{"title":"细胞微生物学的新方法","authors":"Elizabeth L. Hartland","doi":"10.1111/cmi.13369","DOIUrl":null,"url":null,"abstract":"<p>In an era where new viruses can emerge suddenly and anti-microbial resistance is now widespread, a full understanding of the pathogen and host factors that contribute to disease pathology and spread remains a critically important goal. Methods and technologies to study the cell biology of infection are changing rapidly. The interdisciplinary requirements to interrogate host-pathogen interactions are ever more complex and indispensable to identify the cellular and immune factors that lead to the resolution of infection. The ability of a pathogen to replicate in the host likewise needs a thorough knowledge of the pathogen determinants required to cause disease. In this special issue, <i>Cellular Microbiology</i> highlights some of the most cutting-edge approaches and techniques to study pathogen biology and infection.</p><p>Light based and electron microscopic imaging has long played an important role in defining the intracellular mechanisms of infection. Such methods can now be quickly adapted through the design of fluorescent reporters and utilised for high throughout applications. Cortese and Laketa <span>(2021)</span> describe recent advances in high-throughput microscopy and electron microscopy and explain how these were applied during the SARS-CoV-2 outbreak (Cortese & Laketa, <span>2021</span>). For example, the development of a mNeonGreen reporter microscopy-based virus neutralisation assay outperformed standard plaque assays in sensitivity and time in the critical early stages of the COVID-19 pandemic (Muruato et al., <span>2020</span>). Cryo-electron microscopy was instrumental in rapidly defining the structure of the SARS-CoV-2 spike protein in complex with its cellular receptor, angiotensin convertase enzyme 2 (ACE2), directly informing an understanding of neutralising antibodies (Cortese & Laketa, <span>2021</span>). In bacterial pathogens, Dufrêne, Viljoen, Mignolet, and Mathelié-Guinlet (<span>2021</span>) report on the utility of atomic force microscopy (AFM) to provide super-resolution imaging and nanomechanical measurements of bacterial cell surfaces and receptor-ligand interactions. This is providing new insight into the fine structure and biophysical function of adhesins and also informing vaccine and drug development (Dufrêne et al., <span>2021</span>).</p><p>The subcellular host cell compartment occupied by intracellular pathogens is highly specialised to support pathogen replication. Given their role in sampling of extracellular fluid, macropinosomes are hijacked by diverse pathogens to establish entry into the intracellular environment. However, macropinosomes are also highly dynamic organelles with a range of cellular functions. Chang, Enninga, and Stévenin <span>(2021)</span> describe imaging-based and proteomic methods for the tracking and characterisation of these important organelles as they are modified as a niche for invasion and/or replication by bacteria such as <i>Shigella</i>, <i>Salmonella</i>, <i>Brucella</i> and <i>Chlamydia</i> (Chang et al., <span>2021</span>). Similarly, Simeone, Sayes, Lawarée, and Brosch (<span>2021</span>) explore the intracellular niche of Mycobacteria, in particular the bacterial factors that allow phagosomal escape of <i>Mycobacterium tuberculosis</i> into the cell cytosol (Simeone et al., <span>2021</span>). Multiple techniques used in parallel, such as labelling with galectin-3 and ubiquitin to identify damaged phagosomal membranes, as well as FRET-based reporter and cytofluorometric approaches have provided new insight into phagosomal rupture by <i>M</i>. <i>tuberculosis</i>.