Pub Date : 2024-09-16DOI: 10.1101/2024.09.16.612975
Dylan J Enright, Ryan J Quaal, Aishwarya Veerabahu, Anna Nguyen, Jenna Maddox, Sydney I Glassman
A live microbial culture is invaluable to assess traits and functions via 'omics and biophysical assays. However, it is not always logistically feasible to culture immediately from freshly obtained soil, and selecting the proper media for culturing is not trivial. While building a culture collection of pyrophilous microbes obtained from burnt soil, we tested the best 1) method of storing soil to retain culturable viability and 2) media to garner the most microbial diversity. We tested four methods of soil storage (dried, stored at 4°C, stored at -80°C alone or in glycerol) and compared to fresh soil obtained 6 months after a severe California chapparal shrubland wildfire. For bacteria, soil frozen at -80°C with glycerol preserved the greatest diversity (25 species, 13 genera) compared to fresh soil (26 species, 13 genera). For fungi, soil stored at -80°C alone preserved the greatest diversity (10 species, 3 genera) compared to fresh soil (13 species, 7 genera). We also tested 3 media types: rich media (Lysogeny Broth (LB) for bacteria; Malt Yeast Agar (MYA) for fungi), oligotrophic media (Reasoner's 2 Agar (R2A) and media made from pyrogenic organic matter (PyOM). For bacteria, culturing on LB and R2A garnered the greatest diversity (LB = 26 species, 13 genera, R2A = 27 species, 15 genera). For fungi a combination of R2A and PyOM captured the greatest diversity (R2A = 15 species, 8 genera, PyOM = 12 species, 6 genera). For both bacteria and fungi, some species of interest were only captured using the PyOM media. Using a combination of these methods from 2018-2022, we cultured >500 isolates (286 bacteria; 258 fungi) from burned soils of 7 Southern California wildfires.
{"title":"Evaluating Best Practices for Isolating Pyrophilous Bacteria and Fungi from Burned Soil","authors":"Dylan J Enright, Ryan J Quaal, Aishwarya Veerabahu, Anna Nguyen, Jenna Maddox, Sydney I Glassman","doi":"10.1101/2024.09.16.612975","DOIUrl":"https://doi.org/10.1101/2024.09.16.612975","url":null,"abstract":"A live microbial culture is invaluable to assess traits and functions via 'omics and biophysical assays. However, it is not always logistically feasible to culture immediately from freshly obtained soil, and selecting the proper media for culturing is not trivial. While building a culture collection of pyrophilous microbes obtained from burnt soil, we tested the best 1) method of storing soil to retain culturable viability and 2) media to garner the most microbial diversity. We tested four methods of soil storage (dried, stored at 4°C, stored at -80°C alone or in glycerol) and compared to fresh soil obtained 6 months after a severe California chapparal shrubland wildfire. For bacteria, soil frozen at -80°C with glycerol preserved the greatest diversity (25 species, 13 genera) compared to fresh soil (26 species, 13 genera). For fungi, soil stored at -80°C alone preserved the greatest diversity (10 species, 3 genera) compared to fresh soil (13 species, 7 genera). We also tested 3 media types: rich media (Lysogeny Broth (LB) for bacteria; Malt Yeast Agar (MYA) for fungi), oligotrophic media (Reasoner's 2 Agar (R2A) and media made from pyrogenic organic matter (PyOM). For bacteria, culturing on LB and R2A garnered the greatest diversity (LB = 26 species, 13 genera, R2A = 27 species, 15 genera). For fungi a combination of R2A and PyOM captured the greatest diversity (R2A = 15 species, 8 genera, PyOM = 12 species, 6 genera). For both bacteria and fungi, some species of interest were only captured using the PyOM media. Using a combination of these methods from 2018-2022, we cultured >500 isolates (286 bacteria; 258 fungi) from burned soils of 7 Southern California wildfires.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.16.613335
Mallory L Myers, John R Gallagher, DeMarcus D Woolfork, Noah D Khorrami, William B Park, Samantha Maldonado-Puga, Eric Bohrnsen, Benjamin H Schwarz, Derron A Alves, Kevin W Bock, Altaira D Dearborn, Audray K Harris
Development of intranasal vaccines for respiratory viruses has gained popularity. However, currently only a live-attenuated influenza vaccine is FDA-approved for intranasal administration. Here, we focused on influenza virus as it circulates seasonally, has pandemic potential, and has vaccine formulations that present hemagglutinin (HA) in different structural arrangements. These display differences have not been correlated with induction of pan-H1 antibodies or shown to provide intranasal protection. Using electron microscopy, biochemistry and animal studies, we identified HA complexes arranged as lipid discs with multiple trimeric HAs displayed along the perimeter, termed spike nanobicelles (SNB). We utilized a structure-guided approach to synthesize in vitro assembled spiked nanobicelles (IA-SNB) from a classical 1934 H1N1 influenza virus. IA-SNBs elicited pan-H1 antibodies and provided protection against antigenically divergent H1N1 viruses via intranasal immunizations. Viral glycoprotein spikes displayed as SNBs could aid in combating antigenic variation and provide innovative intranasal vaccines to aid universal influenza vaccine development.
