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Shining a light on Candida-induced epithelial damage with a luciferase reporter. 用荧光素酶报告器揭示念珠菌诱导的上皮损伤
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-16 DOI: 10.1128/msphere.00509-24
Millen Tesfamariam, Raghav Vij, Verena Trümper, Bernhard Hube, Sascha Brunke

Host cell damage is a key parameter for research in infection biology, drug testing, and substance safety screening. In this study, we introduce a luciferase reporter system as a new and reliable assay to measure cell damage and validate it with the pathogenic yeast, Candida albicans, as a test case. We transduced human epithelial cell lines with a lentiviral vector to stably express an optimized luciferase enzyme, Nanoluc. Upon cell damage, the release of cytoplasmic luciferase into the extracellular space can be easily detected by a luminometer. We used the luciferase reporter system to investigate the damage caused by C. albicans to different newly generated epithelial reporter cell lines. We found that fungus-induced cell damage, as determined by established methods, correlated tightly with the release of the luciferase. The new luciferase reporter system is a simple, sensitive, robust, and inexpensive method for measuring host cell damage and has a sensitivity comparable to the standard assay, release of lactate dehydrogenase. It is suitable for high-throughput studies of pathogenesis mechanisms of any microbe, for antimicrobial drug screening, and many other applications.IMPORTANCEWe present a quick, easy, inexpensive, and reliable assay to measure damage to mammalian cells. To this end, we created reporter cell lines which artificially express luciferase, an enzyme that can be easily detected in the supernatant when these cells are damaged. We used infections with the well-investigated fungal pathogen of humans, Candida albicans, as a test case of our system. Using our reporter, we were able to recapitulate the known effects of strain variability, gene deletions, and antifungal treatments on host cell damage. This easily adaptable reporter system can be used to screen for damage in infection models with different microbial species, assay cell-damaging potential of substances, discover new non-toxic antibiotics, and many other damage-based applications.

宿主细胞损伤是感染生物学、药物测试和物质安全性筛选研究中的一个关键参数。在本研究中,我们引入了荧光素酶报告系统作为一种新的、可靠的细胞损伤检测方法,并以致病性酵母菌--白色念珠菌--为试验案例进行了验证。我们用慢病毒载体转导人类上皮细胞系,使其稳定表达一种优化的荧光素酶--Nanoluc。细胞受损时,细胞质中的荧光素酶释放到细胞外空间,可以很容易地用荧光计检测到。我们利用荧光素酶报告系统研究了白僵菌对不同新生成的上皮报告细胞系造成的损伤。我们发现,根据既定方法测定的真菌诱导的细胞损伤与荧光素酶的释放密切相关。新的荧光素酶报告系统是一种测量宿主细胞损伤的简单、灵敏、稳健且成本低廉的方法,其灵敏度可与标准检测方法--乳酸脱氢酶的释放相媲美。它适用于任何微生物致病机制的高通量研究、抗菌药物筛选以及许多其他应用。重要意义我们提出了一种快速、简便、廉价且可靠的检测方法来测量哺乳动物细胞的损伤。为此,我们创建了人工表达荧光素酶的报告细胞系,当这些细胞受到破坏时,上清液中很容易检测到这种酶。我们用已调查清楚的人类真菌病原体--白色念珠菌的感染作为我们系统的测试案例。利用我们的报告器,我们能够再现已知的菌株变异、基因缺失和抗真菌治疗对宿主细胞损伤的影响。这种易于改造的报告系统可用于筛选不同微生物物种感染模型中的损伤、检测细胞损伤物质的潜力、发现新的无毒抗生素以及许多其他基于损伤的应用。
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引用次数: 0
The Plasmodium falciparum histone methyltransferase SET10 participates in a chromatin modulation network crucial for intraerythrocytic development. 恶性疟原虫组蛋白甲基转移酶 SET10 参与了对红细胞内发育至关重要的染色质调节网络。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-24 DOI: 10.1128/msphere.00495-24
Jean-Pierre Musabyimana, Sherihan Musa, Janice Manti, Ute Distler, Stefan Tenzer, Che Julius Ngwa, Gabriele Pradel