</p><p>Cell lines and primary cells remain important tools in the study of host-pathogen interactions. However, the development of new models such as human stem cell models and microfluidic ‘organ on chip’ technologies can offer an additional and more physiological perspective. Pellegrino and Gutierrez <span>(2021)</span> report on the use of stem cell-derived models engineered into three-dimensional microenvironments as a novel means to study the biology of infection, with and without other cell types in cellular co-culture (Pellegrino & Gutierrez, <span>2021</span>). These so-called ‘organoids’ are particularly helpful for pre-clinical studies where no small animal model of infection exists and can be adapted to incorporate genome editing tools. Another three-dimensional stem cell approach is ‘organ on-a-chip’ technology which utilises microfluidics and biomaterials to mimic the interface between the pathogen and host tissue during infection. Feaugas and Sauvonnet (<span>2021</span>) provide a timely update on the advantages of using organ on-a-chip and the different elements to consider as well as examples of how it has re-defined our knowledge of host pathogen interactions (Feaugas & Sauvonnet, <span>2021</span>). For example, organ-on-a-chip has provided insight into how mechanical forces in the gut influence <i>Shigella</i> invasion (Grassart et al., <span>2019</span>).</p><p>High throughput screening is an integral part of drug development as well as large scale genomic editing approaches. Subhash and Sundaramurthy describe screening platforms that take the host environment into account (Subhash & Sundaramurthy, <span>2021</span>). This allows the identification of potential host directed therapeutics which may be used as adjunct therapies against chronic intractable infections like tuberculosis, particularly where the pathogen is intracellular. Imaging is a desirable output for many high throughput screening approaches for a number of reasons. An automated image analysis program is discussed by Fisch et al. (<span>2021</span>), HRMAn (Host Response to Microbe Analysis), that uses machine learning and artificial intelligence to assess infection by pathogens in an unbiased manner. Parameters include pathogen growth, pathogen killing and activation of host cell signalling. When incorporated into a functional screen, HRMAn provides unprecedented capacity for high throughput and high-content analysis (Fisch et al., <span>2021</span>).</p><p>In summary, the techniques presented in this special issue of <i>Cellular Microbiology</i> provide inspiration and direction to many investigators studying bacterial, viral and parasite infections. The state-of-the-art approaches explained here will allow transformational research questions to be addressed and deepen our knowledge of host-pathogen interactions that could lead to new disease interventions. We anticipate that further issues of <i>Cellular Microbiology</i> will be dedicated to emerging technologies in order to highlight the most important methodological developments in our field.</p>","PeriodicalId":9844,"journal":{"name":"Cellular Microbiology","volume":"23 7","pages":""},"PeriodicalIF":2.6000,"publicationDate":"2021-06-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1111/cmi.13369","citationCount":"0","resultStr":"{\"title\":\"Emerging methods in cellular microbiology\",\"authors\":\"Elizabeth L. Hartland\",\"doi\":\"10.1111/cmi.13369\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>In an era where new viruses can emerge suddenly and anti-microbial resistance is now widespread, a full understanding of the pathogen and host factors that contribute to disease pathology and spread remains a critically important goal. Methods and technologies to study the cell biology of infection are changing rapidly. The interdisciplinary requirements to interrogate host-pathogen interactions are ever more complex and indispensable to identify the cellular and immune factors that lead to the resolution of infection. The ability of a pathogen to replicate in the host likewise needs a thorough knowledge of the pathogen determinants required to cause disease. In this special issue, <i>Cellular Microbiology</i> highlights some of the most cutting-edge approaches and techniques to study pathogen biology and infection.</p><p>Light based and electron microscopic imaging has long played an important role in defining the intracellular mechanisms of infection. Such methods can now be quickly adapted through the design of fluorescent reporters and utilised for high throughout applications. Cortese and Laketa <span>(2021)</span> describe recent advances in high-throughput microscopy and electron microscopy and explain how these were applied during the SARS-CoV-2 outbreak (Cortese & Laketa, <span>2021</span>). For example, the development of a mNeonGreen reporter microscopy-based virus neutralisation assay outperformed standard plaque assays in sensitivity and time in the critical early stages of