{"title":"Structure-guided assembly of an influenza spike nanobicelle vaccine provides pan H1 intranasal protection","authors":"Mallory L Myers, John R Gallagher, DeMarcus D Woolfork, Noah D Khorrami, William B Park, Samantha Maldonado-Puga, Eric Bohrnsen, Benjamin H Schwarz, Derron A Alves, Kevin W Bock, Altaira D Dearborn, Audray K Harris","doi":"10.1101/2024.09.16.613335","DOIUrl":"https://doi.org/10.1101/2024.09.16.613335","url":null,"abstract":"Development of intranasal vaccines for respiratory viruses has gained popularity. However, currently only a live-attenuated influenza vaccine is FDA-approved for intranasal administration. Here, we focused on influenza virus as it circulates seasonally, has pandemic potential, and has vaccine formulations that present hemagglutinin (HA) in different structural arrangements. These display differences have not been correlated with induction of pan-H1 antibodies or shown to provide intranasal protection. Using electron microscopy, biochemistry and animal studies, we identified HA complexes arranged as lipid discs with multiple trimeric HAs displayed along the perimeter, termed spike nanobicelles (SNB). We utilized a structure-guided approach to synthesize in vitro assembled spiked nanobicelles (IA-SNB) from a classical 1934 H1N1 influenza virus. IA-SNBs elicited pan-H1 antibodies and provided protection against antigenically divergent H1N1 viruses via intranasal immunizations. Viral glycoprotein spikes displayed as SNBs could aid in combating antigenic variation and provide innovative intranasal vaccines to aid universal influenza vaccine development.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259500","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.16.613207
Veronica M Sinotte, Veronica Ramos Viana, Diego Prado Vasquez, Sevgi Mutlu Sirakova, Nabila Rodriguez Valeron, Ana Cuesta Mate, Shannara K Taylor Parkins, Esther Merino Velasco, David Zilber, Rasmus Munk, Sandra B Andersen, Robert R Dunn, Leonie J Jahn
Milk fermentation has a rich history in which food culture, the environment, and microbes intersect. However, the biocultural origins of fermentation practices and microbes have largely been replaced by industrial processes. Here, we consider a historical fermentation originating from Turkey and Bulgaria, ant yogurt. We revisit the traditional practices and modern gastronomic applications that use red wood ants (Formica rufa group) to initiate milk fermentation. Subsequently, we characterize the ants and experimental ant-derived yogurts. We uncover that the ant holobiont, which consists of the ants and their microbes, contributes key acids and enzymes to fermentation. Metabarcoding and culturing revealed that lactic and acetic acid bacteria, including species related to those in conventional yogurt and sourdough, originate from the live ants and proliferate in the milk. The ants and bacteria consequently introduce formic, lactic, and acetic acid, advantageous for yogurt acidification and coagulation. Last, proteases with the potential to act on casein and alter yogurt texture are produced by the ants and bacteria. The ant holobiont thus facilitates fermentation akin to the microbial consortia in other ferments. Our findings highlight the value of integrating traditional, gastronomic, and biological frameworks to uncover the origins and applications of microbes for fermented foods.
