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.     

Identification of CpxR as a positive regulator of icm and dot virulence genes of Legionella pneumophila

To date, 24 Legionella pneumophila genes (icm and dot genes) have been shown to be required for intercellular growth and host cell killing. A previous report indicated that the regulation of these genes is complicated and probably involves several regulatory proteins. In this study, a genetic screen performed in Escherichia coli identified the CpxR response regulator as an activator of the L. pneumophila icmR gene. Construction of an L. pneumophila cpxR insertion mutant showed that the expression of icmR is regulated by CpxR. In addition, a conserved CpxR binding site (GTAAA) was identified in the icmR regulatory region and L. pneumophila His-tagged CpxR protein was shown to bind to the icmR regulatory region using a mobility shift assay. Besides its dramatic effect on the icmR level of expression, the CpxR regulator was also found to affect the expression of the icmV-dotA and icmW-icmX operons, but to a lesser extent. The role of CpxA, the cognate sensor kinase of CpxR, was also examined and its effect on the icmR level of expression was found to be less pronounced than the effect of CpxR. The RpoE sigma factor, which was shown to coregulate genes together with CpxR, was examined as well, but it did not influence icm and dot gene expression. In addition, when the cpxR mutant strain, in which the expression of the icmR gene was dramatically reduced, and the cpxA and rpoE mutant strains were examined for their ability to grow inside Acanthamoeba castellanii and HL-60-derived human macrophages, no intracellular growth defect was observed. This study presents the first evidence for a direct regulator (CpxR) of an icm-dot virulence gene (icmR). The CpxR regulator together with other regulatory factors probably concerts with the expression of icm and dot genes to result in successful infection.     

The response regulator CpxR directly regulates expression of several Legionella pneumophila icm/dot components as well as new translocated substrates

Legionella pneumophila has been shown to utilize the icm/dot type IV secretion system for pathogenesis. This system was shown to be composed of icm/dot complex components and accessory proteins, as well as a large number of translocated substrates. Bioinformatic analysis of the regulatory regions of all the genes revealed that several icm/dot genes, as well as two genes encoding icm/dot translocated substrates, contain the conserved CpxR regulatory element, a regulator that has been shown previously to control the expression of the icmR gene. An experimental analysis, which included a comparison of gene expression in a L. pneumophila wild-type strain and gene expression in a cpxR deletion mutant, construction of mutants with mutations in the CpxR conserved regulatory elements, controlled expression studies, and mobility shift assays, demonstrated the direct relationship between the CpxR regulator and the expression of the genes. Furthermore, genomic analysis identified nine additional genes that contain a putative CpxR regulatory element; five of these genes (two legA genes and three ceg genes) were suggested previously to be putative icm/dot translocated substrates. The three ceg genes identified, which were shown previously to contain a putative PmrA regulatory element, were found here to be regulated by both CpxR and PmrA. The other six genes (two legA genes and four new genes products were found to be regulated by CpxR. Moreover, using the CyaA translocation assay, these nine gene products were found to be translocated into host cells in an Icm/Dot-dependent manner. Our results establish that the CpxR regulator is a fundamental regulator of the icm/dot type IV secretion system in L. pneumophila.     

The Legionella pneumophila CpxRA two‐component regulatory system: new insights into CpxR's function as a dual regulator and its connection to the effectors regulatory network

Legionella pneumophila utilizes the Icm/Dot type‐IV secretion system to translocate approximately 300 effector proteins into host cells, and the CpxRA two‐component system (TCS) was previously shown to regulate the expression of several of these effectors. In this study, we expanded the pool of L. pneumophila CpxR‐regulated genes to 38, including 27 effector‐encoding genes. Our study demonstrates for the first time that the CpxR dual regulator has different requirements for activation and repression of target genes. These differences include the positioning of the CpxR regulatory element relative to the promoter element, and the effect of CpxR phosphate donors on the expression of CpxR target genes. In addition, unlike most response regulators, a mutant form of the L. pneumophila CpxR which cannot be phosphorylated was found to self‐interact, and to repress gene expression similarly to wild‐type CpxR, even though its ability to activate gene expression was reduced. Moreover, the CpxRA TCS was found to activate the expression of LetE which was found to function as a connector protein between the CpxRA TCS and the LetAS‐RsmYZ‐CsrA regulatory cascade. Our results show that CpxR plays a major role in L. pneumophila pathogenesis gene expression and functions as part of a regulatory network.     

IcmR-regulated membrane insertion and efflux by the Legionella pneumophila IcmQ protein

Legionella pneumophila proliferates within alveolar macrophages as a central property of Legionnaires' disease. Intracellular growth involves formation of a replicative phagosome, which requires the bacterial Dot/Icm system, a multiprotein secretion apparatus that translocates proteins from the bacterium across the macrophage plasma membrane. Two components of this system, IcmR and IcmQ, are proposed to exhibit a chaperone/substrate relationship similar to that observed in other protein translocation systems. We report here that IcmQ inserts into lipid membranes and forms pores that allow the efflux of the dye calcein but not Dextran 3000. Both membrane insertion and pore formation were inhibited by IcmR. Trypsin digestion mapping demonstrated that IcmQ is subdivided into two functional domains. The N-terminal region of IcmQ was necessary and sufficient for insertion into lipid membranes and calcein efflux. The C-terminal domain was necessary for efficient association of the protein with lipid bilayers. IcmR was found to bind to the N-terminal portion of the protein thus providing a mechanism for its ability to inhibit IcmQ pore-forming activity. Localization of IcmQ on the surface of the L. pneumophila shortly after infection as well as its pore-forming capacities suggest a role for IcmQ in forming a channel that leads translocated effectors out of the bacterium.     

