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Antibiotic resistant bacteria survive treatment by doubling while shrinking. 抗生素耐药细菌在缩小的同时还能存活一倍。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-20 DOI: 10.1128/mbio.02375-24
Adrian Campey, Urszula Łapińska, Remy Chait, Krasimira Tsaneva-Atanasova, Stefano Pagliara

Many antibiotics that are used in healthcare, farming, and aquaculture end up in environments with different spatial structures that might promote heterogeneity in the emergence of antibiotic resistance. However, the experimental evolution of microbes at sub-inhibitory concentrations of antibiotics has been mainly carried out at the population level which does not allow capturing single-cell responses to antibiotics. Here, we investigate and compare the emergence of resistance to ciprofloxacin in Escherichia coli in well-mixed and structured environments using experimental evolution, genomics, and microfluidics-based time-lapse microscopy. We discover that resistance to ciprofloxacin and cross-resistance to other antibiotics is stronger in the well-mixed environment due to the emergence of target mutations, whereas efflux regulator mutations emerge in the structured environment. The latter mutants also harbor sub-populations of persisters that survive high concentrations of ciprofloxacin that inhibit bacterial growth at the population level. In contrast, genetically resistant bacteria that display target mutations also survive high concentrations of ciprofloxacin that inhibit their growth via population-level antibiotic tolerance. These resistant and tolerant bacteria keep doubling while shrinking in size in the presence of ciprofloxacin and regain their original size after antibiotic removal, which constitutes a newly discovered phenotypic response. This new knowledge sheds light on the diversity of strategies employed by bacteria to survive antibiotics and poses a stepping stone for understanding the link between mutations at the population level and phenotypic single-cell responses.

Importance: The evolution of antimicrobial resistance poses a pressing challenge to global health with an estimated 5 million deaths associated with antimicrobial resistance every year globally. Here, we investigate the diversity of strategies employed by bacteria to survive antibiotics. We discovered that bacteria evolve genetic resistance to antibiotics while simultaneously displaying tolerance to very high doses of antibiotics by doubling while shrinking in size.

许多用于医疗保健、种植业和水产养殖业的抗生素最终会进入具有不同空间结构的环境中,这可能会促进抗生素耐药性的异质性出现。然而,微生物在亚抑制浓度抗生素作用下的实验进化主要是在群体水平上进行的,无法捕捉单细胞对抗生素的反应。在这里,我们利用实验进化、基因组学和基于微流控技术的延时显微镜,研究并比较了大肠埃希菌在混合良好的环境和结构化环境中对环丙沙星产生耐药性的情况。我们发现,在混合良好的环境中,由于目标突变的出现,对环丙沙星的耐药性和对其他抗生素的交叉耐药性更强,而在结构化环境中,则出现了外排调节剂突变。后一种突变体还蕴藏着亚群持久体,它们能在高浓度环丙沙星的作用下存活下来,从而在群体水平上抑制细菌的生长。与此相反,出现目标突变的耐药细菌也能在高浓度环丙沙星的环境中存活下来,而高浓度环丙沙星会在群体水平上抑制细菌的生长。在环丙沙星存在的情况下,这些具有抗药性和耐受性的细菌在不断加倍的同时体积也在缩小,并在去除抗生素后恢复到原来的大小,这是一种新发现的表型反应。这一新知识揭示了细菌在抗生素环境中生存策略的多样性,并为理解群体水平的突变与单细胞表型反应之间的联系提供了一块基石:抗菌药耐药性的演变对全球健康构成了紧迫的挑战,据估计,全球每年有 500 万人死于抗菌药耐药性。在这里,我们研究了细菌为在抗生素中存活而采用的策略的多样性。我们发现,细菌在对抗生素产生遗传抗药性的同时,还能通过将体积缩小一倍来显示对高剂量抗生素的耐受性。
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引用次数: 0
Acarbose impairs gut Bacteroides growth by targeting intracellular glucosidases. 阿卡波糖通过靶向细胞内葡萄糖苷酶抑制肠道乳酸杆菌的生长。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-20 DOI: 10.1128/mbio.01506-24
Haley A Brown, Adeline L Morris, Nicholas A Pudlo, Ashley E Hopkins, Eric C Martens, Jonathan L Golob, Nicole M Koropatkin

Acarbose is a type 2 diabetes medicine that prevents dietary starch breakdown into glucose by inhibiting host amylase and glucosidase enzymes. Numerous gut species in the Bacteroides genus enzymatically break down starch and change in relative abundance within the gut microbiome in acarbose-treated individuals. To mechanistically explain this observation, we used two model starch-degrading Bacteroides, Bacteroides ovatus (Bo), and Bacteroides thetaiotaomicron (Bt). Bt growth on starch polysaccharides is severely impaired by acarbose, whereas Bo growth is much less affected by the drug. The Bacteroides use a starch utilization system (Sus) to grow on starch. We hypothesized that Bo and Bt Sus enzymes are differentially inhibited by acarbose. Instead, we discovered that although acarbose primarily targets the Sus periplasmic GH97 enzymes in both organisms, the drug affects starch processing at multiple other points. Acarbose competes for transport through the TonB-dependent SusC proteins and binds to the Sus transcriptional regulators. Furthermore, Bo expresses a non-Sus GH97 (BoGH97D) when grown in starch with acarbose. The Bt homolog, BtGH97H, is not expressed in the same conditions, nor can overexpression of BoGH97D complement the Bt growth inhibition in the presence of acarbose. This work informs us about unexpected complexities of Sus function and regulation in Bacteroides, including variation between related species. Furthermore, this indicates that the gut microbiome may be a source of variable response to acarbose treatment for diabetes.