{"title":"Making yogurt with the ant holobiont uncovers bacteria, acids, and enzymes for food fermentation","authors":"Veronica M Sinotte, Veronica Ramos Viana, Diego Prado Vasquez, Sevgi Mutlu Sirakova, Nabila Rodriguez Valeron, Ana Cuesta Mate, Shannara K Taylor Parkins, Esther Merino Velasco, David Zilber, Rasmus Munk, Sandra B Andersen, Robert R Dunn, Leonie J Jahn","doi":"10.1101/2024.09.16.613207","DOIUrl":"https://doi.org/10.1101/2024.09.16.613207","url":null,"abstract":"Milk fermentation has a rich history in which food culture, the environment, and microbes intersect. However, the biocultural origins of fermentation practices and microbes have largely been replaced by industrial processes. Here, we consider a historical fermentation originating from Turkey and Bulgaria, ant yogurt. We revisit the traditional practices and modern gastronomic applications that use red wood ants (Formica rufa group) to initiate milk fermentation. Subsequently, we characterize the ants and experimental ant-derived yogurts. We uncover that the ant holobiont, which consists of the ants and their microbes, contributes key acids and enzymes to fermentation. Metabarcoding and culturing revealed that lactic and acetic acid bacteria, including species related to those in conventional yogurt and sourdough, originate from the live ants and proliferate in the milk. The ants and bacteria consequently introduce formic, lactic, and acetic acid, advantageous for yogurt acidification and coagulation. Last, proteases with the potential to act on casein and alter yogurt texture are produced by the ants and bacteria. The ant holobiont thus facilitates fermentation akin to the microbial consortia in other ferments. Our findings highlight the value of integrating traditional, gastronomic, and biological frameworks to uncover the origins and applications of microbes for fermented foods.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.16.613209
Hammam Antar, Nicolas Carraro, Stephan Gruber, Jan Roelof van der Meer
Integrative conjugative elements (ICEs) are mobile DNA that remain integrated within the host bacterial genome until they activate their excision and transfer to recipient cells via conjugation. ICE transfer is initiated in a small subpopulation of cells that undergo a hierarchical gene expression cascade leading to transfer competence formation. In this study, we demonstrate that transfer competence formation of the ICEclc element in stationary phase Pseudomonas putida cells is regulated by the coordinated activity of a two-component transcriptional activator, BisC and BisD. Chromatin immunoprecipitation of tagged BisC or BisD followed by high throughput sequencing showed that both proteins accumulate at similar sites around ICEclc transfer competence promoters in P. putida cells. Genetic dissection and single cell microscopy showed that BisD is a dual domain protein, with a C-terminal gene activator domain and an N-terminal ParB-like domain, forming dimeric DNA clamps that self-load at distant sites and reach target promoters after extensive one-dimensional DNA sliding. Expressed mCherry-BisD in P. putida ICEclc cells form discrete fluorescent foci, dependent on parS-like sequences on the ICE. This focus formation is similar as what is seen with canonical ParB proteins accumulating on chromosomal DNA, but in case of BisD corresponds to both chromosomal and excised ICE-molecules. Sliding of BisD over ~50 kb of ICE-DNA is asymmetric and follows the direction of ICE gene transcription. This may help to establish a temporal order of activation of transfer competence formation, optimizing ICE transfer. Given that ICEclc is activated in stationary phase cells, we hypothesize that BisD is not involved in segregating excised ICE-DNA among daughter cells, but rather in actively directing ICE-DNA molecules towards the (multiple) conjugative systems that are produced in transfer competent cells. BisD thus serves as a twin function protein, integrating gene activation and DNA segregation functions.
整合共轭元件（ICEs）是一种移动 DNA，它一直整合在宿主细菌基因组中，直到激活切除并通过共轭转移到受体细胞。ICE 的转移是在一小部分细胞中开始的，这些细胞经历了分级基因表达级联，最终形成转移能力。在这项研究中，我们证明了静止期假单胞菌细胞中 ICEclc 基因的转移能力形成是由双组分转录激活因子 BisC 和 BisD 的协调活动调控的。对标记的 BisC 或 BisD 进行染色质免疫沉淀，然后进行高通量测序，结果表明这两种蛋白都聚集在假单胞菌细胞中 ICEclc 转移能力启动子周围的相似位点。基因剖析和单细胞显微镜检查表明，BisD 是一种双结构域蛋白，具有 C 端基因激活结构域和 N 端 ParB 样结构域，可形成二聚体 DNA 夹，在远处自我加载，并在广泛的一维 DNA 滑动后到达目标启动子。在 P. putida ICEclc 细胞中表达的 mCherry-BisD 会形成离散的荧光病灶，这取决于 ICE 上的 parS 样序列。这种病灶的形成与染色体 DNA 上聚集的典型 ParB 蛋白类似，但在 BisD 的情况下，它同时与染色体和切除的 ICE 分子相对应。BisD 在约 50 kb 的 ICE-DNA 上的滑动是不对称的，并且与 ICE 基因的转录方向一致。这可能有助于确定转移能力形成的激活时间顺序，优化 ICE 转移。鉴于 ICEclc 在静止期细胞中被激活，我们推测 BisD 并不参与在子细胞中分离切除的 ICE-DNA，而是积极引导 ICE-DNA 分子向转移能力细胞中产生的（多个）共轭系统转移。因此，BisD 是一种双功能蛋白，集基因激活和 DNA 分离功能于一身。