<p><p>The lifecycle progression of the malaria parasite <i>Plasmodium falciparum</i> requires precise tuning of gene expression including histone methylation. The histone methyltransferase <i>Pf</i>SET10 was previously described as an H3K4 methyltransferase involved in <i>var</i> gene regulation, making it a prominent antimalarial target. In this study, we investigated the role of <i>Pf</i>SET10 in the blood stages of <i>P. falciparum</i> in more detail, using tagged <i>Pf</i>SET10-knockout (KO) and -knockdown (KD) lines. We demonstrate a nuclear localization of <i>Pf</i>SET10 with peak protein levels in schizonts. <i>Pf</i>SET10 deficiency reduces intraerythrocytic growth but has no effect on gametocyte commitment and maturation. Screening of the <i>Pf</i>SET10-KO line for histone methylation variations reveals that lack of <i>Pf</i>SET10 renders the parasites unable to mark H3K18me1, while no reduction in the H3K4 methylation status could be observed. Comparative transcriptomic profiling of <i>Pf</i>SET10-KO schizonts shows an upregulation of transcripts particularly encoding proteins linked to red blood cell remodeling and antigenic variation, suggesting a repressive function of the histone methylation mark. TurboID coupled with mass spectrometry further highlights an extensive nuclear <i>Pf</i>SET10 interaction network with roles in transcriptional regulation and mRNA processing, DNA replication and repair, and chromatin remodeling. The main interactors of <i>Pf</i>SET10 include ApiAP2 transcription factors, epigenetic regulators like <i>Pf</i>HDAC1, chromatin modulators like <i>Pf</i>MORC and <i>Pf</i>ISWI, mediators of RNA polymerase II, and DNA replication licensing factors. The combined data pinpoint <i>Pf</i>SET10 as a histone methyltransferase essential for H3K18 methylation that regulates nucleic acid metabolic processes in the <i>P. falciparum</i> blood stages as part of a comprehensive chromatin modulation network.IMPORTANCEThe fine-tuned regulation of DNA replication and transcription is particularly crucial for the rapidly multiplying blood stages of malaria parasites and proteins involved in these processes represent important drug targets. This study demonstrates that contrary to previous reports the histone methyltransferase <i>Pf</i>SET10 of the malaria parasite <i>Plasmodium falciparum</i> promotes the methylation of histone 3 at lysine K18, a histone mark to date not well understood. Deficiency of <i>Pf</i>SET10 due to genetic knockout affects genes involved in intraerythrocytic development. Furthermore, in the nuclei of blood-stage parasites, <i>Pf</i>SET10 interacts with various protein complexes crucial for DNA replication, remodeling, and repair, as well as for transcriptional regulation and mRNA processing. In summary, this study highlights <i>Pf</i>SET10 as a methyltransferase affecting H3K18 methylation with critical functions in chromatin maintenance during the development of <i>P. falciparum</i> in red blood cells.</
恶性疟原虫生命周期的进展需要对包括组蛋白甲基化在内的基因表达进行精确调节。组蛋白甲基转移酶 PfSET10 先前被描述为一种参与变异基因调控的 H3K4 甲基转移酶,因此成为一个重要的抗疟靶标。在这项研究中,我们使用标记的 PfSET10 基因敲除(KO)和基因敲除(KD)系,更详细地研究了 PfSET10 在恶性疟原虫血液阶段的作用。我们证明了 PfSET10 的核定位,其蛋白水平在裂殖体中达到峰值。PfSET10 缺乏会降低红细胞内的生长,但对配子细胞的形成和成熟没有影响。对 PfSET10-KO 株系进行组蛋白甲基化变异筛选后发现,缺乏 PfSET10 会使寄生虫无法标记 H3K18me1,而 H3K4 甲基化状态则不会降低。对 PfSET10-KO 裂殖体进行的转录组比较分析表明,编码与红细胞重塑和抗原变异有关的蛋白质的转录本上调,这表明组蛋白甲基化标记具有抑制功能。TurboID 与质谱联用进一步凸显了广泛的核 PfSET10 相互作用网络,该网络在转录调控和 mRNA 处理、DNA 复制和修复以及染色质重塑中发挥作用。PfSET10 的主要相互作用者包括 ApiAP2 转录因子、PfHDAC1 等表观遗传调节因子、PfMORC 和 PfISWI 等染色质调节因子、RNA 聚合酶 II 的介导因子以及 DNA 复制许可因子。综合数据表明,PfSET10 是一种组蛋白甲基转移酶,对 H3K18 甲基化至关重要,它调节恶性疟原虫血液阶段的核酸代谢过程,是全面染色质调节网络的一部分。重要意义DNA 复制和转录的微调调节对快速繁殖的血液阶段疟疾寄生虫尤为重要,参与这些过程的蛋白质是重要的药物靶标。这项研究表明,与之前的报道相反,恶性疟原虫的组蛋白甲基转移酶 PfSET10 能促进组蛋白 3 赖氨酸 K18 的甲基化,而迄今为止人们对这种组蛋白标记还不甚了解。基因敲除导致的 PfSET10 缺乏会影响参与红细胞内发育的基因。此外,在血期寄生虫的细胞核中,PfSET10 与对 DNA 复制、重塑和修复以及转录调控和 mRNA 处理至关重要的各种蛋白复合物相互作用。总之,本研究强调了 PfSET10 是一种影响 H3K18 甲基化的甲基转移酶,在恶性疟原虫在红细胞中的发育过程中对染色质的维持具有关键作用。
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引用次数: 0
mSphere of Influence: Revisiting the central dogma, again! mSphere of Influence:再次重温中心教条!
