱导的人中性粒细胞趋化的影响。在体内,奥马大环素(2.5-30 毫克/千克)、地塞米松(1 毫克/千克)和阿奇霉素(30 毫克/千克)分别在鼻内 LPS 挑战前 1 小时和挑战后 6 小时给药。在挑战后 24 小时或 48 小时评估支气管肺泡灌洗液(BALF)中的白细胞计数和差异、BALF 和肺匀浆中的炎症介质水平、肺水肿指标以及肺损伤的严重程度。体外使用奥马大环素可显著降低IL-6、CXCL-1和MMP-9的表达,并抑制IL-8诱导的中性粒细胞趋化。在体内,奥马他环素可减少促炎细胞因子和趋化因子的产生以及中性粒细胞向肺部的浸润,并适度改善肺损伤的严重程度,从而产生保护和治疗作用。重要意义结核分枝杆菌,尤其是脓肿分枝杆菌复合体(MABSC)与肺功能恶化有关,是囊性纤维化患者(PwCF)最关心的问题。有效治疗的一大障碍是安全有效的抗生素数量有限。奥马他环素的活性、对肺分泌物的有效渗透、更好的耐受性以及在健康志愿者中良好的口服生物利用度,为治疗囊性纤维化患者的 MABSC 感染提供了潜在的进展。我们的研究首次探讨了奥马他环素在无菌肺部炎症和急性肺损伤模型中的作用。我们发现,奥马他环素不仅具有强大的抗菌特性,而且还具有抗炎作用,能减轻临床前模型中的肺部炎症和损伤。这些发现凸显了奥马大环素作为治疗普氏肺癌肺部感染的双效疗法的潜力,表明它在改善患者预后和指导更有效的抗菌治疗决策方面具有巨大潜力。
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引用次数: 0
Circulating extracellular vesicles from severe COVID-19 patients induce lung inflammation. 来自严重 COVID-19 患者的循环细胞外囊泡会诱发肺部炎症。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/msphere.00764-24
Huifeng Qian, Ruoxi Zang, Ruoyang Zhang, Guoping Zheng, Guanguan Qiu, Jianbiao Meng, Jiangmei Wang, Jie Xia, Ruoqiong Huang, Zhenkai Le, Qiang Shu, Jianguo Xu

Circulating extracellular vesicles (EVs) have been associated with the development of COVID-19 due to their roles in viral infection, inflammatory response, and thrombosis. However, the direct induction of lung inflammation by circulating EVs from severe COVID-19 patients remains unknown. EVs were extracted from the plasma of severe COVID-19 patients admitted to intensive care and healthy controls. To study the effect of COVID-19 EVs on lung inflammation, mice were intratracheally instilled with EVs. To examine the proinflammatory effects of EVs in vitro, bone marrow-derived macrophages were treated with EVs. COVID-19 but not control EVs triggered lung inflammation, as assessed by total protein level, total cell count, neutrophil count, and levels of proinflammatory cytokines in the bronchoalveolar lavage. COVID-19 EVs also promoted M1 polarization of alveolar macrophages in vivo. Treatment of bone marrow-derived macrophages with COVID-19 EVs enhanced the M1 phenotype and augmented the production of IL-1β, IL-6, and TNF-α. In summary, circulating EVs from severe COVID-19 patients induce lung inflammation in mice. EVs could become a potential therapeutic target for alleviating lung injury in COVID-19.

Importance: Extracellular vesicles (EVs) have been reported to facilitate cytokine storm, coagulation, vascular dysfunction, and the spread of the virus in COVID-19. The direct role of circulating EVs from severe COVID-19 patients in lung injury remains unrecognized. Our study demonstrated that plasma EVs obtained from severe COVID-19 patients induced lung inflammation and polarization of alveolar macrophages in vivo. In vitro experiments also revealed the proinflammatory effects of COVID-19 EVs. The present study sheds fresh insight into the mechanisms of COVID-19-induced lung injury, highlighting EVs as a potential therapeutic target in combating the disease.

由于循环细胞外囊泡(EVs)在病毒感染、炎症反应和血栓形成中的作用,它们与 COVID-19 的发病有关。然而,严重COVID-19患者的循环EVs对肺部炎症的直接诱导作用仍然未知。研究人员从接受重症监护的重症 COVID-19 患者和健康对照组的血浆中提取了 EVs。为了研究 COVID-19 EVs 对肺部炎症的影响,给小鼠气管内灌注了 EVs。为了研究 EVs 在体外的促炎作用,用 EVs 处理骨髓衍生的巨噬细胞。根据支气管肺泡灌洗液中的总蛋白水平、总细胞数、中性粒细胞数和促炎细胞因子水平评估,COVID-19 而非对照组 EVs 会引发肺部炎症。COVID-19 EVs 还能促进体内肺泡巨噬细胞的 M1 极化。用 COVID-19 EVs 处理骨髓衍生巨噬细胞可增强 M1 表型,并增加 IL-1β、IL-6 和 TNF-α 的产生。总之,来自严重 COVID-19 患者的循环 EV 可诱导小鼠肺部炎症。EVs可能成为缓解COVID-19肺损伤的潜在治疗靶点:据报道,细胞外囊泡(EVs)可促进细胞因子风暴、凝血、血管功能障碍以及 COVID-19 病毒的传播。重症 COVID-19 患者的循环 EVs 在肺损伤中的直接作用仍未得到确认。我们的研究表明,从重症 COVID-19 患者体内获得的血浆 EV 可诱导肺部炎症和肺泡巨噬细胞的极化。体外实验也揭示了 COVID-19 EVs 的促炎作用。本研究揭示了COVID-19诱导肺损伤的新机制,强调了EVs是抗击该疾病的潜在治疗靶点。
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引用次数: 0
Kinase function of TgTKL1 is essential for its role in Toxoplasma propagation and pathogenesis. TgTKL1 的激酶功能对其在弓形虫繁殖和致病过程中的作用至关重要。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/msphere.00779-24
Dima Hajj Ali, Ramu Anandakrishnan, Vern B Carruthers, Rajshekhar Y Gaji