{"title":"Orchestrated long-distance gene activation by a ParB-like BisD-CTP DNA clamp in low-frequency transfer competence development in Pseudomonas putida","authors":"Hammam Antar, Nicolas Carraro, Stephan Gruber, Jan Roelof van der Meer","doi":"10.1101/2024.09.16.613209","DOIUrl":"https://doi.org/10.1101/2024.09.16.613209","url":null,"abstract":"Integrative conjugative elements (ICEs) are mobile DNA that remain integrated within the host bacterial genome until they activate their excision and transfer to recipient cells via conjugation. ICE transfer is initiated in a small subpopulation of cells that undergo a hierarchical gene expression cascade leading to transfer competence formation. In this study, we demonstrate that transfer competence formation of the ICEclc element in stationary phase Pseudomonas putida cells is regulated by the coordinated activity of a two-component transcriptional activator, BisC and BisD. Chromatin immunoprecipitation of tagged BisC or BisD followed by high throughput sequencing showed that both proteins accumulate at similar sites around ICEclc transfer competence promoters in P. putida cells. Genetic dissection and single cell microscopy showed that BisD is a dual domain protein, with a C-terminal gene activator domain and an N-terminal ParB-like domain, forming dimeric DNA clamps that self-load at distant sites and reach target promoters after extensive one-dimensional DNA sliding. Expressed mCherry-BisD in P. putida ICEclc cells form discrete fluorescent foci, dependent on parS-like sequences on the ICE. This focus formation is similar as what is seen with canonical ParB proteins accumulating on chromosomal DNA, but in case of BisD corresponds to both chromosomal and excised ICE-molecules. Sliding of BisD over ~50 kb of ICE-DNA is asymmetric and follows the direction of ICE gene transcription. This may help to establish a temporal order of activation of transfer competence formation, optimizing ICE transfer. Given that ICEclc is activated in stationary phase cells, we hypothesize that BisD is not involved in segregating excised ICE-DNA among daughter cells, but rather in actively directing ICE-DNA molecules towards the (multiple) conjugative systems that are produced in transfer competent cells. BisD thus serves as a twin function protein, integrating gene activation and DNA segregation functions.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259447","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.16.611982
Jelmer Dijkstra, Anouk C. van Westerhoven, Lucia Gomez-Gil, Carolina Aguilera-Galvez, Giuliana Nakasato-Tagami, Sebastien D. Garnier, Masaya Yamazaki, Tsutomu Arie, Takashi Kamakura, Takayuki Arazoe, Antonio Di Pietro, Michael F. Seidl, Gert H.J. Kema
Filamentous fungi have evolved compartmentalized genomes consisting of conserved core regions and dynamic accessory regions, which aid the adaptation to changing environments including the interaction with host organisms. In the Fusarium oxysporum species complex, accessory regions play an important role during infection and it has been reported that these regions undergo extensive duplications, however, it is currently unknown how such duplications shape accessory regions. Moreover, the function of accessory regions apart from encoding virulence effectors is not completely understood. Here we determined the karyotype of F. oxysporum Tropical Race 4 (TR4), which causes the ongoing pandemic of Fusarium wilt of banana (FWB). We show that the single accessory chromosome of TR4 isolate II5 has undergone extensive intrachromosomal duplications, resulting in triplication of the chromosome size compared to other closely related TR4 strains. By obtaining mutant strains that have lost the accessory chromosome, we demonstrate that this chromosome is dispensable for vegetative growth but is required for full virulence on banana. Lastly, we found that the loss of chromosome 12 co-occurs with structural rearrangements of core chromosomes, which are generally co-linear between members of the F. oxysporum species complex. Together, our results provide new insights into the chromosome dynamics of the banana infecting TR4 lineage of the F. oxysporum species complex.