Importance: Acarbose is a type 2 diabetes medication that works primarily by stopping starch breakdown into glucose in the small intestine. This is accomplished by the inhibition of host enzymes, leading to better blood sugar control via reduced ability to derive glucose from dietary starches. The drug and undigested starch travel to the large intestine where acarbose interferes with the ability of some bacteria to grow on starch. However, little is known about how gut bacteria interact with acarbose, including microbes that can use starch as a carbon source. Here, we show that two gut species, Bacteroides ovatus (Bo) and Bacteroides thetaiotaomicron (Bt), respond differently to acarbose: Bt growth is inhibited by acarbose, while Bo growth is less affected. We reveal a complex set of mechanisms involving differences in starch import and sensing behind the different Bo and Bt responses. This indicates the gut microbiome may be a source of variable response to acarbose treatment for diabetes via complex mechanisms in common gut microbes.

阿卡波糖是一种 2 型糖尿病药物,它通过抑制宿主淀粉酶和葡萄糖苷酶来阻止饮食中的淀粉分解成葡萄糖。乳杆菌属中的许多肠道物种都能酶解淀粉,在阿卡波糖治疗的个体中,这些物种在肠道微生物组中的相对丰度发生了变化。为了从机理上解释这一观察结果,我们使用了两种典型的淀粉降解乳杆菌,即卵形乳杆菌(Bo)和Bt乳杆菌(Bt)。阿卡波糖会严重影响 Bt 在淀粉多糖上的生长,而 Bo 的生长受药物的影响要小得多。杆菌利用淀粉利用系统(Sus)在淀粉上生长。我们假设,阿卡波糖对 Bo 和 Bt 的 Sus 酶有不同的抑制作用。但我们发现,虽然阿卡波糖主要针对这两种生物的 Sus 质外 GH97 酶,但这种药物会影响其他多个点的淀粉加工。阿卡波糖通过依赖于 TonB 的 SusC 蛋白竞争转运,并与 Sus 转录调节因子结合。此外,在含有阿卡波糖的淀粉中生长时,Bo 会表达一种非 Sus GH97(BoGH97D)。Bt 同源物 BtGH97H 在相同条件下不表达,BoGH97D 的过表达也不能补充 Bt 在阿卡波糖存在下的生长抑制。这项工作让我们了解到乳杆菌中 Sus 功能和调控的意外复杂性,包括相关物种之间的差异。此外,这表明肠道微生物组可能是对阿卡波糖治疗糖尿病的不同反应的来源:阿卡波糖是一种 2 型糖尿病药物,主要通过阻止淀粉在小肠中分解为葡萄糖而发挥作用。阿卡波糖是一种 2 型糖尿病药物,其主要作用是阻止淀粉在小肠中分解为葡萄糖,通过抑制宿主酶来实现这一目的,从而降低从膳食淀粉中获取葡萄糖的能力,更好地控制血糖。药物和未消化的淀粉进入大肠后,阿卡波糖会干扰某些细菌在淀粉上生长的能力。然而,人们对肠道细菌如何与阿卡波糖相互作用知之甚少,其中包括可以利用淀粉作为碳源的微生物。在这里,我们展示了两种肠道细菌,即卵形乳杆菌(Bo)和太田乳杆菌(Bt)对阿卡波糖的不同反应:Bt 的生长受到阿卡波糖的抑制,而 Bo 的生长受到的影响较小。我们揭示了一套复杂的机制,其中涉及淀粉输入和感知的差异,而这正是 Bo 和 Bt 不同反应的背后原因。这表明肠道微生物组可能是通过普通肠道微生物的复杂机制对阿卡波糖治疗糖尿病产生不同反应的一个来源。
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引用次数: 0
Staphylococcus epidermidis ST2 strains associated with bloodstream infections contain a unique mobile genetic element encoding a plasmin inhibitor. 与血流感染有关的表皮葡萄球菌 ST2 菌株含有一种独特的移动遗传因子,该遗传因子编码一种血浆蛋白酶抑制剂。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-19 DOI: 10.1128/mbio.01907-24
Amy A Gomez, Clara Kjerfve, Minseo Choi, Wen Liu, Kelly Churion, Sheila Thomas, Holger Rohde, Sam Shelburne, Jon T Skare, Magnus Hook, Srishtee Arora

Staphylococcus epidermidis, a common commensal bacterium, is a leading cause of nosocomial catheter-associated bloodstream infections. S. epidermidis sequence type 2 (ST2) is specifically recognized globally for causing invasive disease. In this study, we identified a novel putative integrated conjugative element, pICE-Sepi-ST2, unique to the genomes of S. epidermidis ST2. Our investigation identified pICE-Sepi-ST2 in all ST2 isolates from bloodstream infections. Meanwhile, ST2 isolates from other infection sources, such as catheters, prosthetic joints, and fracture fixations, showed variable pICE-Sepi-ST2 prevalence. pICE-Sepi-ST2 encodes two putative cell wall anchored proteins that we have designated SesX and SesY. Biochemical characterization of SesY revealed that it binds both plasminogen (Plg) and plasmin (Pln) and inhibits Pln's ability to cleave a chromogenic substrate and degrade fibrin clots. Furthermore, all ST2 isolates containing a pICE-Sepi-ST2 also have a mutated sdrG gene. Thus, all ST2 isolates have two genetic modifications that target distinct steps in the hemostatic pathway. SdrG, which inhibits coagulation, is inactivated, and SesY, which inhibits fibrin, is introduced. These findings suggest that the hemostasis pathway is a strategic target for ST2 S. epidermidis bloodstream pathogenesis.

