嗜肺性军团菌效应蛋白Ceg14的生物学功能研究
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摘要
嗜肺性军团菌是一种胞内寄生菌,以阿米巴原虫为自然宿主,广泛存在于自然界中。人们多因吸入污染的气溶胶而感染,引起以肺部炎症为主要特征的疾病,称为军团杆菌病(legionellosis)。军团菌可以在肺泡巨噬细胞或者单核细胞内复制增殖,军团菌侵入细胞后,会形成一个被生物膜包裹的囊泡结构,称为LCV (legionella containing vacuole)。LCV可以从内吞途径中逃逸出来,募集宿主细胞的组分,最终形成一个表面被内质网成分、核糖体和线粒体成分覆盖的膜结构,军团菌可以在其中复制增殖。LCV的形成和发育取决于一个称为Dot/Icm的Ⅳ型分泌系统,这一分泌系统也是军团菌胞内存活的分子基础。通过Dot/Icm系统,军团菌向胞浆内分泌了大量的效应蛋白,目前已鉴定出至少275个效应蛋白。这些蛋白可以作用于宿主细胞的各个生物学过程,从而有利于军团菌在胞内的复制增殖。通过探究这些效应蛋白的生物学功能,可以为进一步揭示军团菌胞内存活的分子机理提供理论依据。
首先利用酵母系统,筛选可以抑制酵母生长的军团菌效应蛋白,进而利用生物、生化手段对这些效应蛋白进行研究。利用这一方法,获得了一些可以完全抑制酵母生长的效应蛋白,如ankX、 SidI、Ceg14等,本论文重点研究Ceg14的生物学功能。Ceg14可以完全抑制酵母生长,通过随机突变分析,确定了两个氨基酸位点,第234位甘氨酸和第623位的苏氨酸,对Ceg14的生物学功能至关重要。通过抑制子筛选实验,发现高表达酵母前纤维蛋白(profilin)可以抑制Ceg14对酵母的毒性,而前纤维蛋白在肌动蛋白(actin)的组装过程中发挥着重要作用。之前利用蛋白质组学分析LCV的膜成分发现,除了其它细胞组分外,在LCV膜上有许多与细胞骨架组装相关的组分,如肌动蛋白、前纤维蛋白、丝切蛋白(cofilin)等。说明军团菌可以修饰宿主细胞骨架,通过改变细胞骨架的重排,从而实现LCV的转运和胞内的存活。最近发现效应蛋白VipA可以直接结合肌动蛋白,体外分析显示,VipA可以作为成核剂,促进肌动蛋白单体(G-actin)的聚集,从而增强肌动蛋白在体外的聚合,该结果表明Ceg14可能影响宿主细胞骨架的组装。
利用荧光显微镜技术,观察到Ceg14的表达可以破坏酵母的细胞骨架,导致酵母无法出芽；体外利用共沉淀(Co-sedimentation)、荧光分光光度计动态检测肌动蛋白单体聚合等实验方法,发现Ce14可以抑制肌动蛋白单体的自发聚合作用,并且Ceg14G234v和Ceg14T623N失去了这一功能。进一步用荧光显微镜观察单个的肌动蛋白丝,发现Ceg14会导致产生更多的短的肌动蛋白丝。体外的数据显示,Ceg14可以抑制肌动蛋白单体的自发组装,这是首次发现军团菌的效应蛋白可以负调控肌动蛋白的组装,不同于VipA的促进作用,从另一方面也体现了军团菌对宿主细胞修饰的复杂性。此外,同其它大多数效应蛋白一样,敲除Ceg14和VipA的菌株在A/J小鼠的巨噬细胞内和阿米巴原虫内可以正常复制增殖,说明可能有其它更多的效应蛋白参与进来,弥补了Ceg14和VipA的生物学功能。本研究结果为深入解析军团菌的致病机理提供了一定的参考。
Legionella pneumophila is the causative agent of a pneumonia known as Legionnaires' disease. These bacteria are ubiquitous in aquatic environments and the protozoan amebae are their natural host. Inhalation of aerosol contaminated by L. pneumophila introduces the bacterium to alveolar macrophage, within which it replicates by mechanisms similar to those observed in its natural host. After phagocytosis, Legionella pneumophila can form an ER-like compartment, also known as legionella containing vacuole (LCV). The LCV can escape from endocytic pathway and avoid the fusion with lysosome where the bacteria will be degraded. Intracellular bacterial replication entirely depends upon a Type IVB secretion system termed Dot/Icm, which delivers a lager cohort of protein substrates into host cells. These proteins, also called effectors function to modify fundamental cellular process, such as cell death, membrane trafficking, stress response, and cytoskeleton to create an environment permissive for bacterial survival and multiplication. Although functional dissection of some of these effectors has revealed exiciting insights into the intricate manipulation of host activity by L. pneumophila, the activity of most of these genes reamains unknown.
The yeast saccharomyces cerevisiae is a powerful tool in functional study of bacterial effectors. Many effectors of L. pneumophila have been identified by their ability to inhibit yeast growth. In our lab, one of the focuses is to dissect the function of effectors exhibiting strong toxicity to yeast. Here we found Cegl4, an effector of L. pneumophila, completely inhibits yeast growth. By random chemical mutagenesis, we found that residues Gly234or Thr623is critical for yeast toxicity of Ceg14. Further, we found that the yeast profilin gene strongly suppresses the toxicity of Ceg14. Consistent with the suppressor phenotype, we found that wild type Ceg14but not the two mutants, disrupted yeast cytoskeletonal structure.
