嗜肺军团菌II型分泌系统及其底物的研究进展

嗜肺军团菌(Legionella pneumophila,L. pneumophila)是研究病原菌-宿主相互作用的重要模式菌株之一,其独特的分泌系统及底物效应蛋白的结构与功能是病原微生物领域的研究热点。II型分泌系统(type II secretion system,T2SS)对促进细菌在环境和人类宿主中的生存至关重要。嗜肺军团菌Legionella secretion pathway (Lsp)系统是革兰氏阴性病原菌中一个典型的T2SS。本文综述了L. pneumophila的T2SS及其底物效应蛋白的研究进展,重点介绍其结构与功能,为深入了解革兰氏阴性病原菌的T2SS功能和作用机制提供参考。     

嗜肺军团菌效应蛋白之间的调控机制

嗜肺军团菌(Legionella pneumophila)是一种革兰氏阴性致病菌,它可以引起人类军团病。嗜肺军团菌的Dot/Icm分泌系统在其致病过程中至关重要,其向宿主细胞内转运约330种效应蛋白,通过修饰细胞调节因子、抑制细胞凋亡等一系列措施操纵宿主细胞的多种生命活动,以完成自身的增殖与侵染。为避免对宿主生理造成不必要的破坏,嗜肺军团菌已进化出复杂而精细的调控机制来平衡嗜肺军团菌毒力与宿主细胞的稳态,以确保嗜肺军团菌在宿主细胞内的生存。军团菌效应蛋白的功能及分子机制的研究近几年取得突破性进展,嗜肺军团菌效应蛋白之间的作用机理也成为我们进一步研究的热点。该文主要对嗜肺军团菌的致病机制及其效应蛋白间的调控机制进行了综述,为进一步了解嗜肺军团菌致病机制提供了一定的参考。     

嗜肺军团菌毒力基因、致病性及实验诊断研究进展

嗜肺军团菌是引起军团菌肺炎的重要病原体,关于军团菌感染的致病机制目前尚不十分清楚。近来研究发现,军团菌的致病性与其毒力因子和铁代谢等相关,本文对嗜肺军团菌的毒力因子及其作用。致病机制和实验室诊断方法研究进展作一综述。     

细菌三型分泌系统效应蛋白转运的研究进展

三型分泌系统(Type 3 secretion system,T3SS)作为存在于革兰氏阴性菌中的分泌系统之一,对革兰氏阴性菌的致病有重要作用。T3SS的致病作用体现在T3SS能直接将效应蛋白转运至宿主细胞,进而通过效应蛋白调控细胞的一系列通路,促进细菌定殖于细胞。而效应蛋白的转运受到两方面因素的调控,一方面是效应蛋白本身的信号序列,另一方面是T3SS相关蛋白的辅助。本文围绕近年来T3SS的构成、效应蛋白转运机制方面的最新进展进行概要综述。     

嗜肺军团菌毒力岛分子分型及其致病性研究进展

嗜肺军团菌是引起社区获得性和医院内感染性肺炎的重要病原体,中央空调冷凝塔水系统是引发军团菌病的重要传染源,在国内外时有暴发流行,病死率较高。嗜肺军团菌的致病性与其毒力岛基因组密切相关。简要概述了嗜肺军团菌毒力岛、分子分型及其致病性。     

细菌Ⅵ型分泌系统效应蛋白的装配及功能的研究进展

蛋白分泌作为细胞之间传递信号的途径之一,在微生物生存竞争中也扮演着重要的角色。革兰氏阴性菌可以通过Ⅵ型分泌系统(type Ⅵ secretion system, T6SS)将效应蛋白传递至胞外或原核和真核微生物中,从而介导微生物间的竞争或宿主-细菌的相互作用,最终建立竞争优势。本文主要总结了T6SS的结构与组成,并重点对效应蛋白的装配以及其与免疫蛋白的作用机制的研究进展进行阐述,为以后靶向T6SS抗菌药物的研制提供新思路。     

嗜肺军团菌调控宿主非折叠蛋白反应的研究进展

内质网作为细胞内重要的细胞器之一,参与胞内蛋白的成熟及转运,其稳态与细胞的存活及免疫反应密切相关.非折叠蛋白反应是维持内质网稳态的重要机制之一,参与细胞的天然免疫反应,在宿主细胞抵抗病原体入侵中具有重要作用.嗜肺军团菌是一种革兰氏阴性致病菌,能够感染人体肺泡巨噬细胞引起严重肺炎,即军团菌病.在入侵宿主细胞后,嗜肺军团菌通过其Ⅳ型分泌系统将330多个效应蛋白转运至宿主细胞中,干扰宿主细胞的多种细胞进程,以形成其生存和复制所需的场所——含嗜肺军团菌囊泡(Legionella-containing vacuole, LCV).LCV的形成与宿主细胞的内质网密切相关.本文主要从嗜肺军团菌的致病机制、细胞非折叠蛋白反应及其与病原体的关系、军团菌对宿主细胞的非折叠蛋白反应的调控等方面进行综述,以期为揭示病原体与内质网应激之间的关系提供参考.     