Importance: This study uncovers a new virulence mechanism in Staphylococcus epidermidis ST2 bloodstream isolates. We identify a mobile genetic element (MGE) characteristic of an integrated conjugated element (ICE). pICE-Sepi-ST2 carries the genetic information needed to produce a cell wall-anchored (CWA) protein called SesY. The results indicate that SesY binds to plasminogen (Plg) and plasmin (Pln) and inhibits Pln's degradation of fibrin clots. Genetic analysis showed that all ST2 bloodstream isolates can express the plasmin inhibitor SesY and carry a mutation in the SdrG gene, resulting in the expression of inactive SdrG. Thus, we describe a molecular pathway targeting the coagulation pathway that may be required for S. epidermidis ST2 to cause bloodstream infections.

表皮葡萄球菌是一种常见的共生细菌,也是引起院内导管相关血流感染的主要原因。表皮葡萄球菌序列 2 型(ST2)是全球公认的引起侵袭性疾病的特异性细菌。在这项研究中,我们发现了表皮葡萄球菌 ST2 基因组中独有的新型推定整合共轭元件 pICE-Sepi-ST2。我们的调查在所有来自血液感染的 ST2 分离物中都发现了 pICE-Sepi-ST2。pICE-Sepi-ST2 编码两种推定的细胞壁锚定蛋白,我们将其命名为 SesX 和 SesY。SesY 的生化特征显示,它能与纤溶酶原(Plg)和纤溶酶(Pln)结合,并抑制 Pln 裂解显色底物和降解纤维蛋白凝块的能力。此外,所有含有 pICE-Sepi-ST2 的 ST2 分离物的 sdrG 基因也发生了突变。因此,所有 ST2 分离物都有两种针对止血途径中不同步骤的基因修饰。抑制凝血的 SdrG 基因失活,而抑制纤维蛋白的 SesY 基因被引入。这些发现表明,止血途径是 ST2 表皮葡萄球菌血流致病的战略目标:本研究在表皮葡萄球菌 ST2 血流分离株中发现了一种新的致病机制。pICE-Sepi-ST2 携带产生一种名为 SesY 的细胞壁锚定(CWA)蛋白所需的遗传信息。结果表明,SesY 能与纤溶酶原(Plg)和纤溶酶(Pln)结合,并抑制 Pln 对纤维蛋白凝块的降解。遗传分析表明,所有 ST2 血流分离株都能表达纤溶酶原抑制剂 SesY,并携带 SdrG 基因突变,导致表达无活性的 SdrG。因此,我们描述了一种针对凝血途径的分子途径,它可能是表皮葡萄球菌 ST2 引起血流感染所必需的。
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引用次数: 0
Integration of horizontally acquired light-harvesting genes into an ancestral regulatory network in the cyanobacterium Acaryochloris marina MBIC11017. 蓝藻 Acaryochloris marina MBIC11017 中横向获得的采光基因与祖先调控网络的整合。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-18 DOI: 10.1128/mbio.02423-24
Nikea J Ulrich, Scott R Miller

The acquisition of new capabilities by horizontal gene transfer (HGT) shapes the distribution of traits during microbial diversification. In the Chlorophyll (Chl) d-producing cyanobacterium Acaryochloris marina, the genes involved in the production and disassembly of the light-harvesting phycobiliprotein phycocyanin (PC) were lost in the A. marina common ancestor but then subsequently regained via HGT in A. marina strain MBIC11017. However, it remains unknown how the HGT-acquired PC genes in MBIC11017 have been reintegrated into its existing regulatory network after tens of millions of years since their loss. Here, we investigated potential mechanisms of regulatory assimilation of PC genes by comparing the transcriptomes of A. marina strain MBIC11017 and a PC-lacking close relative under both low irradiance far-red light and high irradiance white light. We found that PC assembly and degradation processes have been re-assimilated into a conserved ancestral response to high light. Further, we identified putative regulatory elements that were likely co-transferred with PC genes and could be recognized by A. marina's pre-existing light response machinery. This study offers insights into how HGT-acquired genes can be reintegrated into an existing transcriptional regulatory network that has evolved in their absence.IMPORTANCEHorizontal gene transfer, the asymmetric movement of genetic information between donor and recipient organisms, is an important mechanism for acquiring new traits. In order for newly acquired gene content to be retained, it must be integrated into the genetic repertoire and regulatory networks of the recipient cell. In a strain of the Chlorophyll d-producing cyanobacterium Acaryochloris marina, the recent reacquisition of the genes required to produce the light-harvesting pigment phycocyanin offers a rare opportunity to understand the mechanisms underlying the regulatory assimilation of an acquired complex trait in bacteria. The significance in our research is in characterizing how an ancestrally lost, complex trait can be reintegrated into a conserved regulatory network, even after millions of years.

