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Turning the needle into the haystack: Culture-independent amplification of complex microbial genomes directly from their native environment 大海捞针:不依赖培养基,直接从原生环境中扩增复杂微生物基因组
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-12 DOI: 10.1371/journal.ppat.1012418
Olivia A. Pilling, Sesh A. Sundararaman, Dustin Brisson, Daniel P. Beiting
High-throughput sequencing (HTS) has revolutionized microbiology, but many microbes exist at low abundance in their natural environment and/or are difficult, if not impossible, to culture in the laboratory. This makes it challenging to use HTS to study the genomes of many important microbes and pathogens. In this review, we discuss the development and application of selective whole genome amplification (SWGA) to allow whole or partial genomes to be sequenced for low abundance microbes directly from complex biological samples. We highlight ways in which genomic data generated by SWGA have been used to elucidate the population dynamics of important human pathogens and monitor development of antimicrobial resistance and the emergence of potential outbreaks. We also describe the limitations of this method and propose some potential innovations that could be used to improve the quality of SWGA and lower the barriers to using this method across a wider range of infectious pathogens.
高通量测序(HTS)使微生物学发生了革命性的变化,但许多微生物在自然环境中的丰度很低,而且/或者很难甚至无法在实验室中培养。这使得使用 HTS 研究许多重要微生物和病原体的基因组具有挑战性。在这篇综述中,我们将讨论选择性全基因组扩增(SWGA)的开发和应用,以便直接从复杂的生物样本中对低丰度微生物的全基因组或部分基因组进行测序。我们重点介绍了利用 SWGA 生成的基因组数据阐明重要人类病原体的种群动态、监测抗菌药耐药性的发展以及潜在疫情爆发的方式。我们还介绍了这种方法的局限性,并提出了一些潜在的创新方法,可用于提高 SWGA 的质量,降低在更广泛的传染病病原体中使用这种方法的障碍。
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引用次数: 0
α-Synuclein strain propagation is independent of cellular prion protein expression in a transgenic synucleinopathy mouse model 在转基因突触核蛋白病小鼠模型中,α-突触核蛋白菌株的传播与细胞朊病毒蛋白的表达无关
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-12 DOI: 10.1371/journal.ppat.1012517
Raphaella W. L. So, Genki Amano, Erica Stuart, Aeen Ebrahim Amini, Adriano Aguzzi, Graham L. Collingridge, Joel C. Watts
The cellular prion protein, PrPC, has been postulated to function as a receptor for α-synuclein, potentially facilitating cell-to-cell spreading and/or toxicity of α-synuclein aggregates in neurodegenerative disorders such as Parkinson’s disease. Previously, we generated the “Salt (S)” and “No Salt (NS)” strains of α-synuclein aggregates that cause distinct pathological phenotypes in M83 transgenic mice overexpressing A53T-mutant human α-synuclein. To test the hypothesis that PrPC facilitates the propagation of α-synuclein aggregates, we produced M83 mice that either express or do not express PrPC. Following intracerebral inoculation with the S or NS strain, the absence of PrPC in M83 mice did not prevent disease development and had minimal influence on α-synuclein strain-specified attributes such as the extent of cerebral α-synuclein deposition, selective targeting of specific brain regions and cell types, the morphology of induced α-synuclein deposits, and the structural fingerprints of protease-resistant α-synuclein aggregates. Likewise, there were no appreciable differences in disease manifestation between PrPC-expressing and PrPC-lacking M83 mice following intraperitoneal inoculation of the S strain. Interestingly, intraperitoneal inoculation with the NS strain resulted in two distinct disease phenotypes, indicative of α-synuclein strain evolution, but this was also independent of PrPC expression. Overall, these results suggest that PrPC plays at most a minor role in the propagation, neuroinvasion, and evolution of α-synuclein strains in mice that express A53T-mutant human α-synuclein. Thus, other putative receptors or cell-to-cell propagation mechanisms may have a larger effect on the spread of α-synuclein aggregates during disease.