丝状真菌进化出了分区基因组，由保守的核心区和动态的附属区组成，有助于适应不断变化的环境，包括与宿主生物的相互作用。在氧孢镰刀菌物种群中，附属区在感染过程中发挥着重要作用，有报道称这些区域经历了大量的复制，但目前还不清楚这种复制是如何形成附属区的。此外，除了编码毒力效应因子外，附属区的功能也不完全清楚。在这里，我们测定了导致香蕉镰刀菌枯萎病（FWB）大流行的 F. oxysporum 热带第 4 种族（TR4）的核型。我们发现 TR4 分离物 II5 的单条附属染色体经历了广泛的染色体内复制，与其他近缘 TR4 株系相比，染色体大小增加了两倍。通过获得失去附属染色体的突变株，我们证明了该染色体对无性繁殖是不可或缺的，但对香蕉的完全毒力却是必需的。最后，我们发现 12 号染色体的缺失与核心染色体的结构重排同时发生，这在 F. oxysporum 物种复合体成员之间通常是共线性的。总之，我们的研究结果为我们提供了有关氧孢子菌种群中香蕉感染 TR4 系染色体动态的新见解。
{"title":"Extensive intrachromosomal duplications in a virulence-associated fungal accessory chromosome","authors":"Jelmer Dijkstra, Anouk C. van Westerhoven, Lucia Gomez-Gil, Carolina Aguilera-Galvez, Giuliana Nakasato-Tagami, Sebastien D. Garnier, Masaya Yamazaki, Tsutomu Arie, Takashi Kamakura, Takayuki Arazoe, Antonio Di Pietro, Michael F. Seidl, Gert H.J. Kema","doi":"10.1101/2024.09.16.611982","DOIUrl":"https://doi.org/10.1101/2024.09.16.611982","url":null,"abstract":"Filamentous fungi have evolved compartmentalized genomes consisting of conserved core regions and dynamic accessory regions, which aid the adaptation to changing environments including the interaction with host organisms. In the Fusarium oxysporum species complex, accessory regions play an important role during infection and it has been reported that these regions undergo extensive duplications, however, it is currently unknown how such duplications shape accessory regions. Moreover, the function of accessory regions apart from encoding virulence effectors is not completely understood. Here we determined the karyotype of F. oxysporum Tropical Race 4 (TR4), which causes the ongoing pandemic of Fusarium wilt of banana (FWB). We show that the single accessory chromosome of TR4 isolate II5 has undergone extensive intrachromosomal duplications, resulting in triplication of the chromosome size compared to other closely related TR4 strains. By obtaining mutant strains that have lost the accessory chromosome, we demonstrate that this chromosome is dispensable for vegetative growth but is required for full virulence on banana. Lastly, we found that the loss of chromosome 12 co-occurs with structural rearrangements of core chromosomes, which are generally co-linear between members of the F. oxysporum species complex. Together, our results provide new insights into the chromosome dynamics of the banana infecting TR4 lineage of the F. oxysporum species complex.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259492","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.14.613010
Sarah Lanning, Nayeli Aguilar-Hernández, Vitor Hugo Balasco Serrão, Tomás López, Sara M O'Rourke, Adam Lentz, Lena Ricemeyer, Rafaela Espinosa, Susana López, Carlos Federico Arias, Rebecca M. DuBois
Human astroviruses (HAstVs) are a leading cause of viral childhood diarrhea that infect nearly every individual during their lifetime. Although human astroviruses are highly prevalent, no approved vaccine currently exists. Antibody responses appear to play an important role in protection from HAstV infection, however knowledge about the neutralizing epitope landscape is lacking, as only 3 neutralizing antibody epitopes have previously been determined. Here, we structurally define the epitopes of 3 uncharacterized HAstV-neutralizing monoclonal antibodies: antibody 4B6 with X-ray crystallography to 2.67 Å, and antibodies 3H4 and 3B4 simultaneously with single-particle cryogenic-electron microscopy to 3.33 Å. We assess the epitope locations relative to conserved regions on the capsid spike and find that while antibodies 4B6 and 3B4 target the upper variable loop regions of the HAstV spike protein, antibody 3H4 targets a novel region near the base of the spike that is more conserved. Additionally, we found that all 3 antibodies bind with high affinity, and they compete with receptor FcRn binding to the capsid spike. These studies inform which regions of the HAstV capsid can be targeted by monoclonal antibody therapies and could aid in rational vaccine design.
人类星状病毒（HAstVs）是导致儿童病毒性腹泻的主要原因，几乎每个人一生中都会感染这种病毒。虽然人类哮喘病毒的流行率很高，但目前还没有获得批准的疫苗。抗体反应似乎在保护人们免受 HAstV 感染方面发挥了重要作用，但由于之前只确定了 3 个中和抗体表位，因此缺乏有关中和表位情况的知识。在这里，我们从结构上确定了 3 种未表征的 HAstV 中和单克隆抗体的表位：用 X 射线晶体学方法确定了 2.67 Å 的抗体 4B6，用单粒子低温电子显微镜同时确定了 3.33 Å 的抗体 3H4 和 3B4。我们评估了表位位置与噬菌体尖峰蛋白保守区域的相对关系，发现抗体 4B6 和 3B4 的靶标是 HAstV 尖峰蛋白的上可变环区域，而抗体 3H4 的靶标是尖峰蛋白基部附近的一个新区域，该区域更为保守。此外，我们发现这三种抗体的结合亲和力都很高，它们会与受体FcRn竞争结合到帽状尖峰蛋白上。这些研究为单克隆抗体疗法可以靶向HAstV荚膜的哪些区域提供了信息，有助于合理设计疫苗。