通过水平基因转移(HGT)获得新的能力决定了微生物多样化过程中性状的分布。在叶绿素(Chl)d 生产蓝藻 Acaryochloris marina 中,参与生产和分解采光藻蓝蛋白 phycocyanin(PC)的基因在 A. marina 的共同祖先中丢失,但随后通过 HGT 在 A. marina 菌株 MBIC11017 中重新获得。然而,MBIC11017 中 HGT 获得的 PC 基因在丢失数千万年后是如何重新整合到其现有调控网络中的,目前仍不得而知。在此,我们通过比较栗藻菌株 MBIC11017 和缺乏 PC 的近亲在低辐照度远红光和高辐照度白光下的转录组,研究了 PC 基因调控同化的潜在机制。我们发现,PC 的组装和降解过程已被重新整合为祖先对强光的保守反应。此外,我们还发现了可能与 PC 基因共同转移的推定调控元件,这些元件可被滨海甲藻原有的光反应机制识别。这项研究深入探讨了 HGT 获得的基因如何重新整合到现有的转录调控网络中,而该网络是在没有 HGT 基因的情况下进化而来的。 重要意义水平基因转移,即遗传信息在供体和受体生物之间的非对称移动,是获得新性状的重要机制。为了保留新获得的基因内容,必须将其整合到受体细胞的基因库和调控网络中。在一株叶绿素 d 生产蓝藻 Acaryochloris marina 中,最近重新获得了生产采光色素 phycocyanin 所需的基因,这为我们了解细菌获得的复杂性状的调控同化机制提供了一个难得的机会。我们研究的意义在于,即使经过数百万年的时间,我们也能确定一个从祖先那里丢失的复杂性状是如何被重新整合到一个保守的调控网络中的。
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引用次数: 0
Hidden allies: how extracellular vesicles drive biofilm formation, stress adaptation, and host-immune interactions in human fungal pathogens. 隐藏的盟友:细胞外囊泡如何驱动人类真菌病原体的生物膜形成、应激适应和宿主-免疫相互作用。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-18 DOI: 10.1128/mbio.03045-23
Philipp Brandt, Rima Singha, Iuliana V Ene

Pathogenic fungi pose a significant threat to human health, especially given the rising incidence of invasive fungal infections and the emergence of drug-resistant strains. This requires the development of vaccines and the advancement of antifungal strategies. Recent studies have focused on the roles of fungal extracellular vesicles (EVs) in intercellular communication and host-pathogen interactions. EVs are nanosized, lipid membrane-bound particles that facilitate the transfer of proteins, lipids, and nucleic acids. Here, we review the multifaceted functions of EVs produced by different human fungal pathogens, highlighting their importance in the response of fungal cells to different environmental cues and their interactions with host immune cells. We summarize the current state of research on EVs and how leveraging this knowledge can lead to innovative approaches in vaccine development and antifungal treatment.

致病真菌对人类健康构成了重大威胁,尤其是鉴于侵袭性真菌感染发病率的上升和耐药菌株的出现。这就需要开发疫苗和改进抗真菌策略。最近的研究重点是真菌胞外囊泡(EVs)在细胞间通信和宿主-病原体相互作用中的作用。EVs是一种纳米级的脂质膜结合颗粒,可促进蛋白质、脂质和核酸的转移。在此,我们回顾了不同人类真菌病原体产生的EVs的多方面功能,强调了它们在真菌细胞对不同环境线索的反应及其与宿主免疫细胞的相互作用中的重要性。我们总结了有关 EVs 的研究现状,以及利用这些知识如何在疫苗开发和抗真菌治疗中找到创新方法。
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引用次数: 0
Hyaluronan in COVID-19: a matrix for understanding lung disease. COVID-19中的透明质酸:了解肺部疾病的基质。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-18 DOI: 10.1128/mbio.02609-24
Rebecca J Dodd, Judith E Allen, Anthony J Day

The polysaccharide hyaluronan (HA) is an important component of lung extracellular matrix that increases following infection with influenza or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Hellman et al. (U. Hellman, E. Rosendal, J. Lehrstrand, J. Henriksson, et al., mBio 15:e01303-24, https://doi.org/10.1128/mbio.01303-24) show that fragmented HA accumulates in the lungs of coronavirus disease 2019 (COVID-19) patients, with systemic levels of HA being associated with reduced lung function 3-6 months after infection. This study provides novel insights into HA's role in COVID-19 pathology and its potential utility as a biomarker for disease severity. However, much remains to be understood about the lung HA matrix in COVID-19 and how it compares to other lung conditions. In particular, the role of HA-binding proteins in organizing HA into a crosslinked network is yet to be fully determined at a molecular level. This knowledge is crucial in understanding the inter-relationships between the structure of the HA matrix and the regulation of the immune response, and thus our ability to target HA therapeutically for improved outcomes in COVID-19.

