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Cryo-EM structure of the Mycobacterium smegmatis MmpL5-AcpM complex. 分枝杆菌 MmpL5-AcpM 复合物的冷冻电镜结构。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-31 DOI: 10.1128/mbio.03035-24
Rakesh Maharjan, Zhemin Zhang, Philip A Klenotic, William D Gregor, Georgiana E Purdy, Edward W Yu

Mycobacterium tuberculosis, the causative agent of the airborne infection tuberculosis (TB), contains 13 mycobacterial membrane protein large (MmpL) transporters that can be divided into two distinct subclasses. These MmpL proteins play important functional roles within the mycobacterium and subsequently are considered attractive drug targets to combat TB infection. Previously, we reported both X-ray and cryo-electron microscopy (cryo-EM) structures of the MmpL3 transporter, providing high-resolution structural information for this subclass of the MmpL proteins. Thus far, there is no structural information available for the other subclass, which includes MmpL5, an inner membrane transporter that plays a critical role in iron hemostasis. Here, we report the first cryo-EM structure of the Mycobacterium smegmatis MmpL5 transporter bound with the meromycolate extension acyl carrier protein M (AcpM) to a resolution of 2.81 Å. Our structural data reveals that MmpL5 and AcpM interact in the cytoplasm to form a complex, and this allows us to propose that MmpL5 may also associate with the mycobactin L (MbtL) protein in a similar fashion to form a heterocomplex important for iron acquisition, which enables the survival and replication of the mycobacterium.

Importance: The emergence and spread of multidrug-resistant tuberculosis (TB) present enormous challenges to the global public health. The causative agent, Mycobacterium tuberculosis, has now infected more than one-third of the world's population. Here, we report the first structure of the mycobacterial membrane protein large 5 (MmpL5), an essential transporter for iron acquisition, bound with the meromycolate extension acyl carrier protein M (AcpM), indicating a plausible pathway for mycobactin translocation. Our studies will ultimately inform an era in structure-guided drug design to combat TB infection.

结核分枝杆菌是通过空气传播的结核病(TB)的病原体,它含有 13 个可分为两个不同亚类的大分枝杆菌膜蛋白(MmpL)转运体。这些 MmpL 蛋白在分枝杆菌内发挥着重要的功能作用,因此被认为是抗结核感染的有吸引力的药物靶标。此前,我们报道了 MmpL3 转运体的 X 射线和低温电子显微镜(cryo-EM)结构,为该亚类 MmpL 蛋白提供了高分辨率的结构信息。迄今为止,还没有关于另一个亚类的结构信息,其中包括在铁止血中发挥关键作用的内膜转运体 MmpL5。在此,我们首次报道了分枝杆菌 MmpL5 转运体与美拉美酸延伸酰基载体蛋白 M(AcpM)结合的低温电子显微镜结构,分辨率为 2.81 Å。我们的结构数据显示,MmpL5 和 AcpM 在细胞质中相互作用形成复合物,这使我们能够提出,MmpL5 也可能以类似的方式与分枝杆菌素 L(MbtL)蛋白结合,形成对铁获取非常重要的异质复合物,从而使分枝杆菌得以生存和复制:耐多药结核病(TB)的出现和传播给全球公共卫生带来了巨大挑战。致病菌结核分枝杆菌现已感染了全球三分之一以上的人口。在这里,我们首次报告了分枝杆菌膜蛋白大 5(MmpL5)的结构,它是铁获取的一个重要转运体,与美拉美酸延伸酰基载体蛋白 M(AcpM)结合,表明了分枝杆菌素转运的一个合理途径。我们的研究最终将为结构引导药物设计时代的到来提供信息,以对抗结核病感染。
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引用次数: 0
SydR, a redox-sensing MarR-type regulator of Sinorhizobium meliloti, is crucial for symbiotic infection of Medicago truncatula roots. SydR是瓜萎镰刀菌(Sinorhizobium meliloti)的一种氧化还原传感MarR型调节器,对共生感染美智子(Medicago truncatula)根部至关重要。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-31 DOI: 10.1128/mbio.02275-24
Fanny Nazaret, Davoud Farajzadeh, Joffrey Mejias, Marie Pacoud, Anthony Cosi, Pierre Frendo, Geneviève Alloing, Karine Mandon

Rhizobia associate with legumes and induce the formation of nitrogen-fixing nodules. The regulation of bacterial redox state plays a major role in symbiosis, and reactive oxygen species produced by the plant are known to activate signaling pathways. However, only a few redox-sensing transcriptional regulators (TRs) have been characterized in the microsymbiont. Here, we describe SydR, a novel redox-sensing TR of Sinorhizobium meliloti that is essential for the establishment of symbiosis with Medicago truncatula. SydR, a MarR-type TR, represses the expression of the adjacent gene SMa2023 in growing cultures, and this repression is alleviated by NaOCl, tert-butyl hydroperoxide, or H2O2 treatment. Transcriptional psydR-gfp and pSMa2023-gfp fusions, as well as gel shift assays, showed that SydR binds two independent sites of the sydR-SMa2023 intergenic region. This binding is redox-dependent, and site-directed mutagenesis demonstrated that the conserved C16 is essential for SydR redox sensing. The inactivation of sydR did not alter the sensitivity of S. meliloti to NaOCl, tert-butyl hydroperoxide, or H2O2, nor did it affect the response to oxidants of the roGFP2-Orp1 redox biosensor expressed within bacteria. However, in planta, ΔsydR mutation impaired the formation of root nodules. Microscopic observations and analyses of plant marker gene expression showed that the ΔsydR mutant is defective at an early stage of the bacterial infection process. Altogether, these results demonstrated that SydR is a redox-sensing MarR-type TR that plays a key role in the regulation of nitrogen-fixing symbiosis with M. truncatula.IMPORTANCEThe nitrogen-fixing symbiosis between rhizobia and legumes has an important ecological role in the nitrogen cycle, contributes to nitrogen enrichment of soils, and can improve plant growth in agriculture. This interaction is initiated in the rhizosphere by a molecular dialog between the two partners, resulting in plant root infection and the formation of root nodules, where bacteria reduce the atmospheric nitrogen into ammonium. This symbiosis involves modifications of the bacterial redox state in response to reactive oxygen species produced by the plant partner. Here, we show that SydR, a transcriptional regulator of the Medicago symbiont Sinorhizobium meliloti, acts as a redox-responsive repressor that is crucial for the development of root nodules and contributes to the regulation of bacterial infection in S. meliloti/Medicago truncatula symbiotic interaction.