The Tyrosine Kinase-Like (TKL) family of proteins are a set of poorly studied kinases that have garnered attention in recent years for their role in Toxoplasma biology. The Toxoplasma genome contains eight TKL kinases, of which six have been predicted to be important for parasite propagation. We have previously shown that TgTKL1 is a nuclear kinase that is critical for the parasite lytic cycle and is essential for acute virulence in the animal model. However, the contribution of the kinase domain to the functioning of TgTKL1 was not known. Hence to determine the significance of its catalytic function, we first validated that TgTKL1 is a true kinase using purified recombinant protein. Furthermore, we successfully generated a TgTKL1 kinase mutant strain of Toxoplasma via CRISPR-Cas9 gene editing. Our studies revealed that the kinase mutant of TgTKL1 displays defects in parasite growth and host-cell invasion. Additionally, loss of kinase function alters the transcriptomic profile of the parasite, including downregulation of the invasion-related gene, TgSUB1. Importantly, this dysregulation of TgSUB1 expression leads to defects in post-exocytosis processing of micronemal proteins, an event critical for normal host-cell invasion. Furthermore, the TgTKL1 kinase mutant is completely avirulent in the mouse model of acute toxoplasmosis. Since the loss of kinase function leads to phenotypic manifestations seen previously with TgTKL1 knockout parasites, we conclude that kinase activity is important for TgTKL1 function in Toxoplasma propagation and virulence.

Importance: Toxoplasma gondii is a protozoan parasite that can cause life-threatening disease in humans. Hence, identifying key factors required for parasite growth and pathogenesis is important to develop novel therapeutics. We have previously shown that a member of the TKL protein kinase family, TgTKL1, is a plant-like kinase that is required for effective Toxoplasma growth in vitro and essential for virulence in vivo. Herein, we show that the TgTKL1 is, indeed, a bona fide kinase, and loss of its kinase function in the Toxoplasma leads to similar defects seen in parasites with complete loss of TgTKL1. More specifically, the TgTKL1 kinase mutant exhibits defects in parasite growth, host-cell invasion, gene expression profile, and virulence in the animal model. Together, these findings suggest that TgTKL1 is a true kinase, and loss of its kinase activity leads to disruption of TgTKL1 function in Toxoplasma.

酪氨酸激酶样蛋白(TKL)家族是一组研究较少的激酶,近年来因其在弓形虫生物学中的作用而备受关注。弓形虫基因组包含 8 个 TKL 激酶,其中 6 个被认为对寄生虫的繁殖非常重要。我们之前已经证明,TgTKL1 是一种核激酶,对寄生虫的溶解循环至关重要,而且在动物模型中对急性毒力也至关重要。然而,激酶结构域对 TgTKL1 功能的贡献尚不清楚。因此,为了确定其催化功能的重要性,我们首先利用纯化的重组蛋白验证了 TgTKL1 是一个真正的激酶。此外,我们还通过CRISPR-Cas9基因编辑技术成功生成了弓形虫TgTKL1激酶突变株。我们的研究发现,TgTKL1激酶突变体在寄生虫生长和宿主细胞侵袭方面表现出缺陷。此外,激酶功能的丧失改变了寄生虫的转录组特征,包括侵袭相关基因 TgSUB1 的下调。重要的是,TgSUB1 表达的这种失调会导致微胚层蛋白外渗后处理的缺陷,而微胚层蛋白外渗后处理对于宿主细胞的正常侵袭至关重要。此外,TgTKL1 激酶突变体在急性弓形虫病小鼠模型中完全无毒。由于激酶功能的缺失会导致 TgTKL1 基因敲除寄生虫的表型表现,因此我们得出结论,激酶活性对 TgTKL1 在弓形虫繁殖和毒力方面的功能非常重要:重要意义:弓形虫是一种原生动物寄生虫,可对人类造成危及生命的疾病。因此,确定寄生虫生长和致病所需的关键因素对于开发新型疗法非常重要。我们之前已经证明,TKL 蛋白激酶家族的一个成员 TgTKL1 是一种植物样激酶,它是弓形虫体外有效生长所必需的,也是体内毒力所必需的。在本文中,我们发现 TgTKL1 确实是一种真正的激酶,弓形虫体内激酶功能的缺失会导致完全缺失 TgTKL1 的寄生虫出现类似的缺陷。更具体地说,TgTKL1 激酶突变体在动物模型中表现出寄生虫生长、宿主细胞侵袭、基因表达谱和毒力方面的缺陷。这些发现共同表明,TgTKL1 是一种真正的激酶,其激酶活性的缺失会导致弓形虫体内 TgTKL1 功能的破坏。
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引用次数: 0
Effects of global change drivers on the expression of pathogenicity and stress genes in dryland soil fungi. 全球变化驱动因素对旱地土壤真菌致病基因和应激基因表达的影响。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/msphere.00658-24
Adriana L Romero-Olivares, Andrea Lopez, Jovani Catalan-Dibene, Scott Ferrenberg, Samuel E Jordan, Brooke Osborne