据推测,细胞朊病毒蛋白 PrPC 具有α-突触核蛋白受体的功能,可能会促进α-突触核蛋白聚集体在帕金森病等神经退行性疾病中的细胞间扩散和/或毒性。此前,我们生成了α-突触核蛋白聚集体的 "盐(S)"和 "无盐(NS)"品系,它们会在过表达 A53T 突变人类α-突触核蛋白的 M83 转基因小鼠中引起不同的病理表型。为了验证PrPC促进α-突触核蛋白聚集体扩散的假设,我们培育了表达或不表达PrPC的M83小鼠。在脑内接种 S 株或 NS 株后,M83 小鼠体内缺乏 PrPC 并不能阻止疾病的发展,而且对α-突触核蛋白株指定的属性(如大脑α-突触核蛋白沉积的程度、特定脑区和细胞类型的选择性靶向、诱导的α-突触核蛋白沉积物的形态以及蛋白酶抗性α-突触核蛋白聚集体的结构指纹)的影响微乎其微。同样,腹腔注射S株后,表达PrPC和缺乏PrPC的M83小鼠在疾病表现上没有明显差异。有趣的是,腹腔接种 NS 株会导致两种不同的疾病表型,表明α-突触核蛋白株的演变,但这也与 PrPC 的表达无关。总之,这些结果表明,PrPC 在表达 A53T 突变人类α-突触核蛋白的小鼠中的α-突触核蛋白菌株的传播、神经侵入和进化过程中最多只能发挥微不足道的作用。因此,其他假定受体或细胞间传播机制可能对疾病期间α-突触核蛋白聚集体的扩散有更大的影响。
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引用次数: 0
Drivers of diversification in fungal pathogen populations 真菌病原体种群多样化的驱动因素
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-12 DOI: 10.1371/journal.ppat.1012430
Daniel Murante, Deborah Ann Hogan
To manage and treat chronic fungal diseases effectively, we require an improved understanding of their complexity. There is an increasing appreciation that chronic infection populations are often heterogeneous due to diversification and drift, even within a single microbial species. Genetically diverse populations can contribute to persistence and resistance to treatment by maintaining cells with different phenotypes capable of thriving in these dynamic environments. In chronic infections, fungal pathogens undergo prolonged challenges that can drive trait selection to convergent adapted states through restricted access to critical nutrients, assault by immune effectors, competition with other species, and antifungal drugs. This review first highlights the various genetic and epigenetic mechanisms that promote diversity in pathogenic fungal populations and provide an additional barrier to assessing the actual heterogeneity of fungal infections. We then review existing studies of evolution and genetic heterogeneity in fungal populations from lung infections associated with the genetic disease cystic fibrosis. We conclude with a discussion of open research questions that, once answered, may aid in diagnosing and treating chronic fungal infections.