根瘤菌与豆科植物结合并诱导固氮结节的形成。细菌氧化还原状态的调节在共生中起着重要作用,植物产生的活性氧可激活信号通路。然而,只有少数氧化还原感应转录调节因子(TRs)在微共生体中得到了表征。在这里,我们描述了一种新型的氧化还原感知转录调节因子 SydR,它是瓜萎镰刀菌(Sinorhizobium meliloti)与美迪西(Medicago truncatula)建立共生关系所必需的。SydR是一种MarR型TR,它能抑制生长培养物中相邻基因SMa2023的表达,NaOCl、叔丁基过氧化氢或H2O2处理可减轻这种抑制。转录 psydR-gfp 和 pSMa2023-gfp 融合以及凝胶转移试验表明,SydR 与 sydR-SMa2023 基因间区域的两个独立位点结合。这种结合是氧化还原依赖性的,定点突变证明保守的 C16 是 SydR 氧化还原感应的关键。sydR 失活不会改变 S. meliloti 对 NaOCl、叔丁基过氧化氢或 H2O2 的敏感性,也不会影响细菌内表达的 roGFP2-Orp1 氧化还原生物传感器对氧化剂的反应。然而,在植物体内,ΔsydR 突变会影响根瘤的形成。显微观察和植物标记基因表达分析表明,ΔsydR突变体在细菌感染过程的早期阶段存在缺陷。重要意义根瘤菌与豆科植物之间的固氮共生在氮循环中具有重要的生态作用,有助于土壤的氮富集,并能改善农业植物的生长。根瘤菌与豆科植物之间的这种相互作用是在根瘤层中通过双方的分子对话开始的,从而导致植物根部感染并形成根瘤,细菌在根瘤中将大气中的氮还原成铵。这种共生涉及细菌氧化还原状态的改变,以应对植物伙伴产生的活性氧。在这里,我们发现麦地那龙属共生菌的转录调节因子 SydR 是一种氧化还原反应抑制因子,对根瘤的发育至关重要,并有助于调节麦地那龙属/麦地那龙共生相互作用中的细菌感染。
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引用次数: 0
Emerging mosquito-borne flaviviruses. 新出现的蚊媒黄病毒。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-31 DOI: 10.1128/mbio.02946-24
Amy N Nelson, Alexander Ploss

Flaviviruses comprise a genus of enveloped, positive-sense, single-stranded RNA viruses typically transmitted between susceptible and permissive hosts by arthropod vectors. Established flavivirus threats include dengue viruses (DENV), yellow fever virus (YFV), Zika virus (ZIKV), and West Nile virus (WNV), which continue to cause over 400 million infections annually and are significant global health and economic burdens. Additionally, numerous closely related but largely understudied viruses circulate in animals and can conceivably emerge in human populations. Previous flaviviruses that were recognized to have this potential include ZIKV and WNV, which only became extensively studied after causing major outbreaks in humans. More than 50 species exist within the flavivirus genus, which can be further classified as mosquito-borne, tick-borne, insect-specific, or with no known vector. Historically, many of these flaviviruses originated in Africa and have mainly affected tropical and subtropical regions due to the ecological niche of mosquitoes. However, climate change, as well as vector and host migration, has contributed to geographical expansion, thereby posing a potential risk to global populations. For the purposes of this minireview, we focus on the mosquito-borne subgroup and highlight viruses that cause significant pathology or lethality in at least one animal species and/or have demonstrated an ability to infect humans. We discuss current knowledge of these viruses, existing animal models to study their pathogenesis, and potential future directions. Emerging viruses discussed include Usutu virus (USUV), Wesselsbron virus (WSLV), Spondweni virus (SPOV), Ilheus virus (ILHV), Rocio virus (ROCV), Murray Valley encephalitis virus (MVEV), and Alfuy virus (ALFV).