The impacts of global climate change on dryland fungi have been understudied even though fungi are extremely sensitive to changes in the environment. Considering that many fungi are pathogens of plants and animals, including humans, their responses to anthropogenic change could have important implications for public health and food security. In this study, we investigated the potential physiological responses (i.e., metatranscriptomics) of pathogenicity and stress in dryland fungi exposed to global change drivers, drought, and the physical disturbance associated with land use. Specifically, we wanted to assess if there was an increase in the transcription of genes associated to pathogenicity and stress in response to global change drivers. In addition, we wanted to investigate which pathogenicity and stress genes were consistently differentially expressed under the different global change conditions across the heterogeneous landscape (i.e., microsite) of the Chihuahuan desert. We observed increased transcription of pathogenicity and stress genes, with specific genes being most upregulated in response to global change drivers. Additionally, climatic conditions linked to different microsites, such as those found under patches of vegetation, may play a significant role. We provide evidence supporting the idea that environmental stress caused by global change could contribute to an increase of pathogenicity as global climate changes. Specifically, increases in the transcription of stress and virulence genes, coupled with variations in gene expression, could lead to the onset of pathogenicity. Our work underscores the importance of studying dryland fungi exposed to global climate change and increases in existing fungal pathogens, as well as the emergence of new fungal pathogens, and consequences to public health and food security.

Importance: The effects of global climate change on dryland fungi and consequences to our society have been understudied despite evidence showing that pathogenic fungi increase in abundance under global climate change. Moreover, there is a growing concern that global climate change will contribute to the emergence of new fungal pathogens. Yet, we do not understand what mechanisms might be driving this increase in virulence and the onset of pathogenicity. In this study, we investigate how fungi respond to global change drivers, physical disturbance, and drought, in a dryland ecosystem in terms of pathogenicity and stress. We find that indeed, under global change drivers, there is an increase in the transcription and expression of genes associated to pathogenicity and stress, but that microclimatic conditions matter. Our study shows the importance of investigating dryland fungi exposed to global climate change and impacts on our society, which may include threats to public health and food security.

尽管真菌对环境变化极为敏感,但全球气候变化对旱地真菌影响的研究一直不足。考虑到许多真菌是植物和动物(包括人类)的病原体,它们对人为变化的反应可能会对公共卫生和食品安全产生重要影响。在这项研究中,我们调查了旱地真菌在受到全球变化驱动因素、干旱以及与土地利用相关的物理干扰时,对致病性和压力的潜在生理反应(即元转录组学)。具体来说,我们想评估致病性和应激相关基因的转录是否会因全球变化驱动因素而增加。此外,我们还想研究在奇瓦瓦沙漠不同地貌(即微地貌)的不同全球变化条件下,哪些致病性基因和应激性基因的表达一直存在差异。我们观察到致病基因和应激基因的转录增加,其中特定基因在全球变化驱动因素下的上调幅度最大。此外,与不同微生境相关的气候条件,如植被斑块下的气候条件,也可能起到重要作用。我们提供的证据支持了这样一种观点,即随着全球气候变化,全球变化引起的环境压力可能会导致致病性增加。具体来说,应激基因和毒力基因转录的增加,再加上基因表达的变化,可能会导致致病性的出现。我们的工作强调了研究旱地真菌面临全球气候变化、现有真菌病原体增加、新真菌病原体出现以及对公共卫生和粮食安全的影响的重要性:全球气候变化对旱地真菌的影响及其对我们社会的后果的研究一直不足,尽管有证据表明病原真菌在全球气候变化的情况下数量会增加。此外,人们越来越担心全球气候变化会导致新的真菌病原体的出现。然而,我们并不了解是什么机制在驱动这种毒力的增强和致病性的出现。在这项研究中,我们调查了真菌如何在干旱地区生态系统中对全球变化驱动因素、物理干扰和干旱做出致病性和应激反应。我们发现,在全球变化驱动因素的作用下,与致病性和应激有关的基因转录和表达确实增加了,但小气候条件也很重要。我们的研究表明,研究受全球气候变化影响的旱地真菌及其对社会的影响(可能包括对公共卫生和食品安全的威胁)非常重要。
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引用次数: 0
Unraveling the role of cAMP signaling in Giardia: insights into PKA-mediated regulation of encystation and subcellular interactions. 揭示 cAMP 信号在贾第鞭毛虫中的作用:深入了解 PKA 介导的胞吐调控和亚细胞相互作用。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/msphere.00723-24
Han-Wei Shih, Germain C M Alas, Alexander R Paredez