为了有效管理和治疗慢性真菌病,我们需要进一步了解其复杂性。越来越多的人认识到,由于多样化和漂移,慢性感染群体通常是异质性的,即使在单一微生物物种内部也是如此。基因多样化的种群可以维持不同表型的细胞,使其能够在这些动态环境中茁壮成长,从而有助于持久性和抗药性。在慢性感染中,真菌病原体会经受长期的挑战,这些挑战可通过限制获取关键营养物质、免疫效应物的攻击、与其他物种的竞争以及抗真菌药物的作用,推动性状选择向趋同适应状态发展。本综述首先强调了促进病原真菌种群多样性的各种遗传和表观遗传机制,这些机制为评估真菌感染的实际异质性提供了额外的障碍。然后,我们回顾了与遗传病囊性纤维化相关的肺部感染中真菌种群进化和遗传异质性的现有研究。最后,我们讨论了一些开放性研究问题,这些问题一旦得到解答,将有助于诊断和治疗慢性真菌感染。
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引用次数: 0
Glaesserella parasuis serotype 4 exploits fibronectin via RlpA for tracheal colonization following porcine circovirus type 2 infection 猪 2 型圆环病毒感染后,寄生虫 4 号血清型通过 RlpA 利用纤维粘连蛋白进行气管定植
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-12 DOI: 10.1371/journal.ppat.1012513
Mengru Guo, Yuhui Li, Jinsheng Tang, Qing Wang, Qiancheng Wang, Hong Zhou, Huixing Lin, Zhe Ma, Hongjie Fan
Porcine circovirus type 2 (PCV2) often causes disease through coinfection with other bacterial pathogens, including Glaesserella parasuis (G. parasuis), which causes high morbidity and mortality, but the role played by PCV2 and bacterial and host factors contributing to this process have not been defined. Bacterial attachment is assumed to occur via specific receptor-ligand interactions between adhesins on the bacterial cell and host proteins adsorbed to the implant surface. Mass spectrometry (MS) analysis of PCV2-infected swine tracheal epithelial cells (STEC) revealed that the expression of Extracellular matrix protein (ECM) Fibronectin (Fn) increased significantly on the infected cells surface. Importantly, efficient G. parasuis serotype 4 (GPS4) adherence to STECs was imparted by interactions with Fn. Furthermore, abrogation of adherence was gained by genetic knockout of Fn, Fn and Integrin β1 antibody blocking. Fn is frequently exploited as a receptor for bacterial pathogens. To explore the GPS4 adhesin that interacts with Fn, recombinant Fn N-terminal type I and type II domains were incubated with GPS4, and the interacting proteins were pulled down for MS analysis. Here, we show that rare lipoprotein A (RlpA) directly interacts with host Fibronectin mediating GPS4 adhesion. Finally, we found that PCV2-induced Fibronectin expression and adherence of GPS4 were prevented significantly by TGF-β signaling pathway inhibitor SB431542. Our data suggest the RlpA-Fn interaction to be a potentially promising novel therapeutic target to combat PCV2 and GPS4 coinfection.
猪圆环病毒 2 型(PCV2)经常通过与其他细菌病原体(包括寄生璃泽氏菌(G. parasuis))合并感染而致病,造成很高的发病率和死亡率,但 PCV2 在这一过程中所起的作用以及导致这一过程的细菌和宿主因素尚未明确。据推测,细菌的附着是通过细菌细胞上的粘附素与吸附在种植体表面的宿主蛋白质之间的特异性受体-配体相互作用而发生的。对感染 PCV2 的猪气管上皮细胞(STEC)进行的质谱分析表明,受感染细胞表面细胞外基质蛋白(ECM)纤连蛋白(Fn)的表达量显著增加。重要的是,寄生虫血清型 4(GPS4)通过与 Fn 相互作用而有效地粘附在 STEC 上。此外,通过基因敲除 Fn、Fn 和 Integrin β1 抗体阻断,也能减弱粘附性。Fn 经常被用作细菌病原体的受体。为了探索与Fn相互作用的GPS4粘附蛋白,将重组的Fn N端I型和II型结构域与GPS4孵育,并将相互作用的蛋白拉下来进行质谱分析。在这里,我们发现稀有脂蛋白 A(RlpA)直接与宿主纤连蛋白相互作用,介导 GPS4 的粘附。最后,我们发现 TGF-β 信号通路抑制剂 SB431542 能显著阻止 PCV2 诱导的纤连蛋白表达和 GPS4 的粘附。我们的数据表明,RlpA-Fn 相互作用可能是对抗 PCV2 和 GPS4 协同感染的一个很有前景的新治疗靶点。
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引用次数: 0
Elasticity of the HIV-1 core facilitates nuclear entry and infection HIV-1 核心的弹性有助于核进入和感染
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-11 DOI: 10.1371/journal.ppat.1012537
Akshay Deshpande, Alexander J. Bryer, Jonathan R. Andino-Moncada, Jiong Shi, Jun Hong, Cameron Torres, Shimon Harel, Ashwanth C. Francis, Juan R. Perilla, Christopher Aiken, Itay Rousso
HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacepavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.