多糖透明质酸(HA)是肺细胞外基质的重要组成部分,在感染流感或严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)后会增加。赫尔曼等人(U. Hellman, E. Rosendal, J. Lehrstrand, J. Henriksson, et al., mBio 15:e01303-24, https://doi.org/10.1128/mbio.01303-24)的研究表明,碎裂的HA会在2019年冠状病毒病(COVID-19)患者的肺部积聚,全身HA水平与感染后3-6个月肺功能下降有关。这项研究为了解HA在COVID-19病理学中的作用及其作为疾病严重程度生物标志物的潜在用途提供了新的视角。然而,关于 COVID-19 中的肺 HA 基质及其与其他肺部疾病的比较,仍有许多问题有待了解。特别是,HA 结合蛋白在将 HA 组织成交联网络中的作用尚未在分子水平上完全确定。这些知识对于了解 HA 基质结构与免疫反应调控之间的相互关系至关重要,从而帮助我们以 HA 为治疗靶点,改善 COVID-19 的治疗效果。
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引用次数: 0
Vibrio cholerae CsrA controls ToxR levels by increasing the stability and translation of toxR mRNA. 霍乱弧菌 CsrA 通过增加 toxR mRNA 的稳定性和翻译来控制 ToxR 水平。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-18 DOI: 10.1128/mbio.02853-24
Alexandra R Mey, Charles R Midgett, F Jon Kull, Shelley M Payne

Intestinal colonization and virulence factor production in response to environmental cues is mediated through several regulatory factors in Vibrio cholerae, including the highly conserved RNA-binding global regulatory protein CsrA. We have shown previously that CsrA increases synthesis of the virulence-associated transcription factor ToxR in response to specific amino acids (NRES) and is required for the virulence of V. cholerae in the infant mouse model of cholera. In this study, we mapped the 5' untranslated region (5' UTR) of toxR and showed that CsrA can bind directly to an RNA sequence encompassing the 5' UTR, indicating that the regulation of ToxR levels by CsrA is direct. Consistent with this observation, the 5' UTR of toxR contains multiple putative CsrA binding sequences (GGA motifs), and mutating these motifs disrupted the CsrA-mediated increase in ToxR. Optimal binding of CsrA to a defined RNA oligonucleotide required the bridging of two GGA motifs within a single RNA strand. To determine the mechanism of regulation by CsrA, we assayed toxR transcript levels, stability, and efficiency of translation. Both the amount of toxR mRNA in NRES and the stability of the toxR transcript were increased by CsrA. Using an in vitro translation assay, we further showed that synthesis of ToxR was greatly enhanced in the presence of purified CsrA, suggesting a direct role for CsrA in the translation of toxR mRNA. We propose a model in which CsrA binding to the 5' UTR of the toxR transcript promotes ribosomal access while precluding interactions with RNA-degrading enzymes.IMPORTANCEVibrio cholerae is uniquely adapted to marine environments as well as the human intestinal tract. Global regulators, such as CsrA, which help translate environmental cues into an appropriate cellular response, are critical for switching between these distinct environments. Understanding the pathways involved in relaying environmental signals is essential for understanding both the environmental persistence and the intestinal pathogenesis of this devastating human pathogen. In this study, we demonstrate that CsrA directly regulates the synthesis of ToxR, a key virulence factor of V. cholerae. Under conditions favoring high levels of active CsrA in the cell, such as in the presence of particular amino acids, CsrA increases ToxR protein levels by binding to the toxR transcript and enhancing both its stability and translation. By responding to nutrient availability, CsrA is perfectly poised to activate the virulence gene regulatory cascade at the preferred site of colonization in the human host, the nutrient-rich small intestinal mucosa.

霍乱弧菌的肠道定植和毒力因子的产生是通过几个调控因子介导的,其中包括高度保守的 RNA 结合全局调控蛋白 CsrA。我们之前已经证明,CsrA 会增加毒力相关转录因子 ToxR 对特定氨基酸(NRES)的合成,并且是霍乱婴儿小鼠模型中霍乱弧菌毒力所必需的。在这项研究中,我们绘制了 ToxR 的 5' 非翻译区(5' UTR),并发现 CsrA 可直接与包含 5' UTR 的 RNA 序列结合,这表明 CsrA 对 ToxR 水平的调控是直接的。与这一观察结果相一致的是,ToxR 的 5' UTR 包含多个假定的 CsrA 结合序列(GGA 基序),突变这些基序会破坏 CsrA 介导的 ToxR 的增加。CsrA 与特定 RNA 寡核苷酸的最佳结合需要在单个 RNA 链中桥接两个 GGA 基序。为了确定 CsrA 的调控机制,我们检测了 ToxR 的转录本水平、稳定性和翻译效率。CsrA 增加了 NRES 中 toxR mRNA 的数量和 toxR 转录本的稳定性。通过体外翻译试验,我们进一步发现,在纯化的 CsrA 存在下,ToxR 的合成大大增强,这表明 CsrA 在翻译 toxR mRNA 中起着直接作用。我们提出了一个模型,在该模型中,CsrA 与 toxR 转录本的 5' UTR 结合可促进核糖体的进入,同时排除与 RNA 降解酶的相互作用。CsrA 等全局调节因子有助于将环境线索转化为适当的细胞反应,对于在这些不同环境之间进行切换至关重要。了解传递环境信号的途径对于了解这种毁灭性人类病原体的环境持久性和肠道致病机理至关重要。在这项研究中,我们证明了 CsrA 可直接调控霍乱弧菌关键毒力因子 ToxR 的合成。在有利于细胞内高水平活性 CsrA 的条件下,例如在特定氨基酸存在的情况下,CsrA 会通过与 toxR 转录本结合并增强其稳定性和翻译来提高 ToxR 蛋白水平。通过对营养物质的可用性做出反应,CsrA 完全可以在人类宿主的首选定植部位--营养丰富的小肠粘膜--激活毒力基因调控级联。
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引用次数: 0
RNAseq and targeted metabolomics implicate RIC8 in regulation of energy homeostasis, amino acid compartmentation, and asexual development in Neurospora crassa. RNAseq 和靶向代谢组学表明,RIC8 与十字花科黑孢子属(Neurospora crassa)的能量平衡、氨基酸分区和无性发育的调控有关。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-18 DOI: 10.1128/mbio.03133-24
Monique Quinn, Alexander J Carrillo, Lida Halilovic, Katherine A Borkovich