黄病毒是一种有包膜的正义单链 RNA 病毒,通常通过节肢动物载体在易感宿主和易感宿主之间传播。已有的黄病毒威胁包括登革热病毒(DENV)、黄热病病毒(YFV)、寨卡病毒(ZIKV)和西尼罗河病毒(WNV),这些病毒每年造成超过 4 亿人感染,对全球健康和经济造成重大负担。此外,还有许多密切相关但大多未得到充分研究的病毒在动物中流行,并有可能在人类中出现。以前被认为具有这种潜能的黄病毒包括 ZIKV 和 WNV,它们只是在导致人类重大疾病爆发后才被广泛研究。黄病毒属中有 50 多个种类,可进一步分为蚊媒、蜱媒、昆虫特异性或无已知病媒。历史上,许多黄病毒都起源于非洲,由于蚊子的生态位,它们主要影响热带和亚热带地区。然而,气候变化以及病媒和宿主的迁移造成了地理范围的扩大,从而给全球人口带来了潜在风险。在本小视图中,我们将重点放在蚊子传播的病毒亚群上,并着重介绍至少会对一种动物物种造成重大病理变化或致死和/或已证明有能力感染人类的病毒。我们讨论了目前对这些病毒的了解、研究其致病机理的现有动物模型以及未来可能的研究方向。讨论的新兴病毒包括乌苏图病毒(USUV)、威瑟斯布隆病毒(WSLV)、斯庞德文尼病毒(SPOV)、伊尔休斯病毒(ILHV)、罗西奥病毒(ROCV)、墨累谷脑炎病毒(MVEV)和阿尔富病毒(ALFV)。
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引用次数: 0
Performance of rapid antigen tests to detect SARS-CoV-2 variant diversity and correlation with viral culture positivity: implication for diagnostic development and future public health strategies. 检测 SARS-CoV-2 变异多样性的快速抗原测试的性能以及与病毒培养阳性的相关性:对诊断发展和未来公共卫生战略的影响。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-31 DOI: 10.1128/mbio.02737-24
Heather Goux, Jennetta Green, Andrew Wilson, Shanmuga Sozhamannan, Stephanie A Richard, Rhonda Colombo, David A Lindholm, Milissa U Jones, Brian K Agan, Derek Larson, David L Saunders, Rupal Mody, Jason Cox, Robert Deans, Joseph Walish, Anthony Fries, Mark P Simons, Simon D Pollett, Darci R Smith

Antigen-based rapid diagnostic tests (Ag-RDTs) provide timely results, are simple to use, and are less expensive than molecular assays. Recent studies suggest that antigen-based testing aligns with virus culture-based results (a proxy of contagiousness at the peak viral phase of illness); however, the performance of Ag-RDTs for newer SARS-CoV-2 variants is unclear. In this study, we (i) assessed the performance of Ag-RDTs and diagnostic antibodies to detect a range of SARS-CoV-2 variants and (ii) determined whether Ag-RDT results correlated with culture positivity. We noted only minor differences in the limit of detection by variant for all assays, and we demonstrated consistent antibody affinity to the N protein among the different variants. We observed moderate to high sensitivity (46.8%-83.9%) for Ag-RDTs when compared to PCR positivity (100%), and all variants were assessed on each assay. Ag-RDT sensitivity and PCR Ct showed an inverse correlation with the detection of viable virus. Collectively, our results demonstrate that commercially available Ag-RDTs offer variable sensitivity compared to PCR, show similar diagnostic validity across variants, and may predict the risk of transmissibility. These findings may be used to support more tailored SARS-CoV-2 isolation strategies, particularly if other studies clarify the direct association between Ag-RDT positivity and transmission risk. The apparent trade-off between sensitivity in the detection of any PCR-positive infection and concordance with infectious virus positivity may also inform new RDT diagnostic development strategies for SARS-CoV-2 and other epidemic respiratory pathogens.

Importance: Despite the availability of vaccines, COVID-19 continues to be a major health concern, and antigen-based rapid diagnostic tests (Ag-RDTs) are commonly used as point-of-care or at-home diagnostic tests. In this study, we evaluated the performance of two commercially available Ag-RDTs and a research Ag-RDT to detect multiple SARS-CoV-2 variants using upper respiratory tract swab samples from clinical COVID-19 cases. Furthermore, we determined whether Ag-RDT results correlated with culture positivity, a potential proxy of viral transmissibility. Our results have important implications to inform future testing and response strategies during periods of high COVID-19 transmission with new variants.

基于抗原的快速诊断检测(Ag-RDTs)能及时提供结果,使用简单,而且成本低于分子检测。最近的研究表明,抗原检测结果与病毒培养结果一致(病毒感染高峰期传染性的代表);然而,Ag-RDT 对较新的 SARS-CoV-2 变种的检测效果尚不清楚。在这项研究中,我们(i) 评估了 Ag-RDT 和诊断抗体检测一系列 SARS-CoV-2 变体的性能,(ii) 确定了 Ag-RDT 结果是否与培养阳性相关。我们注意到,在所有检测方法中,不同变异体的检测限仅有细微差别,而且不同变异体对 N 蛋白的抗体亲和力一致。与 PCR 阳性率(100%)相比,我们观察到 Ag-RDT 具有中等到较高的灵敏度(46.8%-83.9%),而且每次检测都对所有变体进行了评估。Ag-RDT 灵敏度和 PCR Ct 与活病毒检测呈反相关。总之,我们的研究结果表明,与 PCR 相比,市售的 Ag-RDT 具有不同的灵敏度,对不同变异株显示出相似的诊断有效性,并可预测传播风险。这些发现可用于支持更有针对性的 SARS-CoV-2 隔离策略,特别是如果其他研究能阐明 Ag-RDT 阳性与传播风险之间的直接联系。在检测任何 PCR 阳性感染的灵敏度与传染性病毒阳性的一致性之间的明显权衡也可为 SARS-CoV-2 和其他流行性呼吸道病原体的新 RDT 诊断开发策略提供参考:尽管有疫苗可用,但 COVID-19 仍是一个主要的健康问题,基于抗原的快速诊断检测(Ag-RDTs)通常用作护理点或家庭诊断检测。在本研究中,我们使用来自临床 COVID-19 病例的上呼吸道拭子样本,评估了两种市售 Ag-RDT 和一种研究型 Ag-RDT 检测多种 SARS-CoV-2 变体的性能。此外,我们还确定了 Ag-RDT 结果是否与培养阳性相关,培养阳性可能代表病毒的传播性。我们的研究结果对未来在 COVID-19 新变种高传播期的检测和应对策略具有重要意义。
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引用次数: 0
PlsX and PlsY: Additional roles beyond glycerophospholipid synthesis in Gram-negative bacteria. PlsX 和 PlsY:革兰氏阴性细菌中甘油磷脂合成以外的其他作用。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/mbio.02969-24
Audrey N Rex, Brent W Simpson, Gregory Bokinsky, M Stephen Trent