{"title":"Discovery of three novel neutralizing antibody epitopes on the human astrovirus capsid spike and mechanistic insights into virus neutralization","authors":"Sarah Lanning, Nayeli Aguilar-Hernández, Vitor Hugo Balasco Serrão, Tomás López, Sara M O'Rourke, Adam Lentz, Lena Ricemeyer, Rafaela Espinosa, Susana López, Carlos Federico Arias, Rebecca M. DuBois","doi":"10.1101/2024.09.14.613010","DOIUrl":"https://doi.org/10.1101/2024.09.14.613010","url":null,"abstract":"Human astroviruses (HAstVs) are a leading cause of viral childhood diarrhea that infect nearly every individual during their lifetime. Although human astroviruses are highly prevalent, no approved vaccine currently exists. Antibody responses appear to play an important role in protection from HAstV infection, however knowledge about the neutralizing epitope landscape is lacking, as only 3 neutralizing antibody epitopes have previously been determined. Here, we structurally define the epitopes of 3 uncharacterized HAstV-neutralizing monoclonal antibodies: antibody 4B6 with X-ray crystallography to 2.67 Å, and antibodies 3H4 and 3B4 simultaneously with single-particle cryogenic-electron microscopy to 3.33 Å. We assess the epitope locations relative to conserved regions on the capsid spike and find that while antibodies 4B6 and 3B4 target the upper variable loop regions of the HAstV spike protein, antibody 3H4 targets a novel region near the base of the spike that is more conserved. Additionally, we found that all 3 antibodies bind with high affinity, and they compete with receptor FcRn binding to the capsid spike. These studies inform which regions of the HAstV capsid can be targeted by monoclonal antibody therapies and could aid in rational vaccine design.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259498","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.15.613162
Xinyue Gu, Alex Gill, Qiong Yang, Perran A Ross, Ella Yeatman, Mel Berran, Monica Stelmach, Sonia Sharma, Paul A. Umina, Ary A. Hoffmann
Aphids are among the world's most economically damaging pests and carry a diverse range of bacterial endosymbionts. There is increasing interest in exploring potential applications of natural and novel strains of endosymbionts in aphid control. One endosymbiont, Rickettsiella viridis, has a large fitness cost following transfer from its natural aphid host Acyrthosiphon pisum into a novel host aphid Myzus persicae. Here, we investigated host impacts after transferring the same Rickettsiella strain to an important cereal aphid, the Russian wheat aphid Diuraphis noxia. Rickettsiella in this host resulted in modest fitness effects, with a minor increase in heat tolerance and a decrease in development time at 25C. The infection persisted in mixed caged populations under different temperatures. Surprisingly, Rickettsiella increased aphid virulence to wheat plants and to two non-crop hosts of D. noxia, barley grass and brome grass. This was evident from sharper decreases in leaf number and leaf area, as well as an increase in chlorotic streaking when plants were exposed to Rickettsiella-infected D. noxia. Rickettsiella also reduced the proportion of alates in aphids held in small cages and in larger mesocosms containing multiple wheat plants where short-distance dispersal of aphids was impacted. These results provide compelling evidence that Rickettsiella can affect virulence - the first case of an endosymbiont transfer directly influencing aphid virulence to host plants - and highlight the species-specific impacts of endosymbiont transfers on aphids which can involve multiple traits.
{"title":"The endosymbiont Rickettsiella viridis increases the virulence of Diuraphis noxia but reduces alate frequency","authors":"Xinyue Gu, Alex Gill, Qiong Yang, Perran A Ross, Ella Yeatman, Mel Berran, Monica Stelmach, Sonia Sharma, Paul A. Umina, Ary A. Hoffmann","doi":"10.1101/2024.09.15.613162","DOIUrl":"https://doi.org/10.1101/2024.09.15.613162","url":null,"abstract":"Aphids are among the world's most economically damaging pests and carry a diverse range of bacterial endosymbionts. There is increasing interest in exploring potential applications of natural and novel strains of endosymbionts in aphid control. One endosymbiont, Rickettsiella viridis, has a large fitness cost following transfer from its natural aphid host Acyrthosiphon pisum into a novel host aphid Myzus persicae. Here, we investigated host impacts after transferring the same Rickettsiella strain to an important cereal aphid, the Russian wheat aphid Diuraphis noxia. Rickettsiella in this host resulted in modest fitness effects, with a minor increase in heat tolerance and a decrease in development time at 25C. The infection persisted in mixed caged populations under different temperatures. Surprisingly, Rickettsiella increased aphid virulence to wheat plants and to two non-crop hosts of D. noxia, barley grass and brome grass. This was evident from sharper decreases in leaf number and leaf area, as well as an increase in chlorotic streaking when plants were exposed to Rickettsiella-infected D. noxia. Rickettsiella also reduced the proportion of alates in aphids held in small cages and in larger mesocosms containing multiple wheat plants where short-distance dispersal of aphids was impacted. These results provide compelling evidence that Rickettsiella can affect virulence - the first case of an endosymbiont transfer directly influencing aphid virulence to host plants - and highlight the species-specific impacts of endosymbiont transfers on aphids which can involve multiple traits.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259494","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.13.612890