Direct analysis of the effects of Ceg14on actin polymerization by biochemical approaches revealed that this protein interferes with actin polymerization, causing the formation of shorter actin filaments. Again, mutations in Gly234or Thr623abolished the effects of Ceg14on actin assembly. These activities are opposite from those observed with VipA, suggesting that the bacterium targets host cytoskeleton in a balanced manner. We also examined the effects of Ceg14on the intracellular growth of L. pneumophila. Similar to most Dot/Icm effectors, deletion of ceg14did not cause detectable defect in intracellular bacterial growth in amoeba or primary mouse macrophages. Mutants lacking both ceg14and vipA also did not display detectable defect in intracellular bacterial growth, suggesting the existence of more effectors involved in targeting the host cytoskeleton. The identification and functional study of such effectors likely will reveal the role of host cytoskeleton in the formation of vacuole supportive for L. pneumophila replication.
首先利用酵母系统,筛选可以抑制酵母生长的军团菌效应蛋白,进而利用生物、生化手段对这些效应蛋白进行研究。利用这一方法,获得了一些可以完全抑制酵母生长的效应蛋白,如ankX、 SidI、Ceg14等,本论文重点研究Ceg14的生物学功能。Ceg14可以完全抑制酵母生长,通过随机突变分析,确定了两个氨基酸位点,第234位甘氨酸和第623位的苏氨酸,对Ceg14的生物学功能至关重要。通过抑制子筛选实验,发现高表达酵母前纤维蛋白(profilin)可以抑制Ceg14对酵母的毒性,而前纤维蛋白在肌动蛋白(actin)的组装过程中发挥着重要作用。之前利用蛋白质组学分析LCV的膜成分发现,除了其它细胞组分外,在LCV膜上有许多与细胞骨架组装相关的组分,如肌动蛋白、前纤维蛋白、丝切蛋白(cofilin)等。说明军团菌可以修饰宿主细胞骨架,通过改变细胞骨架的重排,从而实现LCV的转运和胞内的存活。最近发现效应蛋白VipA可以直接结合肌动蛋白,体外分析显示,VipA可以作为成核剂,促进肌动蛋白单体(G-actin)的聚集,从而增强肌动蛋白在体外的聚合,该结果表明Ceg14可能影响宿主细胞骨架的组装。
利用荧光显微镜技术,观察到Ceg14的表达可以破坏酵母的细胞骨架,导致酵母无法出芽；体外利用共沉淀(Co-sedimentation)、荧光分光光度计动态检测肌动蛋白单体聚合等实验方法,发现Ce14可以抑制肌动蛋白单体的自发聚合作用,并且Ceg14G234v和Ceg14T623N失去了这一功能。进一步用荧光显微镜观察单个的肌动蛋白丝,发现Ceg14会导致产生更多的短的肌动蛋白丝。体外的数据显示,Ceg14可以抑制肌动蛋白单体的自发组装,这是首次发现军团菌的效应蛋白可以负调控肌动蛋白的组装,不同于VipA的促进作用,从另一方面也体现了军团菌对宿主细胞修饰的复杂性。此外,同其它大多数效应蛋白一样,敲除Ceg14和VipA的菌株在A/J小鼠的巨噬细胞内和阿米巴原虫内可以正常复制增殖,说明可能有其它更多的效应蛋白参与进来,弥补了Ceg14和VipA的生物学功能。本研究结果为深入解析军团菌的致病机理提供了一定的参考。
Legionella pneumophila is the causative agent of a pneumonia known as Legionnaires' disease. These bacteria are ubiquitous in aquatic environments and the protozoan amebae are their natural host. Inhalation of aerosol contaminated by L. pneumophila introduces the bacterium to alveolar macrophage, within which it replicates by mechanisms similar to those observed in its natural host. After phagocytosis, Legionella pneumophila can form an ER-like compartment, also known as legionella containing vacuole (LCV). The LCV can escape from endocytic pathway and avoid the fusion with lysosome where the bacteria will be degraded. Intracellular bacterial replication entirely depends upon a Type IVB secretion system termed Dot/Icm, which delivers a lager cohort of protein substrates into host cells. These proteins, also called effectors function to modify fundamental cellular process, such as cell death, membrane trafficking, stress response, and cytoskeleton to create an environment permissive for bacterial survival and multiplication. Although functional dissection of some of these effectors has revealed exiciting insights into the intricate manipulation of host activity by L. pneumophila, the activity of most of these genes reamains unknown.
The yeast saccharomyces cerevisiae is a powerful tool in functional study of bacterial effectors. Many effectors of L. pneumophila have been identified by their ability to inhibit yeast growth. In our lab, one of the focuses is to dissect the function of effectors exhibiting strong toxicity to yeast. Here we found Cegl4, an effector of L. pneumophila, completely inhibits yeast growth. By random chemical mutagenesis, we found that residues Gly234or Thr623is critical for yeast toxicity of Ceg14. Further, we found that the yeast profilin gene strongly suppresses the toxicity of Ceg14. Consistent with the suppressor phenotype, we found that wild type Ceg14but not the two mutants, disrupted yeast cytoskeletonal structure.
Direct analysis of the effects of Ceg14on actin polymerization by biochemical approaches revealed that this protein interferes with actin polymerization, causing the formation of shorter actin filaments. Again, mutations in Gly234or Thr623abolished the effects of Ceg14on actin assembly. These activities are opposite from those observed with VipA, suggesting that the bacterium targets host cytoskeleton in a balanced manner. We also examined the effects of Ceg14on the intracellular growth of L. pneumophila. Similar to most Dot/Icm effectors, deletion of ceg14did not cause detectable defect in intracellular bacterial growth in amoeba or primary mouse macrophages. Mutants lacking both ceg14and vipA also did not display detectable defect in intracellular bacterial growth, suggesting the existence of more effectors involved in targeting the host cytoskeleton. The identification and functional study of such effectors likely will reveal the role of host cytoskeleton in the formation of vacuole supportive for L. pneumophila replication.
引文
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