The unique asymmetry of the Gram-negative outer membrane, with glycerophospholipids (GPLs) in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet, works to resist external stressors and prevent the entry of toxic compounds. Thus, GPL and LPS synthesis must be tightly controlled to maintain the integrity of this essential structure. We sought to decipher why organisms like Escherichia coli possess two redundant pathways-PlsB and PlsX/Y-for synthesis of the GPL precursor lysophosphatidic acid (LPA). LPA is then converted by PlsC to the universal precursor for GPL synthesis, phosphatidic acid (PA). PlsB and PlsC are essential in E. coli, indicating they serve as the major pathway for PA synthesis. While loss of PlsX or PlsY individually has little consequence on the cell, the absence of both was lethal. To understand the synthetic lethality of this seemingly redundant PlsX/Y pathway, we performed a suppressor screen. Suppressor analysis indicated that ∆plsXY requires increased levels of glycerol-3-phosphate (G3P), a GPL precursor. In agreement, ∆plsXY required supplementation with G3P for survival. Furthermore, loss of PlsX dysregulated fatty acid synthesis, resulting in increased long-chain fatty acids. We show that although PlsX/Y together contribute to PA synthesis, they also contribute to the regulation of overall membrane biogenesis. Thus, synthetic lethality of ∆plsXY is multifactorial, suggesting that PlsX/Y has been maintained as a redundant system to fine-tune the synthesis of major lipids and promote cell envelope homeostasis.IMPORTANCEGram-negative bacteria must maintain optimal ratios of glycerophospholipids and lipopolysaccharide within the cell envelope for viability. Maintenance of proper outer membrane asymmetry allows for resistance to toxins and antibiotics. Here, we describe additional roles of PlsX and PlsY in Escherichia coli beyond lysophosphatidic acid synthesis, a key precursor of all glycerophospholipids. These findings suggest that PlsX and PlsY also play a larger role in impacting homeostasis of lipid synthesis.

革兰氏阴性菌的外膜具有独特的不对称性,内叶为甘油磷脂(GPL),外叶为脂多糖(LPS)。因此,必须严格控制 GPL 和 LPS 的合成,以保持这一重要结构的完整性。我们试图破解为什么像大肠杆菌这样的生物拥有两条冗余途径--PlsB 和 PlsX/Y--来合成 GPL 前体溶血磷脂酸(LPA)。LPA 然后由 PlsC 转化为 GPL 合成的通用前体--磷脂酸(PA)。PlsB 和 PlsC 在大肠杆菌中至关重要,表明它们是 PA 合成的主要途径。虽然 PlsX 或 PlsY 的单独缺失对细胞影响不大,但两者的缺失都是致命的。为了了解这种看似多余的 PlsX/Y 通路的合成致死性,我们进行了抑制因子筛选。抑制因子分析表明,∆plsXY 需要增加 GPL 前体甘油-3-磷酸(G3P)的水平。同样,∆plsXY 需要补充 G3P 才能存活。此外,PlsX 的缺失使脂肪酸合成失调,导致长链脂肪酸增加。我们的研究表明,尽管 PlsX/Y 共同促进了 PA 的合成,但它们也有助于调节整个膜的生物生成。因此,∆plsXY 的合成致死率是多因素的,这表明 PlsX/Y 一直作为一个冗余系统维持着,以微调主要脂质的合成并促进细胞膜的平衡。重要意义革兰氏阴性细菌必须在细胞膜内保持甘油磷脂和脂多糖的最佳比例才能存活。维持适当的外膜不对称性可提高对毒素和抗生素的抵抗力。在这里,我们描述了 PlsX 和 PlsY 在大肠杆菌中除了合成溶血磷脂酸(所有甘油磷脂的关键前体)之外的其他作用。这些发现表明,PlsX 和 PlsY 还在影响脂质合成的平衡方面发挥着更大的作用。
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引用次数: 0
Metabolic interplay between Proteus mirabilis and Enterococcus faecalis facilitates polymicrobial biofilm formation and invasive disease. 奇异变形杆菌和粪肠球菌之间的代谢相互作用促进了多微生物生物膜的形成和侵袭性疾病的发生。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/mbio.02164-24
Benjamin C Hunt, Vitus Brix, Joseph Vath, Lauren Beryl Guterman, Steven M Taddei, Namrata Deka, Brian S Learman, Aimee L Brauer, Shichen Shen, Jun Qu, Chelsie E Armbruster