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-16 DOI: 10.1128/msphere.00398-24
Parimal Samir

Dr. Parimal Samir works in the field of host-pathogen interactions. In this mSphere of Influence article, he reflects on how the manuscript entitled "De novo gene synthesis by an antiviral reverse transcriptase" by Samuel Sternberg and colleagues made an impact by reminding him that there is still so much to discover in life sciences.

Parimal Samir 博士从事宿主与病原体相互作用领域的工作。在这篇 mSphere of Influence 文章中,他回顾了塞缪尔-斯特恩伯格(Samuel Sternberg)及其同事撰写的题为 "抗病毒逆转录酶的新基因合成"(De novo gene synthesis by an antiviral reverse transcriptase)的手稿如何对他产生影响,提醒他在生命科学领域还有很多东西有待发现。
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引用次数: 0
Erratum for Hassall et al., "Dissecting Individual Interactions between Pathogenic and Commensal Bacteria within a Multispecies Gut Microbial Community". 对 Hassall 等人 "剖析多物种肠道微生物群落中致病菌与共生菌之间的个体相互作用 "的勘误。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 DOI: 10.1128/msphere.00893-24
Jack Hassall, Jeffery K J Cheng, Meera Unnikrishnan
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引用次数: 0
Chlamydia trachomatis Inc Ct226 is vital for FLI1 and LRRF1 recruitment to the chlamydial inclusion. 沙眼衣原体 Inc Ct226 对 FLI1 和 LRRF1 招募到衣原体包涵体至关重要。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-15 DOI: 10.1128/msphere.00473-24
Natalie A Sturd, Lindsey A Knight, Macy G Wood, Legacy Durham, Scot P Ouellette, Elizabeth A Rucks

The obligate intracellular pathogen, Chlamydia trachomatis, establishes an intracellular niche within a host membrane-derived vacuole called the chlamydial inclusion. From within this inclusion, C. trachomatis orchestrates numerous host-pathogen interactions, in part, by utilizing a family of type III secreted effectors, termed inclusion membrane proteins (Incs). Incs are embedded within the inclusion membrane, and some function to recruit host proteins to the inclusion. Two such recruited host proteins are leucine rich repeat Flightless-1 interacting protein 1 (LRRF1/LRRFIP1) and its binding partner Flightless 1 (FLI1/FLII). Previously, LRRF1 has been shown to interact with Inc protein Ct226/CTL0478. This is the first study to examine interactions of FLI1 with candidate Incs or with LRRF1 during infection. We hypothesized that FLI1 recruitment to the inclusion would be dependent on LRRF1 localization. We demonstrated that FLI1 co-immunoprecipitated with Ct226 but only in the presence of LRRF1. Furthermore, FLI1 localized to the inclusion when LRRF1 was depleted via small interfering RNA, suggesting that FLI1 may have an alternative recruitment mechanism. We further developed a series of CRISPRi knockdown and complementation strains in C. trachomatis serovar L2 targeting ct226 and co-transcribed candidate Incs, ct225 and ct224. Simultaneous knockdown of ct226, ct225, and ct224 prevented localization of both FLI1 and LRRF1 to the inclusion, and only complementation of ct226 restored their localization. Thus, we demonstrated Ct226 is critical for FLI1 and LRRF1 localization to the inclusion. Our results also indicate an LRRF1-independent localization mechanism for FLI1, which likely influence their mechanism(s) of action during chlamydial infection.IMPORTANCEChlamydia trachomatis is a leading cause of both bacterial sexually transmitted infections and preventable infectious blindness worldwide. As an obligate intracellular pathogen, C. trachomatis has evolved multiple ways of manipulating the host to establish a successful infection. As such, it is important to understand host-chlamydial protein-protein interactions as these reveal strategies that C. trachomatis uses to shape its intracellular environment. This study looks in detail at interactions of two host proteins, FLI1 and LRRF1, during chlamydial infection. Importantly, the series of CRISPR inference knockdown and complement strains developed in this study suggest these proteins have both independent and overlapping mechanisms for localization, which ultimately will dictate how these proteins function during chlamydial infection.