Silke Machata, Ute Bertsche, Franziska Hoffmann, Zaher M. Fattal, Franziska Kage, Michal Flak, Alexander N. J. Iliou, Falk Hillmann, Ferdinand von Eggeling, Hortense Slevogt, Axel A. Brakhage, Ilse D. Jacobsen
Aspergillus fumigatus is a saprophytic fungus dwelling in soil and on decaying plant material, but also an opportunistic pathogen in immunocompromised patients. In its environmental niche, A. fumigatus faces competition from other microorganisms including bacteria. Here, we describe the discovery of the first secreted antibacterial protein in A. fumigatus. We identified a secreted fungal endopeptidase, designated CwhA, that cleaves peptidoglycan of Gram-positive bacteria at specific residues within the peptidoglycan stem peptide. Cleavage leads to bacterial lysis and the release of peptidoglycan cleavage products. Expression of cwhA is induced by the presence of bacteria. Furthermore, CwhA is highly abundant in murine lungs during invasive pulmonary aspergillosis and peptidoglycan cleavage products generated by CwhA stimulate cytokine production of human immune cells. Although CwhA does not affect human cells directly, this novel player in fungal-bacterial interactions could affect A. fumigatus infections by inhibiting Gram-positive bacteria in its vicinity, and modulating the immune system.
{"title":"Identification of a fungal antibacterial endopeptidase that modulates immune responses","authors":"Silke Machata, Ute Bertsche, Franziska Hoffmann, Zaher M. Fattal, Franziska Kage, Michal Flak, Alexander N. J. Iliou, Falk Hillmann, Ferdinand von Eggeling, Hortense Slevogt, Axel A. Brakhage, Ilse D. Jacobsen","doi":"10.1101/2024.09.13.612890","DOIUrl":"https://doi.org/10.1101/2024.09.13.612890","url":null,"abstract":"Aspergillus fumigatus is a saprophytic fungus dwelling in soil and on decaying plant material, but also an opportunistic pathogen in immunocompromised patients. In its environmental niche, A. fumigatus faces competition from other microorganisms including bacteria. Here, we describe the discovery of the first secreted antibacterial protein in A. fumigatus. We identified a secreted fungal endopeptidase, designated CwhA, that cleaves peptidoglycan of Gram-positive bacteria at specific residues within the peptidoglycan stem peptide. Cleavage leads to bacterial lysis and the release of peptidoglycan cleavage products. Expression of cwhA is induced by the presence of bacteria. Furthermore, CwhA is highly abundant in murine lungs during invasive pulmonary aspergillosis and peptidoglycan cleavage products generated by CwhA stimulate cytokine production of human immune cells. Although CwhA does not affect human cells directly, this novel player in fungal-bacterial interactions could affect A. fumigatus infections by inhibiting Gram-positive bacteria in its vicinity, and modulating the immune system.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259524","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.16.613245
Inga Kirstein, Marlis Reich, Yanyan Yang, Maike Timmermann, Antje Wichels, Gunnar Gerdts
Fungi play important roles in biofilms, are very versatile in their ecological role, and are considered as plastic degraders. Here we aim to increase the resolution of the fungal members of the Plastisphere, to understand fungal substrate specificities and related potential ecological impacts. Fifteen-month-old fungal Plastisphere communities were investigated on 9 different plastic types and glass in seawater from the North Sea. By integrating scanning electron microscopy (SEM) imaging, ITS-based fingerprinting, and re-evaluated 18S rRNA gene sequence data through a fungal-specific phylogeny-based pipeline, we observed fungal Plastispheres and identified specific characteristics based on morphotypes, phylogeny, and biodiversity across different substrate types. Plastic types selected for specific fungal communities with polyolefine communities indicating significantly higher diversity compared to all other plastic types. Furthermore, specific plastic types may select for specific fungal taxa and their potential hosts, highlighting the complexity of marine biofilm food webs, and related ecological implications.