Biofilms play an important role in the development and pathogenesis of catheter-associated urinary tract infection (CAUTI). Proteus mirabilis and Enterococcus faecalis are common CAUTI pathogens that persistently co-colonize the catheterized urinary tract and form biofilms with increased biomass and antibiotic resistance. In this study, we uncover the metabolic interplay that drives biofilm enhancement and examine the contribution to CAUTI severity. Through compositional and proteomic biofilm analyses, we determined that the increase in biofilm biomass stems from an increase in the protein fraction of the polymicrobial biofilm. We further observed an enrichment in proteins associated with ornithine and arginine metabolism in polymicrobial biofilms compared with single-species biofilms. We show that arginine/ornithine antiport by E. faecalis promotes arginine biosynthesis and metabolism in P. mirabilis, ultimately driving the increase in polymicrobial biofilm protein content without affecting viability of either species. We further show that disrupting E. faecalis ornithine antiport alters the metabolic profile of polymicrobial biofilms and prevents enhancement, and this defect was complemented by supplementation with exogenous ornithine. In a murine model of CAUTI, ornithine antiport did not contribute to E. faecalis colonization but was required for the increased incidence of urinary stone formation and bacteremia that occurs during polymicrobial CAUTI with P. mirabilis. Thus, disrupting metabolic interplay between common co-colonizing species may represent a viable strategy for reducing risk of bacteremia.IMPORTANCEChronic infections often involve the formation of antibiotic-resistant biofilm communities that include multiple different microbes, which pose a challenge for effective treatment. In the catheterized urinary tract, potential pathogens persistently co-colonize for long periods of time and the interactions between them can lead to more severe disease outcomes. In this study, we identified the metabolite L-ornithine as a key mediator of disease-enhancing interactions between two common and challenging pathogens, Enterococcus faecalis and Proteus mirabilis. Disrupting ornithine-mediated interactions may therefore represent a strategy to prevent polymicrobial biofilm formation and decrease risk of severe disease.

生物膜在导尿管相关性尿路感染(CAUTI)的发生和致病过程中发挥着重要作用。mirabilis 变形杆菌和粪肠球菌是常见的 CAUTI 病原体,它们会持续共同定殖导尿管尿路并形成生物膜,生物量和抗生素耐药性都会增加。在本研究中,我们揭示了促使生物膜增强的代谢相互作用,并研究了其对 CAUTI 严重程度的影响。通过对生物膜的成分和蛋白质组分析,我们确定生物膜生物量的增加源于多微生物生物膜蛋白质部分的增加。我们进一步观察到,与单种生物膜相比,多微生物生物膜中与鸟氨酸和精氨酸代谢相关的蛋白质更为丰富。我们的研究表明,粪肠球菌的精氨酸/鸟氨酸反转运体促进了 mirabilis 中精氨酸的生物合成和代谢,最终推动了多微生物生物膜蛋白质含量的增加,而不会影响任何一种生物膜的存活率。我们进一步研究发现,破坏粪肠球菌鸟氨酸反转运口会改变多微生物生物膜的代谢轮廓,并阻止生物膜的增强,而补充外源性鸟氨酸可弥补这一缺陷。在小鼠 CAUTI 模型中,鸟氨酸转运体对粪肠球菌的定植没有作用,但对泌尿系结石形成和菌血症的发生率增加有作用,这种情况在奇异变形杆菌引起的多微生物 CAUTI 中会发生。因此,破坏常见共定植物种之间的新陈代谢相互作用可能是降低菌血症风险的可行策略。在导尿管泌尿道中,潜在的病原体会长期共同定殖,它们之间的相互作用会导致更严重的疾病后果。在这项研究中,我们发现代谢产物 L-鸟氨酸是粪肠球菌和奇异变形杆菌这两种常见且具有挑战性的病原体之间发生疾病增强作用的关键介质。因此,破坏鸟氨酸介导的相互作用可能是防止多微生物生物膜形成和降低严重疾病风险的一种策略。
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引用次数: 0
Unprecedented N2O production by nitrate-ammonifying Geobacteraceae with distinctive N2O isotopocule signatures. 硝酸盐氨化 Geobacteraceae 产生的前所未有的 N2O 具有独特的 N2O 同位素特征。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/mbio.02540-24
Zhenxing Xu, Shohei Hattori, Yoko Masuda, Sakae Toyoda, Keisuke Koba, Pei Yu, Naohiro Yoshida, Zong-Jun Du, Keishi Senoo

Dissimilatory nitrate reduction to ammonium (DNRA), driven by nitrate-ammonifying bacteria, is an increasingly appreciated nitrogen-cycling pathway in terrestrial ecosystems. This process reportedly generates nitrous oxide (N2O), a strong greenhouse gas with ozone-depleting effects. However, it remains poorly understood how N2O is produced by environmental nitrate-ammonifiers and how to identify DNRA-derived N2O. In this study, we characterize two novel enzymatic pathways responsible for N2O production in Geobacteraceae strains, which are predominant nitrate-ammonifying bacteria in paddy soils. The first pathway involves a membrane-bound nitrate reductase (Nar) and a hybrid cluster protein complex (Hcp-Hcr) that catalyzes the conversion of NO2- to NO and subsequently to N2O. The second pathway is observed in Nar-deficient bacteria, where the nitrite reductase (NrfA) generates NO, which is then reduced to N2O by Hcp-Hcr. These enzyme combinations are prevalent across the domain Bacteria. Moreover, we observe distinctive isotopocule signatures of DNRA-derived N2O from other established N2O production pathways, especially through the highest 15N-site preference (SP) values (43.0‰-49.9‰) reported so far, indicating a robust means for N2O source partitioning. Our findings demonstrate two novel N2O production pathways in DNRA that can be isotopically distinguished from other pathways.IMPORTANCEStimulation of DNRA is a promising strategy to improve fertilizer efficiency and reduce N2O emission in agriculture soils. This process converts water-leachable NO3- and NO2- into soil-adsorbable NH4+, thereby alleviating nitrogen loss and N2O emission resulting from denitrification. However, several studies have noted that DNRA can also be a source of N2O, contributing to global warming. This contribution is often masked by other N2O generation processes, leading to a limited understanding of DNRA as an N2O source. Our study reveals two widespread yet overlooked N2O production pathways in Geobacteraceae, the predominant DNRA bacteria in paddy soils, along with their distinctive isotopocule signatures. These findings offer novel insights into the role of the DNRA bacteria in N2O production and underscore the significance of N2O isotopocule signatures in microbial N2O source tracking.