沙眼衣原体(Chlamydia trachomatis)是一种强制性细胞内病原体,它在宿主膜衍生的空泡(称为衣原体包涵体)中建立了一个细胞内生态位。在这种包涵体中,沙眼衣原体通过利用被称为包涵膜蛋白(Incs)的 III 型分泌效应物家族,协调宿主与病原体之间的许多相互作用。包涵膜蛋白(Incs)嵌入包涵膜中,其中一些具有将宿主蛋白招募到包涵膜中的功能。富亮氨酸重复Flightless-1相互作用蛋白1(LRRF1/LRRFIP1)及其结合伙伴Flightless 1(FLI1/FLII)就是这样两种被招募的宿主蛋白。此前,LRRF1 已被证明与 Inc 蛋白 Ct226/CTL0478 相互作用。这是首次研究 FLI1 与候选 Incs 或 LRRF1 在感染过程中的相互作用。我们假设,FLI1 招募到包涵体将取决于 LRRF1 的定位。我们证明了 FLI1 与 Ct226 的共沉淀,但只有在 LRRF1 存在的情况下才能发生。此外,当通过小干扰 RNA 删除 LRRF1 时,FLI1 会定位到包涵体,这表明 FLI1 可能有另一种招募机制。我们进一步在沙眼衣原体血清 L2 中开发了一系列 CRISPRi 基因敲除和互补菌株,靶向 ct226 和共转录候选 Incs(ct225 和 ct224)。同时敲除ct226、ct225和ct224会阻止FLI1和LRRF1在包涵体上的定位,只有对ct226进行互补才能恢复它们的定位。因此,我们证明了 Ct226 对于 FLI1 和 LRRF1 在包涵体上的定位至关重要。我们的研究结果还表明,FLI1 的定位机制与 LRRF1 无关,这可能会影响它们在衣原体感染过程中的作用机制。作为一种强制性细胞内病原体,沙眼衣原体已进化出多种操纵宿主的方法,以成功建立感染。因此,了解宿主-衣原体蛋白质-蛋白质之间的相互作用非常重要,因为这些相互作用揭示了沙眼衣原体用来塑造其细胞内环境的策略。本研究详细研究了衣原体感染过程中两种宿主蛋白 FLI1 和 LRRF1 的相互作用。重要的是,本研究开发的一系列CRISPR推断敲除和互补菌株表明,这些蛋白质具有独立和重叠的定位机制,这最终将决定这些蛋白质在衣原体感染过程中如何发挥作用。
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引用次数: 0
The Plasmodium falciparum histone methyltransferase PfSET10 is dispensable for the regulation of antigenic variation and gene expression in blood-stage parasites. 恶性疟原虫组蛋白甲基转移酶PfSET10对于血期寄生虫抗原变异和基因表达的调控是不可或缺的。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-24 DOI: 10.1128/msphere.00546-24
Matthias Wyss, Abhishek Kanyal, Igor Niederwieser, Richard Bartfai, Till S Voss
<p><p>The malaria parasite <i>Plasmodium falciparum</i> employs antigenic variation of the virulence factor <i>P. falciparum</i> erythrocyte membrane protein 1 (PfEMP1) to escape adaptive immune responses during blood infection. Antigenic variation of PfEMP1 occurs through epigenetic switches in the mutually exclusive expression of individual members of the multi-copy <i>var</i> gene family. <i>var</i> genes are located in perinuclear clusters of transcriptionally inactive heterochromatin. Singular <i>var</i> gene activation is linked to locus repositioning into a dedicated zone at the nuclear periphery and deposition of histone 3 lysine 4 di-/trimethylation (H3K4me2/3) and H3K9 acetylation marks in the promoter region. While previous work identified the putative H3K4-specific methyltransferase PfSET10 as an essential enzyme and positive regulator of <i>var</i> gene expression, a recent study reported conflicting data. Here, we used iterative genome editing to engineer a conditional PfSET10 knockout line tailored to study the function of PfSET10 in <i>var</i> gene regulation. We demonstrate that PfSET10 is not required for mutually exclusive <i>var</i> gene expression and switching. We also show that PfSET10 is dispensable not only for asexual parasite proliferation but also for sexual conversion and gametocyte differentiation. Furthermore, comparative RNA-seq experiments revealed that PfSET10 plays no obvious role in regulating gene expression during asexual parasite development and gametocytogenesis. Interestingly, however, PfSET10 shows different subnuclear localization patterns in asexual and sexual stage parasites and female-specific expression in mature gametocytes. In summary, our work confirms in detail that PfSET10 is not involved in regulating <i>var</i> gene expression and is not required for blood-stage parasite viability, indicating PfSET10 may be important for life cycle progression in the mosquito vector or during liver stage development.IMPORTANCEThe malaria parasite <i>Plasmodium falciparum</i> infects hundreds of millions of people every year. To survive and proliferate in the human bloodstream, the parasites need to escape recognition by the host's immune system. To achieve this, <i>P. falciparum</i> can change the