the COVID-19 pandemic (Muruato et al., <span>2020</span>). Cryo-electron microscopy was instrumental in rapidly defining the structure of the SARS-CoV-2 spike protein in complex with its cellular receptor, angiotensin convertase enzyme 2 (ACE2), directly informing an understanding of neutralising antibodies (Cortese & Laketa, <span>2021</span>). In bacterial pathogens, Dufrêne, Viljoen, Mignolet, and Mathelié-Guinlet (<span>2021</span>) report on the utility of atomic force microscopy (AFM) to provide super-resolution imaging and nanomechanical measurements of bacterial cell surfaces and receptor-ligand interactions. This is providing new insight into the fine structure and biophysical function of adhesins and also informing vaccine and drug development (Dufrêne et al., <span>2021</span>).</p><p>The subcellular host cell compartment occupied by intracellular pathogens is highly specialised to support pathogen replication. Given their role in sampling of extracellular fluid, macropinosomes are hijacked by diverse pathogens to establish entry into the intracellular environment. However, macropinosomes are also highly dynamic organelles with a range of cellular functions. Chang, Enninga, and Stévenin <span>(2021)</span> describe imaging-based and proteomic methods for the tracking and characterisation of these important organelles as they are modified as a niche for invasion and/or replication by bacteria such as <i>Shigella</i>, <i>Salmonella</i>, <i>Brucella</i> and <i>Chlamydia</i> (Chang et al., <span>2021</span>). Similarly, Simeone, Sayes, Lawarée, and Brosch (<span>2021</span>) explore the intracellular niche of Mycobacteria, in particular the bacterial factors that allow phagosomal escape of <i>Mycobacterium tuberculosis</i> into the cell cytosol (Simeone et al., <span>2021</span>). Multiple techniques used in parallel, such as labelling with galectin-3 and ubiquitin to identify damaged phagosomal membranes, as well as FRET-based reporter and cytofluorometric approaches have provided new insight into phagosomal rupture by <i>M</i>. <i>tuberculosis</i>.</p><p>Cell lines and primary cells remain important tools in the study of host-pathogen interactions. However, the development of new models such as human stem cell models and microfluidic ‘organ on chip’ technologies can offer an additional and more physiological perspective. Pellegrino and Gutierrez <span>(2021)</span> report on the use of stem cell-derived models engineered into three-dimensional microenvironments as a novel means to study the biology of infection, with and without other cell types in cellular co-culture (Pellegrino & Gutierrez, <span>2021</span>). These so-called ‘organoids’ are particularly helpful for pre-clinical studies where no small animal model of infection exists and can be adapted to incorporate genome editing tools. Another three-dimensional stem cell approach is ‘organ on-a-chip’ technology which utilises microfluidics and biomaterials to mimic the interface between the pathogen and host tissue during infection. Feaugas and Sauvonnet (<span>2021</span>) provide a timely update on the advantages of using organ on-a-chip and the different elements to consider as well as examples of how it has re-defined our knowledge of host pathogen interactions (Feaugas & Sauvonnet, <span>2021</span>). For example, organ-on-a-chip has provided insight into how mechanical forces in the gut influence <i>Shigella</i> invasion (Grassart et al., <span>2019</span>).</p><p>High throughput screening is an integral part of drug development as well as large scale genomic editing approaches. Subhash and Sundaramurthy describe screening platforms that take the host environment into account (Subhash & Sundaramurthy, <span>2021</span>). This allows the identification of potential host directed therapeutics which may be used as adjunct therapies against chronic intractable infections like tuberculosis, particularly where the pathogen is intracellular. Imaging is a desirable output for many high throughput screening approaches for a number of reasons. An automated image analysis program is discussed by Fisch et al. (<span>2021</span>), HRMAn (Host Response to Microbe Analysis), that uses machine learning and artificial intelligence to assess infection by pathogens in an unbiased manner. Parameters include pathogen growth, pathogen killing and activation of host cell signalling. 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引用次数: 0