HIV-1 病毒感染需要病毒核心通过细胞核孔,这一过程取决于病毒外壳的功能。最近的研究表明,HIV-1 病毒核心在病毒帽分解之前就已进入细胞核。病毒衣壳与核孔复合体的相互作用是核进入的必要条件,但还不够充分,病毒核心穿过大小相当的核孔的机制尚不清楚。在这里,我们证明了 HIV-1 核心具有高弹性,而且这种特性与核进入和感染性有关。通过使用基于原子力显微镜的方法,我们发现纯化的野生型病毒核心在受到严重压缩后会迅速恢复正常的圆锥形形态。独立进行的成熟 HIV-1 胶囊的分子动力学模拟结果也揭示了它的弹性特性。对四种表现出核进入能力受损的 HIV-1 胶囊突变体的分析表明,突变病毒核心很脆。在细胞培养中对其中两种突变病毒进行适应性改造后,又进行了一些替换,从而恢复了弹性,并挽救了病毒的感染性和核进入能力。我们还发现,囊膜靶向化合物 PF74 和抗病毒药物 Lenacepavir 能降低病毒核心的弹性,并在保持病毒核心与核包膜相互作用的浓度下阻止 HIV-1 核进入。我们的研究结果表明,弹性是 HIV-1 核心的一个基本特性,它使核进入成为可能,从而促进感染。这些结果为我们深入了解囊膜在 HIV-1 核进入中的作用以及 HIV-1 囊膜抑制剂的抗病毒机制提供了新的视角。
{"title":"Elasticity of the HIV-1 core facilitates nuclear entry and infection","authors":"Akshay Deshpande, Alexander J. Bryer, Jonathan R. Andino-Moncada, Jiong Shi, Jun Hong, Cameron Torres, Shimon Harel, Ashwanth C. Francis, Juan R. Perilla, Christopher Aiken, Itay Rousso","doi":"10.1371/journal.ppat.1012537","DOIUrl":"https://doi.org/10.1371/journal.ppat.1012537","url":null,"abstract":"HIV-1 infection requires passage of the viral core through the nuclear pore of the cell, a process that depends on functions of the viral capsid. Recent studies have shown that HIV-1 cores enter the nucleus prior to capsid disassembly. Interactions of the viral capsid with the nuclear pore complex are necessary but not sufficient for nuclear entry, and the mechanism by which the viral core traverses the comparably sized nuclear pore is unknown. Here we show that the HIV-1 core is highly elastic and that this property is linked to nuclear entry and infectivity. Using atomic force microscopy-based approaches, we found that purified wild type cores rapidly returned to their normal conical morphology following a severe compression. Results from independently performed molecular dynamic simulations of the mature HIV-1 capsid also revealed its elastic property. Analysis of four HIV-1 capsid mutants that exhibit impaired nuclear entry revealed that the mutant viral cores are brittle. Adaptation of two of the mutant viruses in cell culture resulted in additional substitutions that restored elasticity and rescued infectivity and nuclear entry. We also show that capsid-targeting compound PF74 and the antiviral drug Lenacepavir reduce core elasticity and block HIV-1 nuclear entry at concentrations that preserve interactions between the viral core and the nuclear envelope. Our results indicate that elasticity is a fundamental property of the HIV-1 core that enables nuclear entry, thereby facilitating infection. These results provide new insights into the role of the capsid in HIV-1 nuclear entry and the antiviral mechanisms of HIV-1 capsid inhibitors.","PeriodicalId":20178,"journal":{"name":"PLoS Pathogens","volume":"7 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142202265","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal 从超级延长复合体中释放 P-TEFb 可促进 HIV-1 潜伏期逆转
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-11 DOI: 10.1371/journal.ppat.1012083
William J. Cisneros, Shimaa H. A. Soliman, Miriam Walter, Lacy M. Simons, Daphne Cornish, Simone De Fabritiis, Ariel W. Halle, Eun-Young Kim, Steven M. Wolinsky, Ramon Lorenzo-Redondo, Ali Shilatifard, Judd F. Hultquist
The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.