expression of surface antigens <i>via</i> a process called antigenic variation. This fascinating survival strategy is based on infrequent switches in the expression of single members of the <i>var</i> multigene family. Previous research reported conflicting results on the role of the epigenetic regulator PfSET10 in controlling mutually exclusive <i>var</i> gene expression and switching. Here, we unequivocally demonstrate that PfSET10 is neither required for antigenic variation nor the expression of any other proteins during blood-stage infection. This information is critical in directing our attention toward exploring alternative molecular mechanisms underlying the control of antigenic variation and investigating the
恶性疟原虫利用毒力因子恶性疟原虫红细胞膜蛋白 1(PfEMP1)的抗原变异来逃避血液感染过程中的适应性免疫反应。PfEMP1 的抗原变异是通过多拷贝变异基因家族各成员互斥表达的表观遗传开关发生的。单个 var 基因的激活与基因座重新定位到核外围的专用区域以及启动子区域组蛋白 3 赖氨酸 4 二/三甲基化(H3K4me2/3)和 H3K9 乙酰化标记的沉积有关。之前的研究发现,推测的 H3K4 特异性甲基转移酶 PfSET10 是变异基因表达的必需酶和正调控因子,但最近的一项研究报告了相互矛盾的数据。在这里,我们利用迭代基因组编辑技术设计了一个条件性 PfSET10 基因敲除品系,以研究 PfSET10 在 var 基因调控中的功能。我们证明,PfSET10 并不是相互排斥的变异基因表达和切换所必需的。我们还发现,PfSET10 不仅对无性寄生虫的增殖是不可或缺的,而且对有性转化和配子细胞分化也是不可或缺的。此外,RNA-seq 比较实验显示,PfSET10 在无性寄生虫发育和配子细胞发生过程中没有明显的基因表达调控作用。但有趣的是,PfSET10 在无性寄生虫和有性寄生虫中显示出不同的核下定位模式,并在成熟配子细胞中显示出雌性特异性表达。总之,我们的工作详细证实了 PfSET10 不参与调节变异基因的表达,也不是血期寄生虫存活所必需的,这表明 PfSET10 可能对蚊媒的生命周期进展或肝脏阶段的发育很重要。为了在人体血液中生存和繁殖,寄生虫需要躲避宿主免疫系统的识别。为此,恶性疟原虫可以通过一种叫做抗原变异的过程来改变表面抗原的表达。这种引人入胜的生存策略是基于变异多基因家族中单个成员表达的不频繁切换。以前的研究报告称,表观遗传调节因子 PfSET10 在控制相互排斥的变异基因表达和切换中的作用存在相互矛盾的结果。在这里,我们明确证明了 PfSET10 既不需要抗原变异,也不需要在血期感染过程中表达任何其他蛋白。这一信息对于引导我们探索控制抗原变异的其他分子机制以及研究 PfSET10 在其他生命周期阶段的功能至关重要。
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引用次数: 0
Mouse innate resistance to Neospora caninum infection is driven by early production of IFNγ by NK cells in response to parasite ligands. [公式:见正文]小鼠对犬新孢子虫感染的先天抵抗力是由 NK 细胞对寄生虫配体早期产生的 IFNγ 驱动的。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-24 DOI: 10.1128/msphere.00255-24
R S Coombs, A E Overacre-Delgoffe, A Bhattacharjee, T W Hand, J P Boyle

Toxoplasma gondii is capable of being transmitted by nearly all warm-blooded animals, and rodents are a major source of parasite dissemination, yet mechanisms driving its broad host range are poorly understood. Although a phylogenetically close relative of T. gondii, Neospora caninum differs from T. gondii in that it does not infect mice and only infects a small number of ruminant and canine species. We recently showed that T. gondii and N. caninum grow similarly in mice during the first 24 h post-infection, but only N. caninum induces an IFNγ-driven response within hours that controls the infection. The goal of the present study was to understand the cellular basis of this rapid response to N. caninum. To do this, we compared immune cell populations at the site of infection 4 h after T. gondii or N. caninum infection in mice. We found that both parasites induced similar frequencies of peritoneal monocytes, while macrophages and dendritic cell populations were not increased compared to uninfected mice. Through a series of knockout mouse experiments, we show that B, T, and NKT cells are not required for immediate IFNγ production and ultimate control of N. caninum infection, suggesting that natural killer (NK) cells are the primary inducers of immediate IFNγ in response to N. caninum. N. caninum infections exhibited significantly more IFNγ+ NK cells in the peritoneum compared with T. gondii-infected and uninfected mice. Finally, we demonstrate that differences in early IFNγ production during N. caninum and T. gondii infections in mice are at least partly due to differences in soluble antigen(s) produced by tachyzoites.