摘要
在一个新病毒可能突然出现、抗微生物药物耐药性现在普遍存在的时代,充分了解导致疾病病理和传播的病原体和宿主因素仍然是一个至关重要的目标。研究感染细胞生物学的方法和技术正在迅速变化。研究宿主-病原体相互作用的跨学科要求越来越复杂,对于确定导致感染解决的细胞和免疫因素是必不可少的。病原体在宿主中复制的能力同样需要对引起疾病所需的病原体决定因素有透彻的了解。在本期特刊中,细胞微生物学重点介绍了研究病原体生物学和感染的一些最先进的方法和技术。长期以来,光学和电子显微镜成像在定义细胞内感染机制方面发挥了重要作用。这种方法现在可以通过荧光报告的设计快速适应,并用于高贯穿应用。Cortese和Laketa(2021)描述了高通量显微镜和电子显微镜的最新进展,并解释了如何在SARS-CoV-2爆发期间应用这些技术(Cortese &Laketa, 2021)。例如,在COVID-19大流行的关键早期阶段,基于mNeonGreen报告显微镜的病毒中和试验的开发在灵敏度和时间上优于标准空斑试验(Muruato等人,2020)。冷冻电子显微镜有助于快速确定SARS-CoV-2刺突蛋白及其细胞受体血管紧张素转换酶2 (ACE2)复合物的结构,直接为了解中和抗体提供信息(Cortese &Laketa, 2021)。在细菌病原体中,Dufrêne, Viljoen, Mignolet和matheli<s:1> - guinlet(2021)报告了原子力显微镜(AFM)的应用,以提供细菌细胞表面和受体-配体相互作用的超分辨率成像和纳米力学测量。这为粘附素的精细结构和生物物理功能提供了新的见解,也为疫苗和药物开发提供了信息(Dufrêne等人,2021)。细胞内病原体占据的亚细胞宿主细胞室高度特化以支持病原体复制。鉴于它们在细胞外液取样中的作用,大蛋白酶体被各种病原体劫持以进入细胞内环境。然而,大脂小体也是具有一系列细胞功能的高度动态的细胞器。Chang、Enninga和stsamuvenin(2021)描述了基于成像和蛋白质组学的方法,用于跟踪和表征这些重要的细胞器,因为它们被修改为志贺氏菌、沙门氏菌、布鲁氏菌和衣原体等细菌入侵和/或复制的小环境(Chang等人,2021)。同样,Simeone, Sayes, lawarsamae和Brosch(2021)探索了分枝杆菌的细胞内生态位,特别是允许结核分枝杆菌吞噬体逃逸到细胞质中的细菌因子(Simeone et al., 2021)。同时使用多种技术,如用半乳糖凝集素-3和泛素标记来识别受损的吞噬体膜,以及基于fret的报告细胞和细胞荧光测定方法,为结核分枝杆菌吞噬体破裂提供了新的见解。细胞系和原代细胞仍然是研究宿主-病原体相互作用的重要工具。然而,新模型的发展,如人类干细胞模型和微流体“芯片上器官”技术,可以提供一个额外的和更多的生理角度。Pellegrino和Gutierrez(2021)报告了将干细胞衍生模型设计到三维微环境中,作为研究感染生物学的新手段,在细胞共培养中有或没有其他细胞类型(Pellegrino &古铁雷斯,2021)。这些所谓的“类器官”对临床前研究特别有帮助,因为临床前研究不存在小动物感染模型,并且可以适应基因组编辑工具。另一种三维干细胞方法是“器官芯片”技术,它利用微流体和生物材料来模拟感染过程中病原体和宿主组织之间的界面。Feaugas和Sauvonnet(2021)及时更新了使用器官芯片的优势和需要考虑的不同元素,以及它如何重新定义我们对宿主病原体相互作用的认识的例子(Feaugas &Sauvonnet, 2021)。例如,器官芯片提供了肠道机械力如何影响志贺氏菌入侵的见解(Grassart et al., 2019)。高通量筛选是药物开发和大规模基因组编辑方法的一个组成部分。Subhash和Sundaramurthy描述了将主机环境考虑在内的筛选平台(Subhash &Sundaramurthy, 2021)。 这允许鉴定潜在的宿主定向治疗方法,这些治疗方法可能用作治疗慢性难治性感染(如结核病)的辅助疗法,特别是当病原体在细胞内时。由于许多原因,成像是许多高通量筛选方法的理想输出。Fisch等人(2021)讨论了一个自动图像分析程序,HRMAn(宿主对微生物的反应分析),它使用机器学习和人工智能以公正的方式评估病原体的感染。参数包括病原体生长、病原体杀伤和宿主细胞信号的激活。当整合到功能屏幕中时,HRMAn提供了前所未有的高通量和高含量分析能力(Fisch et al., 2021)。总之,这期《细胞微生物学》特刊中介绍的技术为许多研究细菌、病毒和寄生虫感染的研究者提供了灵感和方向。这里解释的最先进的方法将允许解决变革性的研究问题,并加深我们对宿主-病原体相互作用的认识,这可能导致新的疾病干预措施。我们预计细胞微生物学的进一步问题将致力于新兴技术,以突出我们领域最重要的方法发展。
In an era where new viruses can emerge suddenly and anti-microbial resistance is now widespread, a full understanding of the pathogen and host factors that contribute to disease pathology and spread remains a critically important goal. Methods and technologies to study the cell biology of infection are changing rapidly. The interdisciplinary requirements to interrogate host-pathogen interactions are ever more complex and indispensable to identify the cellular and immune factors that lead to the resolution of infection. The ability of a pathogen to replicate in the host likewise needs a thorough knowledge of the pathogen determinants required to cause disease. In this special issue, Cellular Microbiology highlights some of the most cutting-edge approaches and techniques to study pathogen biology and infection.
Light based and electron microscopic imaging has long played an important role in defining the intracellular mechanisms of infection. Such methods can now be quickly adapted through the design of fluorescent reporters and utilised for high throughout applications. Cortese and Laketa (2021) describe recent advances in high-throughput microscopy and electron microscopy and explain how these were applied during the SARS-CoV-2 outbreak (Cortese & Laketa, 2021). For example, the development of a mNeonGreen reporter microscopy-based virus neutralisation assay outperformed standard plaque assays in sensitivity and time in the critical early stages of the COVID-19 pandemic (Muruato et al., 2020). Cryo-electron microscopy was instrumental in rapidly defining the structure of the SARS-CoV-2 spike protein in complex with its cellular receptor, angiotensin convertase enzyme 2 (ACE2), directly informing an understanding of neutralising antibodies (Cortese & Laketa, 2021). In bacterial pathogens, Dufrêne, Viljoen, Mignolet, and Mathelié-Guinlet (2021) report on the utility of atomic force microscopy (AFM) to provide super-resolution imaging and nanomechanical measurements of bacterial cell surfaces and receptor-ligand interactions. This is providing new insight into the fine structure and biophysical function of adhesins and also informing vaccine and drug development (Dufrêne et al., 2021).