The Legionella pneumophila IcmR protein exhibits chaperone activity for IcmQ by preventing its participation in high-molecular-weight complexes

A key event in legionellosis is the ability of Legionella pneumophila to survive and proliferate inside alveolar macrophages. The dot/icm genes, which are necessary for intracellular growth, show sequence similarity to genes encoding conjugative transfer systems, and it is believed that they are responsible for the formation of a secretion apparatus. Evidence is provided here that the IcmR and IcmQ proteins participate in a chaperone-substrate relationship similar to that observed for translocated proteins in type III and type IV secretion apparatuses. Immobilized IcmQ was found to bind IcmR from crude bacterial extracts efficiently. Furthermore, purified IcmR and IcmQ bind with high affinity. This interaction was also observed in vivo by co-immunoprecipitation. The presence of IcmR was found to affect the physical state of IcmQ directly. In the absence of IcmR, IcmQ formed high-molecular-weight complexes both in vivo and in vitro, whereas IcmR prevented and reversed the formation of these complexes.     

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.     

嗜肺军团菌效应蛋白质介导的新型泛素化过程

泛素化是真核细胞特有的蛋白质翻译后修饰方式,调节真核细胞内多种重要生理过程,例如蛋白质稳态、细胞周期、免疫反应、DNA修复以及囊泡转运等。鉴于泛素化对于生命活动的重要性,病原菌在与宿主细胞的长期进化过程中衍生出一系列针对宿主泛素化过程的效应蛋白质,调控宿主体内泛素化过程,从而构建有利于病原菌自身生长繁殖的内环境。嗜肺军团菌是一种革兰氏阴性菌,是军团菌肺炎的致病菌,能够引起发热和肺部感染,重型病死率高达15%~30%。Dot/Icm Ⅳ型分泌系统是嗜肺军团菌侵染过程中最主要的毒力系统。在侵染宿主细胞的过程中,嗜肺军团菌利用该分泌系统,分泌超过330种效应蛋白质,协助细菌在宿主胞内生存、增殖和逃逸。多种嗜肺军团菌效应蛋白质通过直接或者间接的方式对宿主泛素化过程进行调控。近年的研究发现,多种效应蛋白质可以介导不同于真核生物经典泛素化的新型泛素化过程。本文介绍了嗜肺军团菌效应蛋白质介导的新型泛素化过程的最新研究进展,为理解泛素化过程在嗜肺军团菌致病过程中的重要作用提供参考依据。     

Dynamics of Legionella spp. and bacterial populations during the proliferation of L. pneumophila in a cooling tower facility

The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii, and other Legionella-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.     

Identification of species of the genus Legionella using a cloned rRNA gene from Legionella pneumophila

A cloned EcoRI fragment from Legionella pneumophila, which includes 16S and 23S rRNA genes, was used to identify bacteria belonging to the genus Legionella by hybridization to a series of species specific restriction fragments. Examination of the type strains of 28 species of legionellae gave different band patterns in every case. When further isolates of these species were tested the patterns obtained were usually either identical, or very similar, to those of the respective type strains. Thirty-one coded isolates were examined and of these 29 were allocated to the correct species. The remaining strains (a non-Legionella and a L. pneumophila) could not be identified using this technique. The rRNA gene probe method should be of great value in the identification of legionellae, particularly for those species which are at present very difficult to distinguish serologically.     

辽宁省2006-2016年集中空调冷却水中 嗜肺军团菌毒力岛基因组携带情况

摘要:目的 了解2006?2016年辽宁地区集中空调冷却水中军团菌携带毒力岛基因情况及其致病性。方法 根据GenBank公布的嗜肺军团菌核苷酸序列设计和合成嗜肺军团菌种和毒力岛基因鉴定引物,采用PCR法对2006?2016年辽宁省各大公共场所委托及抽样检测中分离到的军团菌,进行了毒力岛基因组检测,并与血清型进行比较分析,其中嗜肺军团菌15株、非嗜肺军团菌8株。结果 标准菌株ATCC（33152）12个毒力岛基因全阳性;9株LP1型嗜肺军团菌分别检出9~11个毒力岛基因,6株LP2-14型嗜肺军团菌分别检出6~9个毒力岛基因,8株非嗜肺军团菌分别检出2~11个毒力岛基因。结论 辽宁地区军团菌广泛存在公共环境集中空调冷却系统中,以LP1型嗜肺军团菌居多,LP2-14型嗜肺军团菌与非嗜肺军团菌也普遍存在,而且所测菌株均携带毒力岛基因,是细菌感染性肺炎的重要隐患病源之一。     

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.     

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting

Abstract Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time-consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1–16 as well as 21 non- pneumophila isolates could be identified and differentiated at the species level using this system.