嗜肺军团菌核酸疫苗研究进展

嗜肺军团菌通过气溶胶的形式感染人和动物,引起军团菌性肺炎,危害极其严重,但该病目前尚无有效的预防措施。军团菌病疫苗的研究经历了灭活疫苗、减毒活疫苗和蛋白亚单位疫苗不同阶段,但至今尚未能在临床得到应用。核酸疫苗作为一种新型疫苗,能有效诱导机体产生细胞免疫应答,符合兼胞内寄生菌嗜肺军团菌的预防需要,并且还具有诸多其他优点,引起了研究者们的兴趣。对嗜肺军团杆菌核酸疫苗的研究现状进行了简要介绍。     

乙酰化修饰在嗜肺军团菌致病过程中的作用

乙酰化修饰是由乙酰基转移酶、去乙酰化酶介导的可逆的蛋白质翻译后修饰。其中,乙酰基转移酶将乙酰辅酶A的乙酰基团转移至底物蛋白的氨基酸残基,而乙酰基团的去除由去乙酰化酶完成。乙酰化修饰参与许多基本生物学过程的调节作用,越来越多的研究表明,蛋白质乙酰化修饰在病原菌的致病过程中具有重要作用。病原菌,如引起非典型性肺炎的嗜肺军团菌,可以通过分泌具有乙酰基转移酶活性的效应蛋白靶向宿主细胞信号通路的关键蛋白质因子,干扰宿主细胞信号通路及免疫反应。本文主要从嗜肺军团菌的致病机制、乙酰化修饰及乙酰化修饰在病原体致病过程中的调控作用进行综述,突出已知的乙酰化毒力蛋白的例子,并讨论它们如何影响与宿主的相互作用,为理解乙酰化修饰在嗜肺军团菌致病过程中的作用机制提供参考。     

Legionella pneumophila secretes an endoglucanase that belongs to the family-5 of glycosyl hydrolases and is dependent upon type II secretion

Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. Legionella pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium's type II protein secretion (T2S) system. Inactivation of celA , encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli . Thus, CelA is the major secreted endoglucanase of L. pneumophila . Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila . The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophil a and the 17th type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the T2S output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella .     

Twitching motility in Legionella pneumophila

Twitching motility is a form of bacterial translocation over solid or semi-solid surfaces mediated by the extension, tethering, and subsequent retraction of type IV pili. These pili are also known to be involved in virulence, biofilm formation, formation of fruiting bodies, horizontal gene transfer, and protein secretion. We have characterized the presence of twitching motility on agar plates in Legionella pneumophila , the etiological agent of Legionnaires' disease. By examining twitching motility zones, we have demonstrated that twitching motility was dependent on agar thickness/concentration, the chemical composition of the media, the presence of charcoal and cysteine, proximity to other bacteria, and temperature. A knockout mutant of the pilus subunit, pilE , exhibited a total loss of twitching motility at 37 °C, but not at 27 °C, suggesting either the existence of a compensating pilus subunit or of another twitching motility system in this organism.     

嗜肺军团菌调节宿主泛素化途径的效应因子及其分子机制研究进展

嗜肺军团菌是一种胞内寄生菌,其通过特有的Dot/IcmType-IVB分泌系统向胞浆内分泌大量效应因子,其中已知参与宿主泛素化调控的效应因子有十多种.这些效应因子通过对宿主泛素化途径进行调控来达到逃避宿主免疫系统"监视"并大量增殖的目的.参与调控宿主泛素化途径的效应因子包括 AnkB、SidC、LubX、SidH、Le...     

The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii

Type-IV secretion systems are devices present in a wide range of bacteria (including bacterial pathogens) that deliver macromolecules (proteins and single-strand-DNA) across kingdom barriers (as well as between bacteria and into the surroundings). The type-IV secretion systems were divided into two subgroups and Legionella pneumophila and Coxiella burnetii are the only two bacteria known today to utilize a type-IVB secretion system for pathogenesis. In this review we summarized the available information concerning the icm/dot type-IVB secretion systems by comparing the two bacteria that possess this system, the proteins components of their systems as well as the homology of proteins from type-IVB secretion systems to proteins from type-IVA secretion systems. In addition, the phenotypes associated with mutants in the L. pneumophila icm/dot genes, their relations to properties of specific Icm/Dot proteins as well as the protein substrates delivered by this system are described.     