cAMP plays an important role as a second messenger in the stage transition of various protozoan parasites. This signaling pathway relies on multiple effectors, such as protein kinase A (PKA), exchange protein activated by cAMP, and cAMP-response element binding protein transcription factors, to initiate signal transduction in humans. The Giardia genome only contains two adenylate cyclases (ACs), a single phosphodiesterase (PDE) and a single known PKA effector, and the specific functions of these components are not fully understood. In our previous research, we demonstrated the important role of AC2-dependent cAMP signaling in promoting the encystation program. Using the NanoBit technology, we emphasized the significance of AC2-dependent cAMP biosynthesis in regulating the dissociation of the PKA regulatory domain (PKAr) and PKA catalytic domain (PKAc). In this study, our objectives are twofold: first, we used the newly developed Split-Halo to examine subcellular interactions of GlPKAr and GlPKAc in Giardia; and second, we investigated whether PKAc regulates encystation-specific proteins. Our findings revealed distinct subcellular locations where GlPKAr and GlPKAc interacted during the trophozoite stage, including the flagella, basal bodies, and cytoplasm. Upon exposure to encystation stimuli, the interaction shifted from the flagella to the cytosol. Knockdown of GlPKAc resulted in the downregulation of encystation-specific genes, leading to the production of fewer viable and water-resistant cysts indicating a role for PKA in the transcriptional regulation of encystation. These discoveries contribute to a deeper understanding of the cAMP signaling pathway and its important role in governing Giardia's encystation process.

Importance: The precise timing of interactions and subcellular compartmentation play crucial roles in signal transduction. The co-immunoprecipitation assay (CO-IP) has long been utilized to validate protein-protein interactions; however, CO-IPs lack spatial and temporal resolutions. Our recent study used the NanoBit assay, which showcased the reversible protein-protein interaction between PKAr and PKAc in response to cAMP analogs and encystation stimuli. Expanding on this groundwork, this study employs the Split-Halo assay to uncover the subcellular compartments where the PKAr and PKAc protein-protein interactions take place and respond to encystation stimuli. Taken together, these molecular tools provide spatiotemporal information on the protein-protein interaction, which will be useful in the field.

cAMP 作为第二信使在各种原生动物寄生虫的阶段转换中发挥着重要作用。这一信号通路依赖多种效应器,如蛋白激酶 A(PKA)、cAMP 激活的交换蛋白和 cAMP 反应元件结合蛋白转录因子,来启动人体的信号转导。贾第虫基因组只包含两个腺苷酸环化酶(AC)、一个磷酸二酯酶(PDE)和一个已知的 PKA 效应器,这些成分的具体功能还不完全清楚。在我们之前的研究中,我们证明了依赖 AC2 的 cAMP 信号在促进烯化程序中的重要作用。利用 NanoBit 技术,我们强调了 AC2 依赖性 cAMP 生物合成在调节 PKA 调节域(PKAr)和 PKA 催化域(PKAc)解离中的重要作用。在本研究中,我们的目标有两个:首先,我们使用新开发的 Split-Halo 来检测贾第虫中 GlPKAr 和 GlPKAc 的亚细胞相互作用;其次,我们研究了 PKAc 是否调控细胞分裂特异性蛋白。我们的研究结果表明,在滋养体阶段,GlPKAr和GlPKAc在不同的亚细胞位置发生相互作用,包括鞭毛、基底体和胞质。在受到包囊刺激后,相互作用从鞭毛转移到了细胞质。敲除 GlPKAc 会导致胞囊变性特异基因的下调,从而导致产生更少的可存活的耐水囊蚴,这表明 PKA 在胞囊变性的转录调控中发挥作用。这些发现有助于人们更深入地了解 cAMP 信号通路及其在控制贾第虫包囊化过程中的重要作用:相互作用的精确时间和亚细胞区隔在信号转导中起着至关重要的作用。长期以来,共沉淀试验(CO-IP)一直被用来验证蛋白质与蛋白质之间的相互作用;然而,CO-IP 缺乏空间和时间分辨率。我们最近的研究使用了 NanoBit 分析法,该方法展示了 PKAr 和 PKAc 在 cAMP 类似物和瞬时刺激下的可逆蛋白-蛋白相互作用。在此基础上,本研究采用 Split-Halo 分析法揭示了发生 PKAr 和 PKAc 蛋白-蛋白相互作用的亚细胞区室,并揭示了它们对电位刺激的反应。总之,这些分子工具提供了蛋白质-蛋白质相互作用的时空信息,将在该领域大有用武之地。
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引用次数: 0
Exploring the tolerable region for HiBiT tag insertion in the hepatitis B virus genome. 探索乙型肝炎病毒基因组中 HiBiT 标记插入的可容忍区域。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-29 Epub Date: 2024-09-30 DOI: 10.1128/msphere.00518-24
Asako Murayama, Hitomi Igarashi, Norie Yamada, Hussein Hassan Aly, Masaaki Toyama, Masanori Isogawa, Tetsuro Shimakami, Takanobu Kato