在抑制性抗逆转录病毒疗法(ART)期间,HIV-1 在长效潜伏库中的持续存在仍然是功能性治愈的主要障碍之一。转录延伸受阻在维持潜伏状态方面起着核心作用,几种潜伏逆转策略都侧重于释放正转录延伸因子 b(P-TEFb),使其摆脱 7SK 复合物和 BRD4 等负调控复合物的封存。P-TEFb 的另一个主要细胞储库是超级延伸复合体(SEC),它们在宿主基因表达中发挥着广泛的调控作用。然而,从 SECs 中释放 P-TEFb 是否是一种可行的潜伏逆转策略还不得而知。在这里,我们证明原代 CD4+ T 细胞中的 HIV-1 复制不需要 SEC,而且 P-TEFb/SEC 相互作用的小分子抑制剂(称为 KL-2)会增加病毒转录。KL-2 与其他潜伏期逆转剂(LRAs)协同作用,在几种潜伏期细胞系模型中重新激活病毒转录,其方式至少部分依赖于病毒 Tat 蛋白。最后,我们证明了 KL-2 能增强接受抑制性抗逆转录病毒疗法的艾滋病病毒感染者(PLWH)外周血单核细胞(PBMC)中的病毒再活化,尤其是与凋亡抑制蛋白拮抗剂(IAPi)联合使用时。综上所述,这些结果表明,P-TEFb 从细胞 SEC 中的释放可能是 HIV-1 潜伏期重新激活的新途径。
{"title":"Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal","authors":"William J. Cisneros, Shimaa H. A. Soliman, Miriam Walter, Lacy M. Simons, Daphne Cornish, Simone De Fabritiis, Ariel W. Halle, Eun-Young Kim, Steven M. Wolinsky, Ramon Lorenzo-Redondo, Ali Shilatifard, Judd F. Hultquist","doi":"10.1371/journal.ppat.1012083","DOIUrl":"https://doi.org/10.1371/journal.ppat.1012083","url":null,"abstract":"The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation.","PeriodicalId":20178,"journal":{"name":"PLoS Pathogens","volume":"45 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142202235","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The unique Legionella longbeachae capsule favors intracellular replication and immune evasion 独特的长须鲸军团菌胶囊有利于细胞内复制和免疫逃避
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-11 DOI: 10.1371/journal.ppat.1012534
Silke Schmidt, Sonia Mondino, Laura Gomez-Valero, Pedro Escoll, Danielle P. A. Mascarenhas, Augusto Gonçalves, Pedro H. M. Camara, Francisco J. Garcia Rodriguez, Christophe Rusniok, Martin Sachse, Maryse Moya-Nilges, Thierry Fontaine, Dario S. Zamboni, Carmen Buchrieser
Legionella longbeachae and Legionella pneumophila are the most common causative agents of Legionnaires’ disease. While the clinical manifestations caused by both species are similar, species-specific differences exist in environmental niches, disease epidemiology, and genomic content. One such difference is the presence of a genomic locus predicted to encode a capsule. Here, we show that L. longbeachae indeed expresses a capsule in post-exponential growth phase as evidenced by electron microscopy analyses, and that capsule expression is abrogated when deleting a capsule transporter gene. Capsule purification and its analysis via HLPC revealed the presence of a highly anionic polysaccharide that is absent in the capsule mutant. The capsule is important for replication and virulence in vivo in a mouse model of infection and in the natural host Acanthamoeba castellanii. It has anti-phagocytic function when encountering innate immune cells such as human macrophages and it is involved in the low cytokine responses in mice and in human monocyte derived macrophages, thus dampening the innate immune response. Thus, the here characterized L. longbeachae capsule is a novel virulence factor, unique among the known Legionella species, which may aid L. longbeachae to survive in its specific niches and which partly confers L. longbeachae its unique infection characteristics.