由硝酸盐氨化细菌驱动的硝酸盐还原成氨(DNRA)是陆地生态系统中一个日益受到重视的氮循环途径。据报道,这一过程会产生一氧化二氮(N2O),这是一种具有臭氧消耗效应的强温室气体。然而,人们对环境硝酸盐氨化剂如何产生一氧化二氮以及如何识别 DNRA 衍生的一氧化二氮仍然知之甚少。在本研究中,我们描述了水稻田土壤中主要硝酸盐氨化细菌革兰氏菌株产生 N2O 的两种新型酶途径。第一种途径涉及膜结合硝酸还原酶(Nar)和杂交簇蛋白复合物(Hcp-Hcr),它们催化 NO2- 转化为 NO,然后再转化为 N2O。在缺乏 Nar 的细菌中观察到第二种途径,即亚硝酸盐还原酶(NrfA)生成 NO,然后由 Hcp-Hcr 还原成 N2O。这些酶组合在整个细菌领域都很普遍。此外,我们还观察到 DNRA 衍生的 N2O 与其他已建立的 N2O 生产途径具有不同的同位素特征,特别是通过迄今为止报道的最高 15N 位点偏好(SP)值(43.0‰-49.9‰),这表明 N2O 来源分区具有强大的手段。我们的研究结果表明,DNRA 中有两种新的 N2O 生成途径,可以从同位素角度将它们与其他途径区分开来。重要意义对 DNRA 进行优化是提高肥料效率和减少农业土壤中 N2O 排放的一种有前途的策略。这一过程将可渗出水的 NO3- 和 NO2- 转化为可被土壤吸收的 NH4+,从而减少了反硝化作用造成的氮素损失和 N2O 排放。然而,一些研究指出,DNRA 也可能成为一氧化二氮的来源,导致全球变暖。这种贡献往往被其他氧化亚氮生成过程所掩盖,导致人们对 DNRA 作为氧化亚氮来源的认识有限。我们的研究揭示了水稻田土壤中最主要的二硝基氧化还原菌(Geobacteraceae)中两种广泛存在但被忽视的一氧化二氮产生途径,以及它们独特的同位素标记。这些发现为了解 DNRA 细菌在 N2O 生产中的作用提供了新的视角,并强调了 N2O 同位素特征在微生物 N2O 源追踪中的重要性。
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引用次数: 0
Development of KSHV vaccine platforms and chimeric MHV68-K-K8.1 glycoprotein for evaluating the in vivo immunogenicity and efficacy of KSHV vaccine candidates. 开发 KSHV 疫苗平台和嵌合 MHV68-K-K8.1 糖蛋白,用于评估 KSHV 候选疫苗的体内免疫原性和有效性。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/mbio.02913-24
Wan-Shan Yang, Dokyun Kim, Soowon Kang, Chih-Jen Lai, Inho Cha, Pei-Ching Chang, Jae U Jung

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 is an etiological agent of Kaposi's Sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Considering the high seroprevalence reaching up to 80% in sub-Saharan Africa, an effective vaccine is crucial for preventing KSHV infection. However, vaccine development has been limited due to the lack of an effective animal model that supports KSHV infection. Murine Herpesvirus 68 (MHV68), a natural mouse pathogen persisting lifelong post-infection, presents a promising model for KSHV infection. In this study, we developed KSHV vaccine and a chimeric MHV68 carrying the KSHV glycoprotein, serving as a surrogate challenge virus for testing KSHV vaccines in a mouse model. Among KSHV virion glycoproteins, K8.1 is the most abundant envelope glycoprotein with the highest immunogenicity. We developed two K8.1 vaccines: K8.1 mRNA-lipid nanoparticle (LNP) vaccine and K8.126-87-Ferritin (FT) nanoparticle vaccines. Both induced humoral responses in immunized mice, whereas K8.1 mRNA LNP also induced T cell responses. Using BACmid-mediated homologous recombination, the MHV68 M7 (gp150) gene was replaced with KSHV K8.1 gene to generate chimeric MHV68-K-K8.1. MHV68-K-K8.1 established acute and latent infection in the lungs and spleens of infected mice, respectively. Mice immunized with K8.1 mRNA LNP or K8.126-87-FT showed a reduction of MHV68-K-K8.1 titer but not MHV68 wild type (WT) titer in the lung. In addition, viral reactivation of MHV68-K-K8.1 was also significantly reduced in K8.1 mRNA LNP-immunized mice. This study demonstrates the effectiveness of two vaccine candidates in providing immunity against KSHV K8.1 and introduces a surrogate MHV68 system for evaluating vaccine efficacy in vivo.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a prevalent virus that establishes lifelong persistent infection in humans and is linked to several malignancies. While antiretroviral therapy has reduced Kaposi's Sarcoma (KS) complications in people with HIV, KS still affects individuals with well-controlled HIV, older men without HIV, and transplant recipients. Despite its significant impact on human health, however, research on KSHV vaccine has been limited, mainly due to the lack of interest and the absence of a suitable animal model. This study addresses these challenges by developing KSHV K8.1 vaccine with two platforms, mRNA lipid nanoparticle (LNP) and FT nanoparticle. Additionally, chimeric virus, MHV68-K-K8.1, was created to evaluate KSHV vaccine efficacy in vivo. Vaccination of K8.1 mRNA LNP or K8.126-87-FT significantly reduced MHV68-K-K8.1 titers. Developing an effective KSHV vaccine requires an innovative approach to ensure safety and efficacy, especially for the immunocompromised population and people with limited healthcare resources. This study could be a potential blueprint for future KSHV vaccine development.