Heterotrimeric G protein signaling pathways control growth and development in eukaryotes. In the multicellular fungus Neurospora crassa, the guanine nucleotide exchange factor RIC8 regulates heterotrimeric Gα subunits. In this study, we used RNAseq and liquid chromatography-mass spectrometry (LC-MS) to profile the transcriptomes and metabolomes of N. crassa wild type, the Gα subunit mutants Δgna-1 and Δgna-3, and Δric8 strains. These strains exhibit defects in growth and asexual development (conidiation), with wild-type and Δgna-1 mutants producing hyphae in submerged cultures, while Δgna-3 and Δric8 mutants develop conidiophores, particularly in the Δric8 mutant. RNAseq analysis showed that the Δgna-1 mutant possesses 159 mis-regulated genes, while Δgna-3 and Δric8 strains have more than 1,000 each. Many of the mis-regulated genes are involved in energy homeostasis, conidiation, or metabolism. LC-MS revealed changes in levels of primary metabolites in the mutants, with several arginine metabolic intermediates impacted in Δric8 strains. The differences in metabolite levels could not be fully explained by the expression or activity of pathway enzymes. However, transcript levels for two predicted vacuolar arginine transporters were affected in Δric8 mutants. Analysis of arginine and ornithine levels in transporter mutants yielded support for altered compartmentation of arginine and ornithine between the cytosol and vacuole in Δric8 strains. Furthermore, we validated previous reports that arginine and ornithine levels are low in wild-type conidia. Our results suggest that RIC8 regulates asexual sporulation in N. crassa at least in part through altered expression of vacuolar transporter genes and the resultant mis-compartmentation of arginine and ornithine.

Importance: Resistance to inhibitors of cholinesterase-8 (RIC8) is an important regulator of heterotrimeric Gα proteins in eukaryotes. In the filamentous fungus Neurospora crassa, mutants lacking ric8 undergo inappropriate asexual development (macroconidiation) during submerged growth. Our work identifies a role for RIC8 in regulating expression of transporter genes that retain arginine and ornithine in the vacuole (equivalent of the animal lysosome) and relates this function to the developmental defect. Arginine is a critical cellular metabolite, both as an amino acid for protein synthesis and as a precursor for an array of compounds, including proline, ornithine, citrulline, polyamines, creatine phosphate, and nitric oxide. These results have broad relevance to human physiology and disease, as arginine modulates immune, vascular, hormonal, and other functions in humans.

异三聚体 G 蛋白信号通路控制着真核生物的生长和发育。在多细胞真菌蟋蟀神经孢子(Neurospora crassa)中,鸟嘌呤核苷酸交换因子 RIC8 调节着异三聚体 Gα 亚基。在这项研究中,我们使用 RNAseq 和液相色谱-质谱(LC-MS)分析了 N. crassa 野生型、Gα 亚基突变体 Δgna-1 和 Δgna-3 以及 Δric8 株系的转录组和代谢组。这些菌株在生长和无性发育(分生孢子)方面表现出缺陷,野生型和Δgna-1突变体在浸没培养中产生菌丝,而Δgna-3和Δric8突变体则产生分生孢子,尤其是Δric8突变体。RNAseq分析表明,Δgna-1突变体有159个调控不当的基因,而Δgna-3和Δric8株则各有1000多个。许多被错误调控的基因涉及能量平衡、分生孢子或新陈代谢。液相色谱-质谱(LC-MS)显示,突变体中主要代谢物的水平发生了变化,Δric8 株系中的几种精氨酸代谢中间产物受到了影响。代谢物水平的差异不能完全用途径酶的表达或活性来解释。不过,在 Δric8 突变体中,两个预测的液泡精氨酸转运体的转录水平受到了影响。对转运体突变体中精氨酸和鸟氨酸水平的分析表明,Δric8 株系中精氨酸和鸟氨酸在细胞质和液泡之间的分区发生了改变。此外,我们还验证了之前关于野生型分生孢子中精氨酸和鸟氨酸水平较低的报道。我们的研究结果表明,RIC8 至少部分是通过改变液泡转运体基因的表达以及由此导致的精氨酸和鸟氨酸的错配来调节 N. crassa 的无性孢子的:抗胆碱酯酶抑制剂-8(RIC8)是真核生物中异源三聚体 Gα 蛋白的一个重要调节因子。在丝状真菌克氏黑孢子(Neurospora crassa)中,缺乏ric8的突变体在浸没生长过程中会出现不适当的无性发育(大厥)。我们的研究确定了 RIC8 在调节将精氨酸和鸟氨酸保留在液泡(相当于动物溶酶体)中的转运体基因表达中的作用,并将这一功能与发育缺陷联系起来。精氨酸是一种重要的细胞代谢物,既是蛋白质合成的氨基酸,也是脯氨酸、鸟氨酸、瓜氨酸、多胺、磷酸肌酸和一氧化氮等一系列化合物的前体。这些结果与人体生理和疾病有着广泛的关系,因为精氨酸能调节人体的免疫、血管、荷尔蒙和其他功能。
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引用次数: 0
Small but mighty: targeted antifungal liposomes of a smaller size are superior in treating cryptococcal meningitis. 小而强大:体积较小的靶向抗真菌脂质体在治疗隐球菌脑膜炎方面更具优势。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-18 DOI: 10.1128/mbio.02507-24
Tuyetnhu Pham, Peter Zhang, Suresh Ambati, Richard B Meagher, Xiaorong Lin