长臂军团菌和嗜肺军团菌是军团病最常见的致病菌。虽然这两种病菌引起的临床表现相似,但在环境生态位、疾病流行病学和基因组内容方面存在物种特异性差异。其中一个差异是存在一个预测为胶囊编码的基因组位点。在这里,我们通过电子显微镜分析表明,L. longbeachae确实在后生长期表达胶囊,而且当删除胶囊转运体基因时,胶囊的表达也会减弱。通过 HLPC 对胶囊进行纯化和分析,发现胶囊突变体中不存在高阴离子多糖。这种胶囊对小鼠感染模型和自然宿主棘阿米巴的体内复制和毒力非常重要。当遇到先天性免疫细胞(如人类巨噬细胞)时,它具有抗吞噬功能,并参与小鼠和人类单核细胞衍生巨噬细胞的低细胞因子反应,从而抑制先天性免疫反应。因此,长须鲸军团菌胶囊是一种新的毒力因子,在已知的军团菌中是独一无二的,它可能有助于长须鲸军团菌在其特定的壁龛中生存,并在一定程度上赋予了长须鲸军团菌独特的感染特性。
{"title":"The unique Legionella longbeachae capsule favors intracellular replication and immune evasion","authors":"Silke Schmidt, Sonia Mondino, Laura Gomez-Valero, Pedro Escoll, Danielle P. A. Mascarenhas, Augusto Gonçalves, Pedro H. M. Camara, Francisco J. Garcia Rodriguez, Christophe Rusniok, Martin Sachse, Maryse Moya-Nilges, Thierry Fontaine, Dario S. Zamboni, Carmen Buchrieser","doi":"10.1371/journal.ppat.1012534","DOIUrl":"https://doi.org/10.1371/journal.ppat.1012534","url":null,"abstract":"<jats:italic>Legionella longbeachae</jats:italic> and <jats:italic>Legionella pneumophila</jats:italic> are the most common causative agents of Legionnaires’ disease. While the clinical manifestations caused by both species are similar, species-specific differences exist in environmental niches, disease epidemiology, and genomic content. One such difference is the presence of a genomic locus predicted to encode a capsule. Here, we show that <jats:italic>L</jats:italic>. <jats:italic>longbeachae</jats:italic> indeed expresses a capsule in post-exponential growth phase as evidenced by electron microscopy analyses, and that capsule expression is abrogated when deleting a capsule transporter gene. Capsule purification and its analysis <jats:italic>via</jats:italic> HLPC revealed the presence of a highly anionic polysaccharide that is absent in the capsule mutant. The capsule is important for replication and virulence <jats:italic>in vivo</jats:italic> in a mouse model of infection and in the natural host <jats:italic>Acanthamoeba castellanii</jats:italic>. It has anti-phagocytic function when encountering innate immune cells such as human macrophages and it is involved in the low cytokine responses in mice and in human monocyte derived macrophages, thus dampening the innate immune response. Thus, the here characterized <jats:italic>L</jats:italic>. <jats:italic>longbeachae</jats:italic> capsule is a novel virulence factor, unique among the known <jats:italic>Legionella</jats:italic> species, which may aid <jats:italic>L</jats:italic>. <jats:italic>longbeachae</jats:italic> to survive in its specific niches and which partly confers <jats:italic>L</jats:italic>. <jats:italic>longbeachae</jats:italic> its unique infection characteristics.","PeriodicalId":20178,"journal":{"name":"PLoS Pathogens","volume":"11 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142202234","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
NEDD4 family ubiquitin ligase AIP4 interacts with Alix to enable HBV naked capsid egress in an Alix ubiquitination-independent manner NEDD4 家族泛素连接酶 AIP4 与 Alix 相互作用,以不依赖于 Alix 泛素化的方式使 HBV 裸盖蛋白外排
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-11 DOI: 10.1371/journal.ppat.1012485
Sheng Shen, Dawei Cai, Hongyan Liang, Ge Zeng, Wendong Liu, Ran Yan, Xiaoyang Yu, Hu Zhang, Shi Liu, Wanying Li, Rui Deng, Xingyu Lu, Yuanjie Liu, Jian Sun, Haitao Guo
Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle.