卡波西肉瘤相关疱疹病毒(KSHV)/人类疱疹病毒 8 是卡波西肉瘤、多中心卡斯特曼病和原发性渗出淋巴瘤的病原体。考虑到撒哈拉以南非洲地区高达 80% 的血清流行率,有效的疫苗对于预防 KSHV 感染至关重要。然而,由于缺乏支持 KSHV 感染的有效动物模型,疫苗的开发一直受到限制。小鼠疱疹病毒 68(MHV68)是一种天然的小鼠病原体,感染后可终生存活,是一种很有希望的 KSHV 感染模型。在这项研究中,我们开发了 KSHV 疫苗和携带 KSHV 糖蛋白的嵌合 MHV68,作为在小鼠模型中测试 KSHV 疫苗的替代挑战病毒。在 KSHV 病毒糖蛋白中,K8.1 是最丰富的包膜糖蛋白,具有最高的免疫原性。我们开发了两种 K8.1 疫苗:K8.1 mRNA-脂质纳米颗粒(LNP)疫苗和K8.126-87-铁蛋白(FT)纳米颗粒疫苗。这两种疫苗都能诱导免疫小鼠产生体液反应,而 K8.1 mRNA LNP 还能诱导 T 细胞反应。利用 BACmid 媒介同源重组,用 KSHV K8.1 基因替换 MHV68 M7(gp150)基因,生成嵌合型 MHV68-K-K8.1。MHV68-K-K8.1分别在感染小鼠的肺部和脾脏中建立了急性和潜伏感染。用 K8.1 mRNA LNP 或 K8.126-87-FT 免疫小鼠后,肺部的 MHV68-K-K8.1 滴度下降,而 MHV68 野生型(WT)滴度则没有下降。此外,K8.1 mRNA LNP免疫小鼠的MHV68-K-K8.1病毒再活化也显著降低。重要意义卡波西肉瘤相关疱疹病毒(KSHV)是一种流行的病毒,可在人体内形成终身持续感染,并与多种恶性肿瘤有关。虽然抗逆转录病毒疗法减少了艾滋病病毒感染者的卡波西肉瘤(KS)并发症,但 KS 仍然影响着艾滋病病毒控制良好的个体、未感染艾滋病病毒的老年男性和移植受者。尽管 KSHV 对人类健康有重大影响,但有关 KSHV 疫苗的研究一直很有限,主要原因是缺乏兴趣和合适的动物模型。本研究利用 mRNA 脂质纳米颗粒(LNP)和 FT 纳米颗粒这两个平台开发了 KSHV K8.1 疫苗,从而解决了这些难题。此外,研究人员还制作了嵌合病毒 MHV68-K-K8.1,以评估 KSHV 疫苗在体内的效力。接种 K8.1 mRNA LNP 或 K8.126-87-FT 后,MHV68-K-K8.1 滴度明显降低。开发有效的 KSHV 疫苗需要创新的方法来确保其安全性和有效性,尤其是对于免疫力低下的人群和医疗资源有限的人群。这项研究有可能成为未来开发 KSHV 疫苗的蓝图。
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引用次数: 0
Multiple variables influence the finely calibrated antioxidant defenses of Clostridioides difficile. 多种变量影响艰难梭菌精细校准的抗氧化防御能力。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/mbio.02544-24
Erin B Purcell

The obligate anaerobe Clostridioides difficile encodes multiple reductases to detoxify molecular oxygen and reactive oxygen species. Caulat and colleagues have characterized the activity and regulation of four such reductases (L. C. Caulat, A. Lotoux, M. C. Martins, N. Kint, et al., mBio 15:e01591-24, 2024, https://doi.org/10.1128/mbio.01591-24). Each proved critical for clostridial survival in a different range of oxygen concentrations; together, they ameliorate a broad range of oxidative stress levels. Moreover, two previously uncharacterized regulators were found to control reductase gene expression in response to oxidative stress. The genetic repressor Rex and the reductase FdpF are both sensitive to the NAP+:NADH ratio, which is affected by a cell's metabolic state as well as redox activity. While oxygen is known to influence the expression of metabolism genes in C. difficile, the mechanisms for cross-talk between the pathways that respond to oxidative and metabolic stress are not well known. The NADH dependence of Rex and FdpF may represent a newly mapped junction between these pathways.