Importance: Pathogen differences in host range are poorly understood at the molecular level even though even closely related pathogen species can have dramatically distinct host ranges. Here, we study two related parasite species that have a dramatic difference in their ability to infect mice. Here, we show that soluble proteins from these species determine one driver of this difference: induction of interferon gamma by cells of the innate immune system.

弓形虫几乎可以通过所有温血动物传播,而啮齿类动物是寄生虫传播的主要来源,但人们对其广泛宿主范围的机制却知之甚少。尽管在系统发育上与淋病双孢子虫是近亲,但犬新孢子虫与淋病双孢子虫不同,它不感染小鼠,只感染少数反刍动物和犬科动物。我们最近的研究表明,在感染后的头 24 小时内,淋病双孢子虫和犬新孢子虫在小鼠体内的生长情况相似,但只有犬新孢子虫能在数小时内诱导 IFNγ 驱动的反应,从而控制感染。本研究的目的是了解这种对 N. caninum 快速反应的细胞基础。为此,我们比较了小鼠感染 T. gondii 或 N. caninum 4 小时后感染部位的免疫细胞群。我们发现这两种寄生虫诱导的腹膜单核细胞的频率相似,而巨噬细胞和树突状细胞的数量与未感染的小鼠相比并没有增加。通过一系列基因敲除小鼠实验,我们发现B、T和NKT细胞不是产生即时IFNγ和最终控制犬疫母细胞感染所必需的,这表明自然杀伤(NK)细胞是应对犬疫母细胞感染的即时IFNγ的主要诱导因子。与淋球菌感染小鼠和未感染小鼠相比,N. caninum 感染小鼠腹膜中的 IFNγ+ NK 细胞明显增多。最后,我们证明了小鼠感染 N. caninum 和 T. gondii 期间早期 IFNγ 产生的差异至少部分是由于速生虫产生的可溶性抗原的差异:病原体在宿主范围上的差异在分子水平上还鲜为人知,即使是密切相关的病原体物种也可能具有截然不同的宿主范围。在这里,我们研究了两种相关的寄生虫,它们感染小鼠的能力存在巨大差异。在这里,我们发现这两种寄生虫的可溶性蛋白决定了这种差异的一个驱动因素:先天性免疫系统细胞诱导γ干扰素。
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引用次数: 0
Candida glabrata maintains two HAP1 ohnologs, HAP1A and HAP1B, for distinct roles in ergosterol gene regulation to mediate sterol homeostasis under azole and hypoxic conditions. 白色念珠菌有两个 HAP1 同源物,即 HAP1A 和 HAP1B,它们在麦角甾醇基因调控中发挥不同的作用,在偶氮唑和缺氧条件下介导甾醇平衡。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-23 DOI: 10.1128/msphere.00524-24
Debasmita Saha, Justin B Gregor, Smriti Hoda, Katharine E Eastman, Victor A Gutierrez-Schultz, Mindy Navarrete, Jennifer H Wisecaver, Scott D Briggs

Candida glabrata exhibits innate resistance to azole antifungal drugs but also has the propensity to rapidly develop clinical drug resistance. Azole drugs, which target Erg11, is one of the major classes of antifungals used to treat Candida infections. Despite their widespread use, the mechanism controlling azole-induced ERG gene expression and drug resistance in C. glabrata has primarily revolved around Upc2 and/or Pdr1. Phylogenetic and syntenic analyses revealed that C. glabrata, following a whole genome duplication event, maintained HAP1A and HAP1B, whereas Saccharomyces cerevisiae only retained the HAP1A ortholog, HAP1. In this study, we determined the function of two zinc cluster transcription factors, Hap1A and Hap1B, as direct regulators of ERG genes. In S. cerevisiae, Hap1, an ortholog of Hap1A, is a known transcription factor controlling ERG gene expression under aerobic and hypoxic conditions. Interestingly, deleting HAP1 or HAP1B in either S. cerevisiae or C. glabrata, respectively, showed altered susceptibility to azoles. In contrast, the strain deleted for HAP1A did not exhibit azole susceptibility. We also determined that the increased azole susceptibility in a hap1BΔ strain is attributed to decreased azole-induced expression of ERG genes, resulting in decreased levels of total ergosterol. Surprisingly, Hap1A protein expression is barely detected under aerobic conditions but is specifically induced under hypoxic conditions, where Hap1A is required for the repression of ERG genes. However, in the absence of Hap1A, Hap1B can compensate as a transcriptional repressor. Our study shows that Hap1A and Hap1B is utilized by C. glabrata to adapt to specific host and environmental conditions.

