In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography

Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative‐stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type‐specific elaborations and potentially better structural preservation in situ.     

Coxiella burnetii express type IV secretion system proteins that function similarly to components of the Legionella pneumophila Dot/Icm system

Coxiella burnetii is an obligate intracellular pathogen that replicates in large endocytic vacuoles. Genomic sequence data indicate that 21 genes encoding products that are similar to components of the Legionella pneumophila Dot/Icm type IV secretion system are located on a contiguous 35 kb region of the Coxiella chromosome. It was found that several dot/icm genes were expressed by Coxiella during host cell infection and that dot/icm gene expression preceded the formation of large replicative vacuoles. To determine whether these genes encode a functional type IV secretion system, we have amplified the Coxiella dotB, icmQ, icmS and icmW genes and produced the encoded proteins in Legionella mutants in which the native copy of each gene had been deleted. The Coxiella dotB, icmS and icmW products restored dot/icm-dependent growth of Legionella mutants in eukaryotic host cells. The Coxiella IcmQ protein and the Legionella IcmR protein did not interact, which could explain why the Coxiella icmQ gene was unable to restore growth to a Legionella icmQ mutant. Thus, Coxiella encodes functional components of a type IV secretion system expressed in vivo that is mechanistically related to the Legionella Dot/Icm apparatus. These studies suggest that a dot/icm-related secretion system could play an important role in creating the specialized vacuole that supports Coxiella replication.     

胶体金渗滤法检测贝氏柯克斯体的研究

本文建立一种快速、敏感、适于基层应用的贝氏柯克斯体（俗称Ｑ热立克次体）的检测方法。将Ｑ热立克次体多克隆抗体点于硝酸膜上,用以捕获待检标本中的Ｑ热立克次体抗原,通过胶体金标记的鼠抗Ｑ热立克次体单克隆抗体直接显色,阳性者出现红色斑点。结果表明,用该法检测Ｑ热立克次休实验感染豚鼠血液,小鼠肝或脾,蜱血淋巴等标本取得了较满意的结果,整个过程仅需５－６分钟。与其他病原体无交叉反应,敏感度不低于５０ｎｇ立克次     

Identification and characterization of arginine finger-like motifs,and endosome-lysosome basolateral sorting signals within the Coxiella burnetii type IV secreted effector protein CirA

Coxiella burnetii is an obligate intracellular pathogen that replicates in an endolysosome-like compartment termed the Coxiella-containing vacuole (CCV). Formation of this unique replicative niche requires delivery of bacterial effector proteins into the host cytosol where they mediate crucial interactions with the host. We previously identified an essential Dot/Icm effector, CirA that is required for intracellular replication and CCV formation. Furthermore, CirA was shown to stimulate the GTPase activity of RhoA in vitro. In the current study, we used a bioinformatics-guided approach and identified three arginine finger-like motifs, often found in Rho GTPase-activating proteins (GAPs) and endosome–lysosome basolateral sorting signals associated with vesicle trafficking. When expressed in mammalian cells, mutation of either endosome-lysosome-basolateral sorting signals or the arginine finger-like motifs rescued stress phenotypes and decreased plasma membrane localization of ectopically expressed CirA. We further demonstrate that endosome–lysosome sorting signals are required for co-localization with Rab5 and Rab7. Collectively our data indicate that arginine finger-like motifs and endosome-lysosome-basolateral sorting signals within CirA are essential for interaction with the host cytoskeleton.     

一个细菌的致命之旅——嗜肺军团菌分泌系统及效应蛋白的研究进展

嗜肺军团菌是引起军团菌肺炎以及庞蒂亚克热的革兰氏阴性胞内病原细菌,嗜肺军团菌侵染宿主的主要特点是可以通过其IVB型毒力分泌系统,向宿主细胞内分泌超过150种的底物效应蛋白。通过这些效应蛋白的作用,嗜肺军团菌能够调整宿主细胞的胞内运输途径,改变内外环境来伪装自己的吞噬泡,干扰宿主的细胞周期,抑制宿主细胞的凋亡,从而有效逃避宿主细胞的防御功能,创造出理想的胞内增殖环境。最后,效应蛋白还可以帮助军团菌从宿主细胞中逃逸。目前,嗜肺军团菌已经成为"病原菌-宿主相互作用"的重要研究模型,其毒力分泌系统及其底物效应蛋白的功能也成为细胞微生物学的研究热点。对嗜肺军团菌分泌系统及效应蛋白的研究不仅能够帮助阐明病原细菌的致病机理,还有助于推动对宿主免疫机制的更深层次的研究。文章主要针对嗜肺军团菌的毒力分泌系统,尤其是IVB型分泌系统的结构和功能,以及底物效应蛋白的研究进展进行了综述,向读者展示出一个小小的细菌所拥有的那令人惊叹的、如此狡猾的生存策略和它精致的杀伤武器。     