A cell culture system that allows the reproduction of the hepatitis B virus (HBV) life cycle is indispensable to exploring novel anti-HBV agents. To establish the screening system for anti-HBV agents, we exploited the high affinity and bright luminescence (HiBiT) tag and comprehensively explored the regions in the HBV genome where the HiBiT tag could be inserted. The plasmids for the HiBiT-tagged HBV molecular clones with a 1.38-fold HBV genome length were prepared. The HiBiT tag was inserted into five regions: preS1, preS2, hepatitis B e (HBe), hepatitis B X (HBx), and hepatitis B polymerase (HB pol). HiBiT-tagged HBVs were obtained by transfecting the prepared plasmids into sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells, and their infectivity was evaluated in human primary hepatocytes and HepG2/NTCP cells. Among the evaluated viruses, the infection of HiBiT-tagged HBVs in the preS1 or the HB pol regions exhibited a time-dependent increase of the hepatitis B surface antigen (HBsAg) level after infection to HepG2/NTCP cells as well as human primary hepatocytes. Immunostaining of the hepatitis B core (HBc) antigen in infected cells confirmed these viruses are infectious to those cells. However, the time-dependent increase of the HiBiT signal was only detected after infection with the HiBiT-tagged HBV in the preS1 region. The inhibition of this HiBiT-tagged HBV infection in human primary hepatocytes and HepG2/NTCP cells by the preS1 peptide could be detected by measuring the HiBiT signal. The infection system with the HiBiT-tagged HBV in HepG2/NTCP cells facilitates easy, sensitive, and high-throughput screening of anti-HBV agents and will be a useful tool for assessing the viral life cycle and exploring antiviral agents.

Importance: Hepatitis B virus (HBV) is the principal causative agent of chronic hepatitis. Despite the availability of vaccines in many countries, HBV infection has spread worldwide and caused chronic infection. In chronic hepatitis B patients, liver inflammation leads to cirrhosis, and the accumulation of viral genome integration into host chromosomes leads to the development of hepatocellular carcinoma. The currently available treatment strategy cannot expect the eradication of HBV. To explore novel anti-HBV agents, a cell culture system that can detect HBV infection easily is indispensable. In this study, we examined the regions in the HBV genome where the high affinity and bright luminescence (HiBiT) tag could be inserted and established an HBV infection system to monitor infection by measuring the HiBiT signal by infecting the HiBiT-tagged HBV in sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. This system can contribute to screening for novel anti-HBV agents.

要探索新型抗乙型肝炎病毒(HBV)制剂,就必须建立一个能够复制乙型肝炎病毒(HBV)生命周期的细胞培养系统。为了建立抗 HBV 药物的筛选系统,我们利用了高亲和力和明亮发光(HiBiT)标签,并全面探索了 HBV 基因组中可插入 HiBiT 标签的区域。我们制备了 HBV 基因组长度为 1.38 倍的 HiBiT 标记 HBV 分子克隆质粒。HiBiT 标记被插入五个区域:preS1、preS2、乙肝 e(HBe)、乙肝 X(HBx)和乙肝聚合酶(HB pol)。通过将制备好的质粒转染到牛磺胆酸钠共转运多肽转导的 HepG2(HepG2/NTCP)细胞中,获得了 HiBiT 标记的 HBV,并在人类原代肝细胞和 HepG2/NTCP 细胞中评估了它们的感染性。在评估的病毒中,前S1区或HB pol区的HiBiT标记的HBV感染HepG2/NTCP细胞和人类原代肝细胞后,乙肝表面抗原(HBsAg)水平的升高与时间有关。感染细胞中乙肝核心抗原(HBc)的免疫染色证实了这些病毒对这些细胞具有传染性。然而,只有在感染前 S1 区的 HiBiT 标记 HBV 后,才能检测到 HiBiT 信号随时间的增加。通过测量 HiBiT 信号,可以检测到 preS1 肽对人类原代肝细胞和 HepG2/NTCP 细胞中 HiBiT 标记 HBV 感染的抑制作用。HiBiT标记的HBV在HepG2/NTCP细胞中的感染系统有助于简便、灵敏和高通量地筛选抗HBV药物,并将成为评估病毒生命周期和探索抗病毒药物的有用工具:乙型肝炎病毒(HBV)是慢性肝炎的主要病原体。尽管许多国家都有疫苗,但乙型肝炎病毒感染已在全球蔓延并造成慢性感染。慢性乙型肝炎患者的肝脏炎症会导致肝硬化,病毒基因组整合到宿主染色体上的积累会导致肝细胞癌的发生。目前现有的治疗策略无法指望根除 HBV。要探索新型抗 HBV 药物,就必须建立一个能轻松检测 HBV 感染的细胞培养系统。在这项研究中,我们研究了 HBV 基因组中可插入高亲和力和明亮发光(HiBiT)标签的区域,并建立了一个 HBV 感染系统,通过在牛磺胆酸钠共转运多肽转导的 HepG2(HepG2/NTCP)细胞中感染 HiBiT 标签的 HBV,测量 HiBiT 信号来监测感染情况。该系统有助于筛选新型抗 HBV 药物。
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引用次数: 0
Adhesion of Crithidia fasciculata promotes a rapid change in developmental fate driven by cAMP signaling. 束带栉水母的粘附促进了由 cAMP 信号驱动的发育命运的快速改变。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-29 Epub Date: 2024-09-24 DOI: 10.1128/msphere.00617-24
Shane Denecke, Madeline F Malfara, Kelly R Hodges, Nikki A Holmes, Andre R Williams, Julia H Gallagher-Teske, Julia M Pascarella, Abigail M Daniels, Geert Jan Sterk, Rob Leurs, Gordon Ruthel, Rachel Hoang, Megan L Povelones, Michael Povelones