Structural and functional study of Legionella pneumophila effector RavA

Legionella pneumophila is an intracellular pathogen that causes Legionnaire''s disease in humans. This bacterium can be found in freshwater environments as a free‐living organism, but it is also an intracellular parasite of protozoa. Human infection occurs when inhaled aerosolized pathogen comes into contact with the alveolar mucosa and replicates in alveolar macrophages. Legionella enters the host cell by phagocytosis and redirects the Legionella‐containing phagosomes from the phagocytic maturation pathway. These nascent phagosomes fuse with ER‐derived secretory vesicles and membranes forming the Legionella‐containing vacuole. Legionella subverts many host cellular processes by secreting over 300 effector proteins into the host cell via the Dot/Icm type IV secretion system. The cellular function for many Dot/Icm effectors is still unknown. Here, we present a structural and functional study of L. pneumophila effector RavA (Lpg0008). Structural analysis revealed that the RavA consists of four ~85 residue long α‐helical domains with similar folds, which show only a low level of structural similarity to other protein domains. The ~90 residues long C‐terminal segment is predicted to be natively unfolded. We show that during L. pneumophila infection of human cells, RavA localizes to the Golgi apparatus and to the plasma membrane. The same localization is observed when RavA is expressed in human cells. The localization signal resides within the C‐terminal sequence C⁴⁰⁹WTSFCGLF⁴¹⁷. Yeast‐two‐hybrid screen using RavA as bait identified RAB11A as a potential binding partner. RavA is present in L. pneumophila strains but only distant homologs are found in other Legionella species, where the number of repeats varies.     

Localization of Legionella bacteria within ribosome-studded phagosomes is not restricted to Legionella pneumophila

In this report, we investigate the intracellular fate of selected members of the genus Legionella within the monocytic cell line Mono Mac 6 cells. By means of electron microscopy and immunocytochemistry, we could show that Legionella pneumophila as well as Legionella longbeachae are able to induce ribosome-studded phagosomes which associate with the rough endoplasmic reticulum (RER), whereas Legionella micdadei remains to be located within smooth phagosomes but also shows signs of RER association. In addition, we could demonstrate a remarkable correlation between the phagosome type and the morphological phenotype of intracellular bacteria: within ribosome-studded phagosomes, bacteria generally lacked the outer coat of low electron density whereas bacteria within the smooth phagosomes still possessed this outer coat. The virulence factors responsible for inhibition of phagosome maturation and their distribution within the genus Legionella as well as the biological significance of the morphological difference of bacteria within smooth and ER-associated phagosomes remain to be investigated.     

Isolation and characterization of a lipopolysaccharide mutant of Legionella pneumophila

A mutant unable to bind a monoclonal antibody (mAb 1E6) directed against serogroup 1 lipopolysaccharide (LPS) was isolated from L. pneumophila strain Philadelphia-1. SDS-PAGE analysis of isolated LPS from the mutant and wild type revealed that there were no obvious structural differences between the two LPS. The results from Western-blot experiments showed that the mutant LPS was unable to bind mAb 1E6 but retained the ability to bind polyclonal serogroup 1 antibodies. Loss of the LPS epitope recognized by mAb 1E6 did not alter the ability of the mutant to multiply in human monocyte-like U937 cells. Also, the mutant, like wild type, was resistant to killing by normal human serum. These results show that a minor change in the antigenic composition of serogroup 1 LPS has no effect on the virulence properties of strain Philadelphia-1. Additionally, this mutant may be useful for molecular genetic analysis of serogroup 1 LPS biosynthesis and assembly.     

Differences in the respiratory burst of human polymorphonuclear leukocytes induced by virulent and avirulent Legionella pneumophila Serogroup 1

Two strains of Legionella pneumophila of different virulence were examined for their influence on the metabolic oxidative activity of human polymorphonuclear leukocytes. The leukocytes exhibited decreased rates of oxygen consumption and diminished chemiluminescence activity following phagocytosis of a virulent strain of L. pneumophila serogroup 1. In contrast, phagocytosis of its multipassaged derivative rendered avirulent, was accompanied by increased rates of both oxygen consumption and chemiluminescence activity. Although no differences were observed in oxygen uptake induced by the virulent legionellae compared to leukocytes at rest, statistically significant differences were observed in the chemiluminescence responses. These observations were not unexpected, since the luminol-enhanced chemiluminescence assay, is more sensitive than the oxygen uptake assay. In spite of decreased metabolic activity of PMN in the presence of virulent legionellae, electron microscope studies showed higher numbers of intracellular L. pneumophila than the avirulent subtype. Thus, virulent and avirulent L. pneumophila can be differentiated on the basis of oxygen consumption and chemiluminescence assays.     

Lipopolysaccharides as a marker system in the epidemiology of Legionella pneumophila serogroup 12

Abstract Lipopolysaccharides (LPS) isolated from Legionella pneumophila serogroup 12 strains were studied for their usefulness as epidemiological markers. L. pneumophila serogroup 12 was cultured from 3 patients infected at home, in another hospital and abroad, as well as from hot tapwater in their quarters. LPS isolated from these strains showed 2 distinct patterns and 4 different colours in silver-stained polyacrylamide gels. LPS of the first pattern stained dark-grey, those of the second pattern were either orange-brown, dark-brown or black-brown. These differences were reproducible. By comparing patterns and colours of isolated LPS molecules, similarity could be demonstrated between strains from the first patient and from hot water in his home, as well as between strains from the second patient and from hot water in the hosoptal where he became infected. None of the strains displayed LPS of the same colour as that of the third patient, who was admitted with pneumonia from Spain. Patterns and colours of LPS isolated from L. pneumophila serogroup 12 in ilver-stained gels can be used as a marker system in epidemiological studies.