The subcellular host cell compartment occupied by intracellular pathogens is highly specialised to support pathogen replication. Given their role in sampling of extracellular fluid, macropinosomes are hijacked by diverse pathogens to establish entry into the intracellular environment. However, macropinosomes are also highly dynamic organelles with a range of cellular functions. Chang, Enninga, and Stévenin (2021) describe imaging-based and proteomic methods for the tracking and characterisation of these important organelles as they are modified as a niche for invasion and/or replication by bacteria such as Shigella, Salmonella, Brucella and Chlamydia (Chang et al., 2021). Similarly, Simeone, Sayes, Lawarée, and Brosch (2021) explore the intracellular niche of Mycobacteria, in particular the bacterial factors that allow phagosomal escape of Mycobacterium tuberculosis into the cell cytosol (Simeone et al., 2021). Multiple techniques used in parallel, such as labelling with galectin-3 and ubiquitin to identify damaged phagosomal membranes, as well as FRET-based reporter and cytofluorometric approaches have provided new insight into phagosomal rupture by M. tuberculosis.
Cell lines and primary cells remain important tools in the study of host-pathogen interactions. However, the development of new models such as human stem cell models and microfluidic ‘organ on chip’ technologies can offer an additional and more physiological perspective. Pellegrino and Gutierrez (2021) report on the use of stem cell-derived models engineered into three-dimensional microenvironments as a novel means to study the biology of infection, with and without other cell types in cellular co-culture (Pellegrino & Gutierrez, 2021). These so-called ‘organoids’ are particularly helpful for pre-clinical studies where no small animal model of infection exists and can be adapted to incorporate genome editing tools. Another three-dimensional stem cell approach is ‘organ on-a-chip’ technology which utilises microfluidics and biomaterials to mimic the interface between the pathogen and host tissue during infection. Feaugas and Sauvonnet (2021) provide a timely update on the advantages of using organ on-a-chip and the different elements to consider as well as examples of how it has re-defined our knowledge of host pathogen interactions (Feaugas & Sauvonnet, 2021). For example, organ-on-a-chip has provided insight into how mechanical forces in the gut influence Shigella invasion (Grassart et al., 2019).
High throughput screening is an integral part of drug development as well as large scale genomic editing approaches. Subhash and Sundaramurthy describe screening platforms that take the host environment into account (Subhash & Sundaramurthy, 2021). This allows the identification of potential host directed therapeutics which may be used as adjunct therapies against chronic intractable infections like tuberculosis, particularly where the pathogen is intracellular. Imaging is a desirable output for many high throughput screening approaches for a number of reasons. An automated image analysis program is discussed by Fisch et al. (2021), HRMAn (Host Response to Microbe Analysis), that uses machine learning and artificial intelligence to assess infection by pathogens in an unbiased manner. Parameters include pathogen growth, pathogen killing and activation of host cell signalling. When incorporated into a functional screen, HRMAn provides unprecedented capacity for high throughput and high-content analysis (Fisch et al., 2021).
In summary, the techniques presented in this special issue of Cellular Microbiology provide inspiration and direction to many investigators studying bacterial, viral and parasite infections. The state-of-the-art approaches explained here will allow transformational research questions to be addressed and deepen our knowledge of host-pathogen interactions that could lead to new disease interventions. We anticipate that further issues of Cellular Microbiology will be dedicated to emerging technologies in order to highlight the most important methodological developments in our field.
期刊介绍:
Cellular Microbiology aims to publish outstanding contributions to the understanding of interactions between microbes, prokaryotes and eukaryotes, and their host in the context of pathogenic or mutualistic relationships, including co-infections and microbiota. We welcome studies on single cells, animals and plants, and encourage the use of model hosts and organoid cultures. Submission on cell and molecular biological aspects of microbes, such as their intracellular organization or the establishment and maintenance of their architecture in relation to virulence and pathogenicity are also encouraged. Contributions must provide mechanistic insights supported by quantitative data obtained through imaging, cellular, biochemical, structural or genetic approaches.