Trypanosomatids are single-celled parasites responsible for human and animal disease. Typically, colonization of an insect host is required for transmission. Stable attachment of parasites to insect tissues via their single flagellum coincides with differentiation and morphological changes. Although attachment is a conserved stage in trypanosomatid life cycles, the molecular mechanisms are not well understood. To study this process, we elaborate upon an in vitro model in which the swimming form of the trypanosomatid Crithidia fasciculata rapidly differentiates following adhesion to artificial substrates. Live imaging of cells transitioning from swimming to attached shows parasites undergoing a defined sequence of events, including an initial adhesion near the base of the flagellum immediately followed by flagellar shortening, cell rounding, and the formation of a hemidesmosome-like attachment plaque between the tip of the shortened flagellum and the substrate. Quantitative proteomics of swimming versus attached parasites suggests differential regulation of cyclic adenosine monophosphate (cAMP)-based signaling proteins. We have localized two of these proteins to the flagellum of swimming C. fasciculata; however, both are absent from the shortened flagellum of attached cells. Pharmacological inhibition of cAMP phosphodiesterases increased cAMP levels in the cell and prevented attachment. Further, treatment with inhibitor did not affect the growth rate of either swimming or established attached cells, indicating that its effect is limited to a critical window during the early stages of adhesion. These data suggest that cAMP signaling is required for attachment of C. fasciculata and that flagellar signaling domains may be reorganized during differentiation and attachment.IMPORTANCETrypanosomatid parasites cause significant disease burden worldwide and require insect vectors for transmission. In the insect, parasites attach to tissues, sometimes dividing as attached cells or producing motile, infectious forms. The significance and cellular mechanisms of attachment are relatively unexplored. Here, we exploit a model trypanosomatid that attaches robustly to artificial surfaces to better understand this process. This attachment recapitulates that observed in vivo and can be used to define the stages and morphological features of attachment as well as conditions that impact attachment efficiency. We have identified proteins that are enriched in either swimming or attached parasites, supporting a role for the cyclic AMP signaling pathway in the transition from swimming to attached. As this pathway has already been implicated in environmental sensing and developmental transitions in trypanosomatids, our data provide new insights into activities required for parasite survival in their insect hosts.

锥虫是导致人类和动物疾病的单细胞寄生虫。通常情况下,昆虫宿主的定殖是传播的必要条件。寄生虫通过单鞭毛稳定地附着在昆虫组织上,同时发生分化和形态变化。虽然附着是锥虫生命周期中的一个保守阶段,但其分子机制还不十分清楚。为了研究这一过程,我们详细阐述了一个体外模型,在该模型中,锥虫Crithidia fasciculata的游动形态在粘附到人工基底后迅速分化。对从游动型过渡到附着型细胞的实时成像显示,寄生虫经历了一系列确定的事件,包括鞭毛基部附近的初始附着,紧接着是鞭毛缩短、细胞变圆,以及在缩短的鞭毛顶端和基质之间形成类似半膜的附着斑。游动寄生虫与附着寄生虫的定量蛋白质组学研究表明,以环磷酸腺苷(cAMP)为基础的信号蛋白的调控存在差异。我们在游动的 C. fasciculata 的鞭毛上定位到了其中两种蛋白,但在附着细胞的缩短鞭毛上却发现这两种蛋白都不存在。药物抑制 cAMP 磷酸二酯酶可增加细胞中的 cAMP 水平并阻止附着。此外,用抑制剂处理不会影响游动细胞或已附着细胞的生长速度,这表明抑制剂的作用仅限于附着早期的一个关键窗口。这些数据表明,C. fasciculata 的附着需要 cAMP 信号传导,在分化和附着过程中,鞭毛信号传导结构域可能会发生重组。在昆虫体内,寄生虫附着在组织上,有时作为附着细胞分裂,或产生运动性、传染性形式。对附着的意义和细胞机制的研究相对较少。在这里,我们利用一种能牢固附着在人造表面上的锥虫模型来更好地了解这一过程。这种附着再现了在体内观察到的情况,可用于确定附着的阶段和形态特征以及影响附着效率的条件。我们发现了在游动或附着的寄生虫中都富集的蛋白质,支持环磷酸腺苷信号通路在从游动到附着的过渡中发挥作用。由于这一途径已经与锥虫的环境感知和发育过渡有关,我们的数据为寄生虫在昆虫宿主体内生存所需的活动提供了新的见解。
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引用次数: 0
Pseudomonas aeruginosa kills Staphylococcus aureus in a polyphosphate-dependent manner. 铜绿假单胞菌以多磷酸盐依赖的方式杀死金黄色葡萄球菌。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-29 Epub Date: 2024-10-04 DOI: 10.1128/msphere.00686-24
Ritika Shah, Julius Kwesi Narh, Magdalena Urlaub, Olivia Jankiewicz, Colton Johnson, Barry Livingston, Jan-Ulrik Dahl