{"title":"The Plastisphere: Marine fungi communities in the plastics age","authors":"Inga Kirstein, Marlis Reich, Yanyan Yang, Maike Timmermann, Antje Wichels, Gunnar Gerdts","doi":"10.1101/2024.09.16.613245","DOIUrl":"https://doi.org/10.1101/2024.09.16.613245","url":null,"abstract":"Fungi play important roles in biofilms, are very versatile in their ecological role, and are considered as plastic degraders. Here we aim to increase the resolution of the fungal members of the Plastisphere, to understand fungal substrate specificities and related potential ecological impacts. Fifteen-month-old fungal Plastisphere communities were investigated on 9 different plastic types and glass in seawater from the North Sea. By integrating scanning electron microscopy (SEM) imaging, ITS-based fingerprinting, and re-evaluated 18S rRNA gene sequence data through a fungal-specific phylogeny-based pipeline, we observed fungal Plastispheres and identified specific characteristics based on morphotypes, phylogeny, and biodiversity across different substrate types. Plastic types selected for specific fungal communities with polyolefine communities indicating significantly higher diversity compared to all other plastic types. Furthermore, specific plastic types may select for specific fungal taxa and their potential hosts, highlighting the complexity of marine biofilm food webs, and related ecological implications.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-09-16DOI: 10.1101/2024.09.16.613323
Mohammad Sadik, Imran Moin, Saif Ullah, M. Sayeedur Rahman, Oliver H. Voss
The key cellular processes required for rickettsial obligate intracellular lifestyle, include internalization by phagocytosis, regulation of intracellular trafficking, and evasion of lysosomal destruction to establish an intracytosolic replication niche, remain poorly defined. Recent reports showed that rickettsial phospholipases play an important role in vacuolar escape, but their functions are dispensable depending on the host cell-type. Here, we report the identification of a highly conserved putative lipase containing a Serine hydrolase motif (GXSXG), named RLip (Rickettsia Lipase). Our work reveals that RLip expression is cytotoxic to yeast cells, a genetically tractable heterologous model system. We demonstrate that RLip possesses lipase enzymatic activity and show a lipid specificity towards phosphoinositide (PI)(3), PI(3,4,5)P3, and PI(3,4)P2, and to a lesser extent PI(4,5)P2. Further, we found that RLip expression is induced during infection of pathogenic R. rickettsii, while its expression is low or undetectable for R. parkeri (mild-pathogenic) and R. montanensis (non-pathogenic), respectively, during host invasion. Intriguingly, RLip is highly enriched in the cytoplasmic fraction of host cells, however, minimally retained by the rickettsiae themselves, suggesting RLip is synthesized during infection and then secreted into the host cell cytoplasm. Neutralization of RLip activity, by antibody-blocking, significantly abrogated R. rickettsii escape from bactericidal phagolysosomal fusion, suggesting RLip plays a critical role in facilitating the intracytosolic colonization of pathogenic R. rickettsii.
{"title":"Rickettsia rickettsii encodes a secretory lipase that facilitates intracytosolic colonization in host cells.","authors":"Mohammad Sadik, Imran Moin, Saif Ullah, M. Sayeedur Rahman, Oliver H. Voss","doi":"10.1101/2024.09.16.613323","DOIUrl":"https://doi.org/10.1101/2024.09.16.613323","url":null,"abstract":"The key cellular processes required for rickettsial obligate intracellular lifestyle, include internalization by phagocytosis, regulation of intracellular trafficking, and evasion of lysosomal destruction to establish an intracytosolic replication niche, remain poorly defined. Recent reports showed that rickettsial phospholipases play an important role in vacuolar escape, but their functions are dispensable depending on the host cell-type. Here, we report the identification of a highly conserved putative lipase containing a Serine hydrolase motif (GXSXG), named RLip (<em>Rickettsia</em> Lipase). Our work reveals that RLip expression is cytotoxic to yeast cells, a genetically tractable heterologous model system. We demonstrate that RLip possesses lipase enzymatic activity and show a lipid specificity towards phosphoinositide (PI)(3), PI(3,4,5)P<sub>3</sub>, and PI(3,4)P<sub>2</sub>, and to a lesser extent PI(4,5)P<sub>2</sub>. Further, we found that RLip expression is induced during infection of pathogenic <em>R. rickettsii</em>, while its expression is low or undetectable for <em>R. parkeri</em> (mild-pathogenic) and <em>R. montanensis</em> (non-pathogenic), respectively, during host invasion. Intriguingly, RLip is highly enriched in the cytoplasmic fraction of host cells, however, minimally retained by the rickettsiae themselves, suggesting RLip is synthesized during infection and then secreted into the host cell cytoplasm. Neutralization of RLip activity, by antibody-blocking, significantly abrogated <em>R. rickettsii</em> escape from bactericidal phagolysosomal fusion, suggesting RLip plays a critical role in facilitating the intracytosolic colonization of pathogenic <em>R. rickettsii</em>.","PeriodicalId":501357,"journal":{"name":"bioRxiv - Microbiology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142259488","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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