Cryptococcal meningoencephalitis (CME) is deadly. CME is responsible for 19% of deaths in AIDS patients, and its global mortality is greater than 60%. The recommended CME therapy requires amphotericin B (AmB), a fungicidal drug targeting fungal ergosterol. AmB also binds to the host's cholesterol and is highly toxic. Liposomal AmB (AmB-LLs), relative to deoxycholate-solubilized AmB, has lower toxicity and longer tissue retention, but it requires high doses for treatment and its efficacy in treating CME remains unsatisfactory. To improve the effectiveness of AmB-LLs, we previously developed DectiSomes-targeted AmB-LLs decorated with host dectins that recognize fungal polysaccharides. DectiSomes, relative to untargeted AmB-LLs, modestly improve efficacy against systemic cryptococcosis, in contrast to the drastic improvement observed in candidiasis or aspergillosis models. We speculated that limited tissue penetration of the regular-sized DectiSomes might have contributed to the modest improvement in treating systemic cryptococcosis. Here, we discovered that DectiSomes of a smaller size (~50 nm), compared with DectiSomes of the regular size (~100 nm) or untargeted AmB-LLs of either size, had a much better capability in reducing cryptococcal burden of various organs including the brain and in prolonging the survival of mice with systemic cryptococcosis. The performance of small DectiSomes was far superior to all other groups at two different doses of AmB tested. Furthermore, no kidney toxicity was observed in any of the treatment regimens tested. Taken together, our findings indicate that small DectiSomes can be a powerful antifungal delivery platform to drastically improve therapies against the deadly CME.

Importance: Systemic cryptococcosis is fatal even with antifungal interventions. The most effective drug against this disease is amphotericin B (AmB). However, AmB is highly toxic as it binds to fungal ergosterol and also mammalian cholesterol. Liposomal AmB was introduced to the clinic in 1990s because it showed reduced toxicity and longer retention in various organs. However, the dose of AmB required for treatment using liposomal formulation is high and the outcome is far from satisfactory. In our previous work, we generated DectiSomes, dectin-decorated liposomes loaded with AmB that more effectively deliver the drug to the pathogen and enhance antifungal efficacy. However, the improvement in treating systemic cryptococcosis, compared with candidiasis and aspergillosis, is modest. Here, we generated DectiSomes that are half their regular size to improve tissue penetration. We discovered that small DectiSomes are superior in reducing fungal burden in various organs including the brain and in prolonging animal survival.

隐球菌脑膜脑炎(CME)是致命的。19%的艾滋病患者死于隐球菌脑膜脑炎,其全球死亡率超过60%。推荐的 CME 治疗需要两性霉素 B(AmB),这是一种针对真菌麦角固醇的杀真菌药物。AmB 也会与宿主的胆固醇结合,毒性很强。相对于脱氧胆固醇溶解的 AmB,脂质体 AmB(AmB-LLs)的毒性更低,组织保留时间更长,但其治疗需要高剂量,治疗 CME 的疗效仍不令人满意。为了提高AmB-LLs的疗效,我们之前开发出了DectiSomes靶向AmB-LLs,该靶向AmB-LLs上装饰有能识别真菌多糖的宿主右旋糖蛋白。与未靶向的 AmB-LLs 相比,DectiSomes 对全身性隐球菌病的疗效改善不大,而在念珠菌病或曲霉病模型中观察到的疗效改善则非常明显。我们推测,普通大小的 DectiSomes 的组织穿透力有限,可能是导致治疗全身性隐球菌病的疗效改善不大的原因。在这里,我们发现,与常规尺寸(约 100 nm)的 DectiSomes 或任何尺寸的非靶向 AmB-LLs 相比,较小尺寸(约 50 nm)的 DectiSomes 在减少包括大脑在内的各器官的隐球菌负担和延长全身性隐球菌病小鼠的存活时间方面具有更好的能力。在两种不同剂量的 AmB 试验中,小型 DectiSomes 的表现远远优于所有其他组别。此外,在测试的任何治疗方案中都没有观察到肾脏毒性。总之,我们的研究结果表明,小 DectiSomes 可以成为一种强大的抗真菌递送平台,大幅改善针对致命隐球菌病的疗法:重要意义:即使采取抗真菌干预措施,全身性隐球菌病也是致命的。治疗这种疾病最有效的药物是两性霉素 B(AmB)。然而,两性霉素 B 有剧毒,因为它会与真菌麦角固醇和哺乳动物胆固醇结合。20 世纪 90 年代,脂质体 AmB 被引入临床,因为它的毒性较低,在各器官中的存留时间较长。然而,使用脂质体制剂治疗所需的 AmB 剂量很高,效果也不尽如人意。在我们之前的工作中,我们生成了 DectiSomes,这是一种装有 AmB 的去克汀饰脂质体,它能更有效地将药物输送到病原体并增强抗真菌效果。然而,与念珠菌病和曲霉病相比,治疗全身性隐球菌病的效果改善不大。在这里,我们生成了只有正常大小一半的 DectiSomes,以提高组织穿透力。我们发现,小的 DectiSomes 在减少包括大脑在内的各种器官的真菌负担和延长动物存活时间方面具有优势。
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引用次数: 0
Whole-blood model reveals granulocytes as key sites of dengue virus propagation, expanding understanding of disease pathogenesis. 全血模型揭示了粒细胞是登革热病毒传播的关键场所,拓展了对疾病发病机制的认识。
IF 5.1 1区 生物学 Q1 MICROBIOLOGY Pub Date : 2024-11-14 DOI: 10.1128/mbio.01505-24
Hansa Praneechit, Somchai Thiemmeca, Dararat Prayongkul, Kessiri Kongmanas, Dumrong Mairiang, Nuntaya Punyadee, Adisak Songjaeng, Nattaya Tangthawornchaikul, Nasikarn Angkasekwinai, Kanokwan Sriruksa, Yupin Suputtamongkol, Wannee Limpitikul, John P Atkinson, Panisadee Avirutnan