Due to their frequent coexistence in many polymicrobial infections, including in patients with cystic fibrosis or burn/chronic wounds, many studies have investigated the mechanistic details of the interaction between the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus. P. aeruginosa rapidly outcompetes S. aureus under in vitro cocultivation conditions, which is mediated by several of P. aeruginosa's virulence factors. Here, we report that polyphosphate (polyP), an efficient stress defense system and virulence factor in P. aeruginosa, plays a role in the pathogen's ability to inhibit and kill S. aureus in a contact-independent manner. We show that P. aeruginosa cells characterized by low polyP levels are less detrimental to S. aureus growth and survival while the Gram-positive pathogen is significantly more compromised by the presence of P. aeruginosa cells that produce high levels of polyP. The polyP-dependent phenotype of P. aeruginosa-mediated killing of S. aureus could at least in part be direct, as polyP was detected in the spent media and causes significant damage to the S. aureus cell envelope. However, more likely is that polyP's effects are indirect through modulating the production of one of P. aeruginosa's virulence factors, pyocyanin. We show that pyocyanin production in P. aeruginosa occurs polyP-dependently and harms S. aureus through membrane damage and potentially the generation of reactive oxygen species, resulting in the increased expression of antioxidant enzymes. In summary, our study adds a new component to the list of biomolecules that the Gram-negative pathogen P. aeruginosa generates to compete with S. aureus for resources.IMPORTANCEHow do interactions between microorganisms shape the course of polymicrobial infections? Previous studies have provided evidence that the two opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus generate molecules that modulate their interaction with potentially significant impact on disease outcomes. Our study identified the biopolymer polyphosphate (polyP) as a new effector molecule that impacts P. aeruginosa's interaction with S. aureus. We show that P. aeruginosa kills S. aureus in a polyP-dependent manner, which occurs primarily through the polyP-dependent production of the P. aeruginosa virulence factor pyocyanin. Our findings add a new role for polyP to an already extensive list of functions. A more in-depth understanding of how polyP influences interspecies interactions is critical, as targeting polyP synthesis in bacteria such as P. aeruginosa may have a significant impact on other microorganisms and potentially result in dynamic changes in the microbial composition.

由于铜绿假单胞菌和金黄色葡萄球菌在许多多微生物感染(包括囊性纤维化患者或烧伤/慢性伤口患者)中频繁共存,许多研究对机会性病原体铜绿假单胞菌和金黄色葡萄球菌之间相互作用的机理细节进行了调查。在体外共同培养条件下,铜绿假单胞菌能迅速超越金黄色葡萄球菌,这是由铜绿假单胞菌的几种毒力因子介导的。在这里,我们报告了聚磷酸盐(polyP),它是铜绿假单胞菌的一种高效应激防御系统和毒力因子,在病原体以非接触方式抑制和杀死金黄色葡萄球菌的能力中发挥了作用。我们的研究表明,polyP 含量低的铜绿假单胞菌细胞对金黄色葡萄球菌的生长和存活不利,而polyP 含量高的铜绿假单胞菌细胞则对革兰氏阳性病原体的危害更大。铜绿假单胞菌介导的杀死金黄色葡萄球菌的多聚酶依赖表型至少部分是直接的,因为在废培养基中检测到了多聚酶,并对金黄色葡萄球菌的细胞膜造成了严重破坏。不过,更有可能的情况是,polyP 通过调节铜绿假单胞菌的毒力因子之一 "脓青素 "的产生而产生间接影响。我们的研究表明,铜绿假单胞菌产生的焦花青素依赖于 polyP,并通过膜损伤和可能产生的活性氧危害金黄色葡萄球菌,导致抗氧化酶的表达增加。总之,我们的研究为革兰氏阴性病原体铜绿假单胞菌为与金黄色葡萄球菌争夺资源而产生的生物分子清单增加了一个新的组成部分。 重要意义 微生物之间的相互作用如何影响多微生物感染的过程?以前的研究已经提供了证据,证明铜绿假单胞菌和金黄色葡萄球菌这两种机会性病原体会产生一些分子来调节它们之间的相互作用,从而对疾病结果产生潜在的重大影响。我们的研究发现,生物聚合物聚磷酸盐(polyP)是一种影响铜绿假单胞菌与金黄色葡萄球菌相互作用的新效应分子。我们的研究表明,铜绿假单胞菌以依赖聚磷酸盐的方式杀死金黄色葡萄球菌,这主要是通过依赖聚磷酸盐产生铜绿假单胞菌毒力因子脓青素来实现的。我们的发现为 polyP 已经十分广泛的功能清单又增添了一个新的角色。更深入地了解 polyP 如何影响种间相互作用至关重要,因为针对铜绿假单胞菌等细菌的 polyP 合成可能会对其他微生物产生重大影响,并可能导致微生物组成的动态变化。
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引用次数: 0
Sialic acid and PirB are not required for targeting of neural circuits by neurotropic mammalian orthoreovirus. 神经性哺乳动物正交病毒靶向神经回路不需要Sialic酸和PirB。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-10-29 Epub Date: 2024-09-25 DOI: 10.1128/msphere.00629-24
Kira A Griswold, Iaroslavna Vasylieva, Megan C Smith, Kay L Fiske, Olivia L Welsh, Alexa N Roth, Alan M Watson, Simon C Watkins, Danica M Sutherland, Terence S Dermody