Legionella pneumophila Promotes Functional Interactions between Plasma Membrane Syntaxins and Sec22b

Biogenesis of a specialized organelle that supports intracellular replication of Legionella pneumophila involves the fusion of secretory vesicles exiting the endoplasmic reticulum (ER) with phagosomes containing this bacterial pathogen. Here, we investigated host plasma membrane SNARE proteins to determine whether they play a role in trafficking of vacuoles containing L. pneumophila. Depletion of plasma membrane syntaxins by RNA interference resulted in delayed acquisition of the resident ER protein calnexin and enhanced retention of Rab1 on phagosomes containing virulent L. pneumophila, suggesting that these SNARE proteins are involved in vacuole biogenesis. Plasma membrane‐localized SNARE proteins syntaxin 2, syntaxin 3, syntaxin 4 and SNAP23 localized to vacuoles containing L. pneumophila. The ER‐localized SNARE protein Sec22b was found to interact with plasma membrane SNAREs on vacuoles containing virulent L. pneumophila, but not on vacuoles containing avirulent mutants of L. pneumophila. The addition of α‐SNAP and N‐ethylmaleimide‐sensitive factor (NSF) to the plasma membrane SNARE complexes formed by virulent L. pneumophila resulted in the dissociation of Sec22b, indicating functional pairing between these SNAREs. Thus, L. pneumophila stimulates the non‐canonical pairing of plasma membrane t‐SNAREs with the v‐SNARE Sec22b to promote fusion of the phagosome with ER‐derived vesicles. The mechanism by which L. pneumophila promotes pairing of plasma membrane syntaxins and Sec22b could provide unique insight into how the secretory vesicles could provide an additional membrane reserve subverted during phagosome maturation.     

细菌的IV型分泌系统

细菌的分泌系统与细菌的生存及致病性密切相关。细菌的分泌系统包括I-VI型,其中,IV型分泌系统是与细菌接合机制有关的一类分泌系统。IV型分泌系统不但可以转运DNA,还可以转运蛋白质及核糖核蛋白复合物等大分子物质,这点区别于其他几种分泌系统。IV型分泌系统介导基因水平转移,通过细菌间接合作用,传递抗性基因和毒力基因,有利于细菌进化;另一方面,IV型分泌系统转运效应蛋白质分子到宿主细胞,参与细菌致病。本文着重从IV型分泌系统几种主要类型的分泌机制等方面对IV型分泌系统进行概述。     

感染小鼠组织中Q热立克次体的分子病理学检测

腹腔注射Q热立克次体悬液感染BALB／c小鼠,发病后解剖,取主要组织脏器,应用免疫组化和原位杂交检测感染小鼠体内Q热立克次体的抗原分布和DNA在靶细胞中的表达,探讨Q热病变规律及致病机理。结果显示阳性信号多位于单核-巨噬细胞系统细胞胞浆内。结果提示免疫组化和原位杂交技术可以作为Q热特异性的诊断方法,并为致病机理的研究提供线索。     

The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation

Many gram-negative pathogens use a type IV secretion system (T4SS) to deliver effector proteins into eukaryotic host cells. The fidelity of protein translocation depends on the efficient recognition of effector proteins by the T4SS. Legionella pneumophila delivers a large number of effector proteins into eukaryotic cells using the Dot/Icm T4SS. How the Dot/Icm system is able to recognize and control the delivery of effectors is poorly understood. Recent studies suggest that the IcmS and IcmW proteins interact to form a stable complex that facilitates translocation of effector proteins by the Dot/Icm system by an unknown mechanism. Here we demonstrate that the IcmSW complex is necessary for the productive translocation of multiple Dot/Icm effector proteins. Effector proteins that were able to bind IcmSW in vitro required icmS and icmW for efficient translocation into eukaryotic cells during L. pneumophila infection. We identified regions in the effector protein SidG involved in icmSW-dependent translocation. Although the full-length SidG protein was translocated by an icmSW-dependent mechanism, deletion of amino terminal regions in the SidG protein resulted in icmSW-independent translocation, indicating that the IcmSW complex is not contributing directly to recognition of effector proteins by the Dot/Icm system. Biochemical and genetic studies showed that the IcmSW complex interacts with a central region of the SidG protein. The IcmSW interaction resulted in a conformational change in the SidG protein as determined by differences in protease sensitivity in vitro. These data suggest that IcmSW binding to effectors could enhance effector protein delivery by mediating a conformational change that facilitates T4SS recognition of a translocation domain located in the carboxyl region of the effector protein.     