难辨梭状芽孢杆菌(Clostridioides difficile)为多种还原酶编码,用于对分子氧和活性氧进行解毒。Caulat 及其同事描述了四种此类还原酶的活性和调控(L. C. Caulat, A. Lotoux, M. C. Martins, N. Kint, et al., mBio 15:e01591-24, 2024, https://doi.org/10.1128/mbio.01591-24)。事实证明,在不同的氧气浓度范围内,每种物质都对梭状芽孢杆菌的生存至关重要;它们共同改善了广泛的氧化应激水平。此外,研究还发现了两种以前未表征的调节因子,它们控制还原酶基因的表达以应对氧化应激。基因抑制因子 Rex 和还原酶 FdpF 都对 NAP+:NADH 比率敏感,而 NAP+:NADH 比率受细胞代谢状态和氧化还原活动的影响。虽然已知氧气会影响艰难梭菌中代谢基因的表达,但对氧化应激和代谢应激做出反应的途径之间的交叉机制还不十分清楚。Rex 和 FdpF 对 NADH 的依赖性可能代表了这些途径之间新绘制的连接点。
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引用次数: 0
On the possibility of yet a third kinetochore system in the protist phylum Euglenozoa. 关于原生动物门中可能存在第三个动核系统的问题。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-10-30 DOI: 10.1128/mbio.02936-24
Corinna Benz, Maximilian W D Raas, Pragya Tripathi, Drahomíra Faktorová, Eelco C Tromer, Bungo Akiyoshi, Julius Lukeš
<p><p>Transmission of genetic material from one generation to the next is a fundamental feature of all living cells. In eukaryotes, a macromolecular complex called the kinetochore plays crucial roles during chromosome segregation by linking chromosomes to spindle microtubules. Little is known about this process in evolutionarily diverse protists. Within the supergroup Discoba, Euglenozoa forms a speciose group of unicellular flagellates-kinetoplastids, euglenids, and diplonemids. Kinetoplastids have an unconventional kinetochore system, while euglenids have subunits that are conserved among most eukaryotes. For diplonemids, a group of extremely diverse and abundant marine flagellates, it remains unclear what kind of kinetochores are present. Here, we employed deep homology detection protocols using profile-versus-profile Hidden Markov Model searches and AlphaFold-based structural comparisons to detect homologies that might have been previously missed. Interestingly, we still could not detect orthologs for most of the kinetoplastid or canonical kinetochore subunits with few exceptions including a putative centromere-specific histone H3 variant (cenH3/CENP-A), the spindle checkpoint protein Mad2, the chromosomal passenger complex members Aurora and INCENP, and broadly conserved proteins like CLK kinase and the meiotic synaptonemal complex proteins SYCP2/3 that also function at kinetoplastid kinetochores. We examined the localization of five candidate kinetochore-associated proteins in the model diplonemid, <i>Paradiplonema papillatum. Pp</i>CENP-A shows discrete dots in the nucleus, implying that it is likely a kinetochore component. <i>Pp</i>Mad2, <i>Pp</i>CLK<sup>KKT10/19</sup>, <i>Pp</i>SYCP2L1<sup>KKT17/18</sup>, and <i>Pp</i>INCENP reside in the nucleus, but no clear kinetochore localization was observed. Altogether, these results point to the possibility that diplonemids evolved a hitherto unknown type of kinetochore system.</p><p><strong>Importance: </strong>A macromolecular assembly called the kinetochore is essential for the segregation of genetic material during eukaryotic cell division. Therefore, characterization of kinetochores across species is essential for understanding the mechanisms involved in this key process across the eukaryotic tree of life. In particular, little is known about kinetochores in divergent protists such as Euglenozoa, a group of unicellular flagellates that includes kinetoplastids, euglenids, and diplonemids, the latter being a highly diverse and abundant component of marine plankton. While kinetoplastids have an unconventional kinetochore system and euglenids have a canonical one similar to traditional model eukaryotes, preliminary searches detected neither unconventional nor canonical kinetochore components in diplonemids. Here, we employed state-of-the-art deep homology detection protocols but still could not detect orthologs for the bulk of kinetoplastid-specific nor canonical kinetochore proteins in diplone
遗传物质代代相传是所有活细胞的基本特征。在真核生物中,一种被称为动核的大分子复合物通过将染色体与纺锤体微管连接起来,在染色体分离过程中发挥着至关重要的作用。人们对进化过程中多种多样的原生生物的这一过程知之甚少。在 Discoba 超群中,Euglenozoa 形成了一个由单细胞鞭毛虫--动核细胞、Euglenid 和 diplonemids 组成的特殊群体。内生鞭毛虫有一个非常规的动核系统,而外生鞭毛虫的亚基在大多数真核生物中都是保守的。双鞭毛虫是一类种类繁多、数量巨大的海洋鞭毛虫,目前还不清楚它们的动核是哪一种。在这里,我们采用了深度同源性检测协议,利用profile-vers-us-profile隐马尔可夫模型搜索和基于AlphaFold的结构比较来检测以前可能被忽略的同源性。有趣的是,我们仍然无法检测到大多数动点细胞或典型动点支链亚基的同源物,只有少数例外,包括推测的中心粒特异性组蛋白 H3 变体(cenH3/CENP-A)、纺锤体检查点蛋白 Mad2、染色体客体复合体成员 Aurora 和 INCENP,以及广泛保守的蛋白,如 CLK 激酶和减数分裂突触复合体蛋白 SYCP2/3,它们也在动点细胞的动点支链上发挥作用。我们研究了五种候选动点相关蛋白在模式双鞭毛目(Paradiplonema papillatum)中的定位情况。PpCENP-A 在细胞核中显示出离散的小点,这意味着它很可能是一个动点元件。PpMad2、PpCLKKKT10/19、PpSYCP2L1KKT17/18 和 PpINCENP 位于细胞核中,但没有观察到明确的动点定位。总之,这些结果表明,双子叶植物可能进化出了一种迄今未知的动核系统:重要性:在真核细胞分裂过程中,遗传物质的分离离不开一种被称为动核的大分子组装体。因此,要了解真核生命树中这一关键过程所涉及的机制,就必须确定不同物种动核的特征。特别是,人们对不同原生动物(如优格伦动物)中的动核知之甚少,优格伦动物是一类单细胞鞭毛虫,包括动核细胞、优格伦动物和双鞭毛虫,后者是海洋浮游生物中种类繁多、数量巨大的组成部分。动核鞭毛虫有一个非常规的动核系统,而尤格林鞭毛虫有一个与传统模式真核生物类似的标准动核系统,但初步搜索发现双鞭毛虫中既没有非常规的也没有标准的动核元件。在这里,我们采用了最先进的深度同源性检测方案,但除了一种推测的中心粒特异性组蛋白 H3 变体外,仍然无法在双膜动物中检测到大部分动点细胞特异性或标准动点核心蛋白的同源物。我们的研究结果表明,双螺旋虫进化出的动核与之前已知的动核并不相似。
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