Importance: Invasive and drug-resistant fungal infections pose a significant public health concern. Candida glabrata, a human fungal pathogen, is often difficult to treat due to its intrinsic resistance to azole antifungal drugs and its capacity to develop clinical drug resistance. Therefore, understanding the pathways that facilitate fungal growth and environmental adaptation may lead to novel drug targets and/or more efficacious antifungal therapies. While the mechanisms of azole resistance in Candida species have been extensively studied, the roles of zinc cluster transcription factors, such as Hap1A and Hap1B, in C. glabrata have remained largely unexplored until now. Our research shows that these factors play distinct yet crucial roles in regulating ergosterol homeostasis under azole drug treatment and oxygen-limiting growth conditions. These findings offer new insights into how this pathogen adapts to different environmental conditions and enhances our understanding of factors that alter drug susceptibility and/or resistance.

胶状念珠菌(Candida glabrata)对唑类抗真菌药物具有先天耐药性,但也有迅速产生临床耐药性的倾向。针对 Erg11 的唑类药物是治疗念珠菌感染的主要抗真菌药物之一。尽管唑类药物被广泛使用,但控制唑类药物诱导的 ERG 基因表达和光滑念珠菌耐药性的机制主要围绕着 Upc2 和/或 Pdr1。系统发育和同源分析表明,在全基因组复制事件后,草履虫保留了 HAP1A 和 HAP1B,而酿酒酵母只保留了 HAP1A 的直向同源物 HAP1。在这项研究中,我们确定了两个锌簇转录因子 Hap1A 和 Hap1B 作为 ERG 基因直接调控因子的功能。在 S. cerevisiae 中,Hap1A 的同源物 Hap1 是一种已知的转录因子,在有氧和缺氧条件下控制 ERG 基因的表达。有趣的是,在 S. cerevisiae 或 C. glabrata 中分别删除 HAP1 或 HAP1B 会改变对唑类的敏感性。相比之下,缺失 HAP1A 的菌株对唑类没有敏感性。我们还确定,hap1BΔ菌株的唑敏感性增加是由于唑诱导的ERG基因表达减少,导致麦角甾醇总量减少。令人惊讶的是,在有氧条件下几乎检测不到 Hap1A 蛋白的表达,但在缺氧条件下,Hap1A 蛋白会被特异性诱导,而在缺氧条件下,Hap1A 是抑制 ERG 基因所必需的。然而,在缺乏 Hap1A 的情况下,Hap1B 可以作为转录抑制因子进行补偿。我们的研究表明,Hap1A和Hap1B可被C. glabrata利用来适应特定的宿主和环境条件:侵袭性和耐药性真菌感染是一个重大的公共卫生问题。由于对唑类抗真菌药物的固有耐药性及其产生临床耐药性的能力,人类真菌病原体--白色念珠菌往往难以治疗。因此,了解促进真菌生长和环境适应的途径可能会发现新的药物靶点和/或更有效的抗真菌疗法。虽然对念珠菌的唑类抗药性机制进行了广泛的研究,但迄今为止,锌簇转录因子(如 Hap1A 和 Hap1B)在草绿色念珠菌中的作用在很大程度上仍未得到探索。我们的研究表明,在唑类药物治疗和限氧生长条件下,这些因子在调节麦角固醇稳态方面发挥着独特而关键的作用。这些发现为我们了解这种病原体如何适应不同的环境条件提供了新的视角,并加深了我们对改变药物敏感性和/或耐药性的因素的理解。
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引用次数: 0
Effect of C-to-T transition at CpG sites on tumor suppressor genes in tumor development in cattle evaluated by somatic mutation analysis in enzootic bovine leukosis. 通过对牛白血病的体细胞突变分析,评估CpG位点的C-T转换对肿瘤抑制基因在牛肿瘤发生中的影响。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-15 DOI: 10.1128/msphere.00216-24
Asami Nishimori, Kiyohiko Andoh, Yuichi Matsuura, Tomohiro Okagawa, Satoru Konnai

Oncogenic transformation of normal cells is caused by mutations and chromosomal abnormalities in cancer-related genes. Enzootic bovine leukosis (EBL) is a malignant B-cell lymphoma caused by bovine leukemia virus (BLV) infection in cattle. Although a small fraction of BLV-infected cattle develops EBL after a long latent period, the mechanisms for oncogenesis in EBL cattle remain largely unknown. In this study, we analyzed the types and patterns of somatic mutations in cancer cells from 36 EBL cases, targeting 21 cancer-related genes. Various somatic mutations