Large-scale identification of Legionella pneumophila Dot/Icm substrates that modulate host cell vesicle trafficking pathways

The bacterial pathogen Legionella pneumophila replicates in a specialized vacuole within host cells. Establishment of the replication vacuole depends on the Dot/Icm translocation system that delivers a large number of protein substrates into the host cell. The functions of most substrates are unknown. Here, we analysed a defined set of 127 confirmed or candidate Dot/Icm substrates for their effect on host cell processes using yeast as a model system. Expression of 79 candidates caused significant yeast growth defects, indicating that these proteins impact essential host cell pathways. Notably, a group of 21 candidates interfered with the trafficking of secretory proteins to the yeast vacuole. Three candidates that caused yeast secretory defects (SetA, Ceg19 and Ceg9) were investigated further. These proteins impinged upon vesicle trafficking at distinct stages and had signals that allowed translocation into host cells by the Dot/Icm system. Ectopically produced SetA, Ceg19 and Ceg9 localized to secretory organelles in mammalian cells, consistent with a role for these proteins in modulating host cell vesicle trafficking. Interestingly, the ability of SetA to cause yeast phenotypes was dependent upon a functional glycosyltransferase domain. We hypothesize that SetA may glycosylate a component of the host cell vesicle trafficking machinery during L. pneumophila infection.     

Comprehensive identification of protein substrates of the Dot/Icm type IV transporter of Legionella pneumophila

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter.     

Modulation of ubiquitin dynamics and suppression of DALIS formation by the Legionella pneumophila Dot/Icm system

Legionella pneumophila is an intracellular pathogen that uses effector proteins translocated by the Dot/Icm type IV secretion system to modulate host cellular processes. Here we investigate the dynamics of subcellular structures containing ubiquitin during L. pneumophila infection of phagocytic host cells. The Dot/Icm system mediated the formation of K48 and K63 poly-ubiquitin conjugates to proteins associated with L. pneumophila -containing vacuoles in macrophages and dendritic cells, suggesting that regulatory events and degradative events involving ubiquitin are regulated by bacterial effectors during infection. Stimulation of TLR2 on the surface of macrophages and dendritic cells by L. pneumophila- derived molecules resulted in the production of ubiquitin-rich dendritic cell aggresome-like structures (DALIS). Cells infected by L. pneumophila with a functional Dot/Icm system, however, failed to produce DALIS. Suppression of DALIS formation did not affect the accumulation of ubiquitinated proteins on vacuoles containing L. pneumophila. Examining other species of Legionella revealed that Legionella jordanis was unable to suppress DALIS formation after creating a ubiquitin-decorated vacuole. Thus, the L. pneumophila Dot/Icm system has the ability to modulate host processes to promote K48 and K63 ubiquitin conjugates on proteins at the vacuole membrane, and independently suppress cellular events required for the formation of DALIS.     

Type IVB secretion by intracellular pathogens

A growing number of pathogens are being found to possess specialized secretion systems which they use in various ways to subvert host defenses. One class, called type IV, are defined as having homology to the conjugal transfer systems of naturally occurring plasmids. It has been proposed that pathogens with type IV secretion systems have acquired and adapted the conjugal transfer systems of plasmids and now use them to export toxins. Several well-characterized intracellular pathogens, including Legionella pneumophila , Coxiella burnetii , Brucella abortus , and Rickettsia prowazekii , contain type IV systems which are known or suspected to be of critical importance in their ability to cause disease. Specifically, these systems are believed to be the key factors determining intracellular fate, and thus the ability to replicate and cause disease.     

The response regulator PmrA is a major regulator of the icm/dot type IV secretion system in Legionella pneumophila and Coxiella burnetii

Legionella pneumophila and Coxiella burnetii have been shown to utilize the icm/dot type IV secretion system for pathogenesis and recently a large number of icm/dot-translocated substrates were identified in L. pneumophila. Bioinformatic analysis has revealed that 13 of the genes encoding for L. pneumophila-translocated substrates and five of the C. burnetii icm/dot genes, contain a conserved regulatory element that resembles the target sequence of the PmrA response regulator. Experimental analysis which included the construction of a L. pneumophila pmrA deletion mutant, intracellular growth analysis, comparison of gene expression between L. pneumophila wild type and the pmrA mutant, construction of mutations in the PmrA conserved regulatory element, controlled expression studies as well as mobility shift assays, demonstrated the direct relation between the PmrA regulator and the expression of L. pneumophila icm/dot-translocated substrates and several C. burnetii icm/dot genes. Furthermore, genomic analysis identified 35 L. pneumophila and 68 C. burnetii unique genes that contain the PmrA regulatory element and few of these genes from L. pneumophila were found to be new icm/dot-translocated substrates. Our results establish the PmrA regulator as a fundamental regulator of the icm/dot type IV secretion system in these two bacteria.     

A Screen of Coxiella burnetii Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis

Coxiella burnetii is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of C. burnetii NMII transposon insertion mutants to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type C. burnetii displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the cig2 mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type C. burnetii resulted in a multi-vacuolar phenotype similar to that displayed by the cig2 mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.