乙型肝炎病毒(HBV)利用运输所需的内体分拣复合物(ESCRT)/多泡体(MVB)途径进行病毒出芽。除了包膜病毒外,HBV 复制细胞还能独立于整体 ESCRT 机制非lytically 地释放非包膜(裸体)病毒盖,但确切的分泌机制仍然难以捉摸。在此,我们提供了有关裸盖体的存在、特征以及病毒和宿主对裸盖体排出的调控的更多详细信息。HBV 荚膜/核心蛋白有两个高度保守的赖氨酸残基(K7/K96),这两个残基在随后的生物学事件中可能会发生各种类型的翻译后修饰。突变研究发现,K96残基对裸噬菌体的排出至关重要,细胞内有能力排出的噬菌体与泛素化的宿主蛋白相关。与之前的报道一致,ESCRT-III结合蛋白Alix及其Bro1结构域通过与细胞内的噬菌体结合,是裸噬菌体分泌所必需的,而且我们进一步发现泛素化的Alix能与野生型噬菌体结合,而不能与K96R突变体结合。此外,通过对 NEDD4 E3 泛素连接酶家族成员的筛选发现,AIP4 能刺激裸盖蛋白的释放,而裸盖蛋白的释放依赖于 AIP4 蛋白的完整性和 E3 连接酶的活性。我们进一步证实,AIP4 与 Alix 相互作用并促进其泛素化,AIP4 对 Alix 介导的裸盖蛋白分泌至关重要。然而,Alix的Bro1结构域是非泛素化的,这表明AIP4诱导的裸盖蛋白分泌并不绝对需要Alix泛素化。综上所述,我们的研究为HBV裸盖蛋白在病毒生命周期中的排出机制提供了新的启示。
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引用次数: 0
Prion diseases disrupt glutamate/glutamine metabolism in skeletal muscle 朊病毒疾病会破坏骨骼肌中谷氨酸/谷氨酰胺的新陈代谢
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-11 DOI: 10.1371/journal.ppat.1012552
Davide Caredio, Maruša Koderman, Karl J. Frontzek, Silvia Sorce, Mario Nuvolone, Juliane Bremer, Giovanni Mariutti, Petra Schwarz, Lidia Madrigal, Marija Mitrovic, Stefano Sellitto, Nathalie Streichenberger, Claudia Scheckel, Adriano Aguzzi
In prion diseases (PrDs), aggregates of misfolded prion protein (PrPSc) accumulate not only in the brain but also in extraneural organs. This raises the question whether prion-specific pathologies arise also extraneurally. Here we sequenced mRNA transcripts in skeletal muscle, spleen and blood of prion-inoculated mice at eight timepoints during disease progression. We detected gene-expression changes in all three organs, with skeletal muscle showing the most consistent alterations. The glutamate-ammonia ligase (GLUL) gene exhibited uniform upregulation in skeletal muscles of mice infected with three distinct scrapie prion strains (RML, ME7, and 22L) and in victims of human sporadic Creutzfeldt-Jakob disease. GLUL dysregulation was accompanied by changes in glutamate/glutamine metabolism, leading to reduced glutamate levels in skeletal muscle. None of these changes were observed in skeletal muscle of humans with amyotrophic lateral sclerosis, Alzheimer’s disease, or dementia with Lewy bodies, suggesting that they are specific to prion diseases. These findings reveal an unexpected metabolic dimension of prion infections and point to a potential role for GLUL dysregulation in the glutamate/glutamine metabolism in prion-affected skeletal muscle.