were identified in eight genes, TP53, KMT2D, CREBBP, KRAS, PTEN, NOTCH1, MYD88, and CARD11. In addition, TP53 gene was found to be mutated in 69.4% of EBL cases, with most being biallelic mutations. In some cases, associations were observed between the ages at which cattle had developed EBL and somatic mutation patterns; young onset of EBL possibly occurs due to high impact mutations affecting protein translation and biallelic mutations. Furthermore, nucleotide substitution patterns indicated that cytosine at CpG sites tended to be converted to thymine in many EBL cases, which was considered to be the result of spontaneous deamination of 5-methylcytosine. These results demonstrate how somatic mutations have occurred in cancer cells leading to EBL development, thereby explaining its pathogenic mechanism. These findings will contribute to a better understanding and future elucidation of disease progression in BLV infection.IMPORTANCEEnzootic bovine leukosis (EBL) is a malignant and lethal disease in cattle. Currently, there are no effective vaccines or therapeutic methods against bovine leukemia virus (BLV) infection, resulting in severe economic losses in livestock industry. This study provides a renewed hypothesis to explain the general mechanisms of EBL onset by combining the previous finding that several integration sites of BLV provirus can affect the increase in survival and proliferation of infected cells. We demonstrate that two additional random events are necessary for oncogenic transformation in infected cell clones, elucidating the reason why only few infected cattle develop EBL. Further exploration of somatic mutation and BLV integration sites could support this hypothesis more firmly, potentially contributing to the development of novel control methods for EBL onset.

正常细胞的致癌转化是由癌症相关基因的突变和染色体异常引起的。牛白血病(EBL)是一种由牛白血病病毒(BLV)感染引起的恶性 B 细胞淋巴瘤。虽然一小部分感染了BLV的牛经过长期潜伏后会患上EBL,但EBL牛的肿瘤发生机制在很大程度上仍是未知的。在这项研究中,我们针对 21 个癌症相关基因,分析了 36 例 EBL 病例癌细胞中体细胞突变的类型和模式。我们在 TP53、KMT2D、CREBBP、KRAS、PTEN、NOTCH1、MYD88 和 CARD11 这 8 个基因中发现了各种体细胞突变。此外,在69.4%的EBL病例中发现TP53基因发生了突变,其中大多数为双拷贝突变。在某些病例中,观察到牛发生 EBL 的年龄与体细胞突变模式之间存在关联;EBL 的年轻发病可能是由于影响蛋白质翻译的高影响突变和双倍突变。此外,核苷酸替换模式表明,在许多 EBL 病例中,CpG 位点上的胞嘧啶倾向于转化为胸腺嘧啶,这被认为是 5-甲基胞嘧啶自发脱氨基的结果。这些结果表明了体细胞突变是如何导致 EBL 发生的,从而解释了其致病机制。重要意义牛白血病(EBL)是牛的一种恶性致命疾病。目前,针对牛白血病病毒(BLV)感染尚无有效的疫苗或治疗方法,给畜牧业造成了严重的经济损失。本研究结合之前发现的 BLV 前病毒的几个整合位点会影响感染细胞的存活率和增殖率的增加,提出了一个新的假设来解释 EBL 发病的一般机制。我们证明,受感染细胞克隆的致癌转化还需要另外两个随机事件,从而阐明了为什么只有少数受感染的牛会发生 EBL。对体细胞突变和 BLV 整合位点的进一步研究可以更有力地支持这一假说,并有可能促进 EBL 发病的新型控制方法的开发。
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引用次数: 0
Erratum for Hasan et al., "Role of glycogen metabolism in Clostridioides difficile virulence". 对 Hasan 等人 "艰难梭菌毒力中糖原代谢的作用 "的更正。
IF 3.7 2区 生物学 Q2 MICROBIOLOGY Pub Date : 2024-11-21 Epub Date: 2024-10-22 DOI: 10.1128/msphere.00835-24
Md Kamrul Hasan, Marjorie Pizarro-Guajardo, Javier Sanchez, Revathi Govind
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引用次数: 0
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