Serotype 3 (T3) strains of mammalian orthoreovirus (reovirus) spread to the central nervous system to infect the brain and cause lethal encephalitis in newborn mice. Although reovirus targets several regions in the brain, susceptibility to infection is not uniformly distributed. The neuronal subtypes and anatomic sites targeted throughout the brain are not precisely known. Reovirus binds several attachment factors and entry receptors, including sialic acid (SA)-containing glycans and paired immunoglobulin-like receptor B (PirB). While these receptors are not required for infection of some types of neurons, reovirus engagement of these receptors can influence neuronal infection in certain contexts. To identify patterns of T3 neurotropism, we used microbial identification after passive tissue clearance and hybridization chain reaction to stain reovirus-infected cells throughout intact, optically transparent brains of newborn mice. Three-dimensional reconstructions revealed in detail the sites targeted by reovirus throughout the brain volume, including dense infection of the midbrain and hindbrain. Using reovirus mutants incapable of binding SA and mice lacking PirB expression, we found that neither SA nor PirB is required for the infection of various brain regions. However, SA may confer minor differences in infection that vary by region. Collectively, these studies indicate that many regions in the brain of newborn mice are susceptible to reovirus and that patterns of reovirus infection are not dependent on reovirus receptors SA and PirB.IMPORTANCENeurotropic viruses invade the central nervous system (CNS) and target various cell types to cause disease manifestations, such as meningitis, myelitis, or encephalitis. Infections of the CNS are often difficult to treat and can lead to lasting sequelae or death. Mammalian orthoreovirus (reovirus) causes age-dependent lethal encephalitis in many young mammals. Reovirus infects neurons in several different regions of the brain. However, the complete pattern of CNS infection is not understood. We found that reovirus targets almost all regions of the brain and that patterns of tropism are not dependent on receptors sialic acid and paired immunoglobulin-like receptor B. These studies confirm that two known reovirus receptors do not completely explain the cell types infected in brain tissue and establish strategies that can be used to understand complete patterns of viral tropism in an intact brain.

血清型 3(T3)哺乳动物正始病毒(雷奥病毒)毒株会扩散到中枢神经系统,感染大脑并导致新生小鼠患上致命性脑炎。尽管再病毒以大脑中的多个区域为目标,但感染易感性的分布并不均匀。目前还不清楚整个大脑的神经元亚型和解剖部位。Reovirus 与几种附着因子和进入受体结合,包括含唾液酸(SA)的聚糖和成对的免疫球蛋白样受体 B(PirB)。虽然感染某些类型的神经元并不需要这些受体,但在某些情况下,雷诺病毒与这些受体的结合会影响神经元的感染。为了确定 T3 神经滋养模式,我们利用被动组织清除后的微生物鉴定和杂交链反应对新生小鼠完整、光学透明的大脑中受重病毒感染的细胞进行染色。三维重建详细显示了整个脑容量中被再病毒感染的部位,包括中脑和后脑的密集感染。通过使用无法结合 SA 的再病毒突变体和缺乏 PirB 表达的小鼠,我们发现 SA 和 PirB 都不是感染不同脑区所必需的。不过,SA 可能会使不同区域的感染略有不同。重要意义神经病毒入侵中枢神经系统(CNS)并以各种细胞类型为目标,导致脑膜炎、脊髓炎或脑炎等疾病表现。中枢神经系统感染通常难以治疗,并可能导致持久的后遗症或死亡。哺乳动物正粘病毒(Reovirus)会导致许多幼年哺乳动物出现年龄依赖性致死性脑炎。Reovirus 会感染大脑多个不同区域的神经元。然而,中枢神经系统感染的完整模式尚不清楚。这些研究证实,两种已知的雷诺病毒受体并不能完全解释脑组织中受感染的细胞类型,并建立了可用于了解完整大脑中病毒向性的完整模式的策略。
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