在朊病毒疾病(PrDs)中,折叠错误的朊病毒蛋白(PrPSc)聚集体不仅会在大脑中积聚,还会在脑外器官中积聚。这就提出了一个问题:朊病毒特异性病症是否也会在外部发生?在这里,我们对朊病毒接种小鼠骨骼肌、脾脏和血液中的 mRNA 转录本进行了测序,测序时间为疾病进展的八个时间点。我们在这三个器官中都检测到了基因表达的变化,其中骨骼肌的变化最为一致。在感染了三种不同的豚鼠朊病毒株(RML、ME7和22L)的小鼠和人类散发性克雅氏病患者的骨骼肌中,谷氨酸氨连接酶(GLUL)基因表现出一致的上调。GLUL 失调伴随着谷氨酸/谷氨酰胺代谢的变化,导致骨骼肌中谷氨酸水平降低。在患有肌萎缩侧索硬化症、阿尔茨海默病或路易体痴呆症的人体骨骼肌中均未观察到这些变化,这表明它们是朊病毒疾病所特有的。这些发现揭示了朊病毒感染的一个意想不到的代谢层面,并指出了GLUL在受朊病毒影响的骨骼肌谷氨酸/谷氨酰胺代谢中的潜在作用。
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引用次数: 0
A comprehensive study of SARS-CoV-2 mfigain protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system 对在基于 VSV 的系统中筛选出的 SARS-CoV-2 mfigain 蛋白酶 (Mpro) 抑制剂抗性突变体的全面研究
IF 6.7 1区 医学 Q1 Immunology and Microbiology Pub Date : 2024-09-11 DOI: 10.1371/journal.ppat.1012522
Francesco Costacurta, Andrea Dodaro, David Bante, Helge Schöppe, Ju-Yi Peng, Bernhard Sprenger, Xi He, Seyed Arad Moghadasi, Lisa Maria Egger, Jakob Fleischmann, Matteo Pavan, Davide Bassani, Silvia Menin, Stefanie Rauch, Laura Krismer, Anna Sauerwein, Anne Heberle, Toni Rabensteiner, Joses Ho, Reuben S. Harris, Eduard Stefan, Rainer Schneider, Theresia Dunzendorfer-Matt, Andreas Naschberger, Dai Wang, Teresa Kaserer, Stefano Moro, Dorothee von Laer, Emmanuel Heilmann
Nirmatrelvir was the first protease inhibitor specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available protease inhibitors (nirmatrelvir and ensitrelvir) with cell-based, biochemical and SARS-CoV-2 replicon assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease inhibitor resistance mechanisms and show the relevance of specific mutations, thereby informing treatment decisions.
Nirmatrelvir 是首个专门针对 SARS-CoV-2 主要蛋白酶(3CLpro/Mpro)开发并获得临床使用许可的蛋白酶抑制剂。随着 SARS-CoV-2 的继续传播,很可能会出现对 nirmatrelvir 和其他现有治疗方法产生抗药性的变种。本研究旨在确定对尼马瑞韦产生耐药性的突变并描述其特征。为了安全地产生 Mpro 抗性突变,我们将先前开发的嵌合型水泡性口炎病毒(VSV-Mpro)与浓度不断增加但又不太理想的 nirmatrelvir 进行了传代。利用武汉-1 和 Omicron Mpro 变体,我们筛选出了一大批突变体。有些突变在 GISAID 中经常出现,这表明它们与 SARS-CoV-2 有关。通过细胞、生化和 SARS-CoV-2 复制子检测,我们确定了一部分突变体对临床上可用的蛋白酶抑制剂(nirmatrelvir 和 ensitrelvir)的耐药性表型。此外,我们还根据硅学分子模型展示了抗药性的假定分子机制。这些发现对下一代 Mpro 抑制剂的开发具有重要意义,将有助于了解 SARS-CoV-2 蛋白酶抑制剂的耐药机制,并显示特定突变的相关性,从而为治疗决策提供信息。
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