Dengue virus (DENV) infection poses a significant global health threat, yet our understanding of its immunopathogenesis remains incomplete due to limitations of existing models. Here, we establish an in vitro whole-blood model using hirudin, an anticoagulant that preserves complement activity and cellular interactions, to study DENV infection. Our model reveals the susceptibility of all major leukocyte populations to DENV infection, with monocytes and granulocytes demonstrating high permissiveness and production of infectious virus progeny. Notably, granulocytes emerge as previously unrecognized targets of DENV infection, highlighting the importance of studying viral tropism within a physiologically relevant context. We also observed efficient DENV binding to B cells, but limited production of infectious virus, suggesting a potential role in viral sequestration or immune dysregulation. Interestingly, both NK and T cells, while less permissive, were also found to be susceptible to DENV infection. Our ex vivo analysis of whole blood from DENV-infected patients confirms the susceptibility of granulocytes, monocytes, B cells, natural killer cells, and T cells to infection, further validating the clinical relevance of our model. Additionally, we observed dynamic changes in circulating blood cell populations during acute dengue, potentially reflecting both direct virus-mediated effects and immune responses. This whole-blood model offers a valuable tool for investigating the complex interplay between DENV and host factors, facilitating a deeper understanding of dengue pathogenesis and ultimately contributing to the development of novel therapeutic strategies.IMPORTANCEDengue virus (DENV) infection is a significant global health threat, with increasing incidence in endemic regions and expanding geographic range due to factors like global warming. Current models for studying DENV pathogenesis often lack the complexity of the human immune system, hindering the development of effective therapies and vaccines. To address this, we have established the first in vitro whole-blood model using hirudin, preserving critical immune components and cellular interactions. This model reveals granulocytes as previously unrecognized targets of productive DENV infection, challenging existing paradigms of viral tropism. Our ex vivo analysis of patient blood samples confirms the clinical relevance of this finding and validates our model's utility. This unique model offers a powerful platform for future studies to dissect the complex interactions between DENV and the host immune system, including the roles of different leukocyte populations, ultimately informing the development of novel therapeutic strategies to combat this devastating disease.

登革病毒(DENV)感染对全球健康构成重大威胁,但由于现有模型的局限性,我们对其免疫发病机制的了解仍不全面。在这里,我们建立了一个体外全血模型,使用水蛭素(一种保留补体活性和细胞相互作用的抗凝血剂)来研究登革热病毒感染。我们的模型揭示了所有主要白细胞群对 DENV 感染的易感性,其中单核细胞和粒细胞表现出高度的易感性并产生传染性病毒后代。值得注意的是,粒细胞成为以前未曾认识到的DENV感染目标,这凸显了在生理相关背景下研究病毒趋向性的重要性。我们还观察到 DENV 与 B 细胞的高效结合,但感染性病毒的产生却很有限,这表明它可能在病毒螯合或免疫失调中发挥作用。有趣的是,我们还发现NK细胞和T细胞虽然不那么敏感,但也易受DENV感染。我们对感染 DENV 患者的全血进行的体内外分析证实了粒细胞、单核细胞、B 细胞、自然杀伤细胞和 T 细胞对感染的易感性,进一步验证了我们模型的临床相关性。此外,我们还观察到急性登革热期间循环血细胞群的动态变化,这可能反映了病毒介导的直接效应和免疫反应。这种全血模型为研究登革热病毒与宿主因素之间复杂的相互作用提供了一种宝贵的工具,有助于加深对登革热发病机制的理解,并最终促进新型治疗策略的开发。重要意义登革热病毒(DENV)感染是一种严重的全球健康威胁,其在流行地区的发病率不断上升,并且由于全球变暖等因素,其地理分布范围不断扩大。目前研究 DENV 发病机制的模型往往缺乏人类免疫系统的复杂性,从而阻碍了有效疗法和疫苗的开发。为了解决这个问题,我们建立了首个使用水蛭素的体外全血模型,保留了关键的免疫成分和细胞相互作用。该模型揭示了粒细胞是以前未认识到的生产性 DENV 感染的目标,对现有的病毒滋养范式提出了挑战。我们对患者血液样本的体内外分析证实了这一发现的临床意义,并验证了我们模型的实用性。这种独特的模型为今后的研究提供了一个强大的平台,以剖析 DENV 与宿主免疫系统之间复杂的相互作用,包括不同白细胞群的作用,最终为开发新型治疗策略提供信息,以对抗这种毁灭性疾病。
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