Distinct difference of flaA genotypes of Legionella pneumophila between isolates from bath water and cooling tower water

To investigate the genetic difference of Legionella pneumophila in human‐made environments, we collected isolates of L. pneumophila from bath water (n = 167) and cooling tower water (n = 128) primarily in the Kanto region in 2001 and 2005. The environmental isolates were serogrouped and sequenced for a target region of flaA. A total of 14 types of flaA genotypes were found: 10 from cooling tower water and nine from bath water. The flaA genotypes of isolates from cooling tower water were quite different from those of bath water.     

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.     

嗜肺军团菌生物膜形成及其影响因素

嗜肺军团菌在自然环境和人工供水系统中普遍存在,能够在阿米巴原虫和其他原生动物体内繁殖,所引起的军团菌病主要表现为严重的呼吸系统疾病。但在自然环境中,嗜肺军团菌的生存和繁殖受到多菌种生物膜形成和繁殖的影响,一些军团菌病的暴发与生物膜的存在相关。因此,阻止自然环境和人工水系统中生物膜的形成显然已成为降低水污染的有效策略之一。根据近年来的报道分别对影响嗜肺军团菌生物膜形成的生化因子和嗜肺军团菌的毒力因子,以及其他生物物种在嗜肺军团菌生物膜形成过程中所起的不同作用等进行综述。     

The Lly protein is essential for p-hydroxyphenylpyruvate dioxygenase activity in Legionella pneumophila

The lly locus confers fluorescence, haemolysis, brown pigmentation and an increased resistance to light in Legionella pneumophila. In this study, we correlated the pigment production of two lly-positive L. pneumophila isolates and a recombinant lly-positive Escherichia coli strain with the presence of homogentisic acid (HGA) in the culture supernatant. The detection of HGA by high performance liquid chromatography and the data analysis of the deduced amino acid sequence of the lly gene indicate that the lly locus codes for a p-hydroxyphenylpyruvate dioxygenase (HPPD). This enzyme catalyses the transformation of p-hydroxyphenylpyruvate into HGA, which subsequently oxidises and polymerises into a melanin-like pigment. One open reading frame (ORF 1) in the lly region exhibited homologies with genes of Synechocystis sp., Petroselium crispum and Streptomyces mycarofaciens that code for methyltransferases. By screening a genomic library of L. pneumophila (serogroup 1) strain Corby with a monoclonal antibody against the legiolysin (lly), we identified two recombinant E. coli clones that did not produce the brown pigment and showed no haemolysis and fluorescence. DNA sequencing revealed that both clones contained 874 nucleotides of the N-terminal part of the lly gene. The recombinant strains expressed truncated legiolysin proteins of 39.5 and 35.7 kDa and did not produce HGA. Considering the highly conserved structure of legiolysin-like HPPD genes from other organisms, we suggest that the C-terminus of the legiolysin may be essential for the enzymatic activity that conferred pigmentation via HGA polymerisation, haemolysis and fluorescence.     

Identification and serotyping of atypical Legionella pneumophila strains isolated from human and environmental sources

AIMS: To validate identification methods for Legionella pneumophila strains that cannot be serotyped into the known serogroups and to characterize their antigenic diversity. METHODS AND RESULTS: Fifty L. pneumophila strains that could not be serogrouped, but which had been confirmed as L. pneumophila by mip gene sequencing, were further identified phenotypically. We used (i) MONOFLUO anti-Legionella Staining Reagent (Bio-Rad) (50/50), (ii) an in-house prepared immunoblot assay for the detection of L. pneumophila- specific Mip protein epitope (50/50), (iii) fatty acid analysis using the Microbial Identifications System (MIDI) (47/50) and (iv) Oxoid agglutination tests (44/50). The serological diversity was further characterized by testing with five serogroup-cross-reactive monoclonal antibodies, resulting in nine phenons. CONCLUSIONS: The division of L. pneumophila into 15 serogroups does not reflect the serogroup heterogeneity. Results of these tests indicate that there are more serogroups. SIGNIFICANCE AND IMPACT OF THE STUDY: MONOFLUO anti-Legionella Staining Reagent is the only commercially available tool for identifying atypical strains of L. pneumophila. If necessary for epidemiological purposes, the antigenic heterogeneity of these strains can be analysed by monoclonal antibodies.     

嗜肺军团菌实时荧光定量PCR快速检测方法的建立

目的：建立针对嗜肺军团菌Mip基因的实时荧光定量TaqMan PCR检测方法,并进行自来水和空调冷却水模拟标本的检测评价。方法：根据嗜肺军团菌Mip基因的特异性序列设计引物和TaqMan探针,建立嗜肺军团菌的实时荧光定量TaqMan PCR快速检测方法,对方法进行灵敏度及特异性评价,并对自来水和空调冷却水模拟标本中的嗜肺军团菌进行检测。结果：建立的方法对嗜肺军团菌的检测具有高度特异性,与3种非嗜肺军团菌和6种其他呼吸道病原均没有交叉反应;基因组DNA的检测灵敏度为1.6pg／μL,模拟自来水和空调冷却水标本的检测灵敏度为10CFU／mL。结论：建立的TaqMan荧光定量PCR方法特异、灵敏、快速,适于嗜肺军团菌的日常监测和暴发疫情的应急诊断。     

Structure-function analysis of the C-terminus of IcmT of Legionella pneumophila in pore formation-mediated egress from macrophages

We have recently shown an essential role of the 32 amino acids C-terminus domain of IcmT of Legionella pneumophila in bacterial egress from macrophages. Mutants expressing an IcmT protein with a truncation in the C-terminus, replicate intracellularly but are defective in pore formation-mediated egress. The C-terminus domain of IcmT is the only hydrophilic domain of IcmT that is predicted to be in the cytoplasm while the rest of the protein is in the cytoplasmic membrane. In order to characterize the structure-function of the C-terminus of IcmT in the pore-forming activity and bacterial egress, we constructed 10 icmT missense mutant alleles differing by a single amino acid in the C-terminus of icmT and introduced them into the null icmT mutant. The H58Q, W69L, R71I, R79I and R86I icmT mutant alleles showed significantly lower pore-forming activity as measured by hemolysis of sRBC. The Y59S, R68L and S77L mutant alleles showed significantly lower cytopathogenicity to U937 macrophages. All 10 mutant alleles enabled the icmT null mutant to replicate intracellularly as efficiently as icmT null mutant harboring the wild-type icmT. Seven of the icmT alleles enabled the icmT null mutant to egress from infected macrophages as efficiently as icmT null mutant harboring the wild-type icmT. The other 3 substitutions conferred a partial defect in hemolysis and two of them also conferred a defect in egress from macrophages. Thus, two amino acid residues in the C-terminus of IcmT are required for both pore formation and bacterial egress. However, certain single amino acid substitutions in the C-terminus reduce the pore-forming activity when tested in vitro, but may or may not have a detectable effect on egress of L. pneumophila from U937 macrophages.     

Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm^?2) or 5.34 ± 0.33 log (gu cm^?2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ～300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.     

Micro- and macromethod assays for the ecological study of Legionella pneumophila

The survival of a strain of Legionella pneumophila (Lp-1) inoculated in artificial water microcosms was investigated with and without an amoebal host and varying environmental conditions, such as biofilm formation, amount of nutrients and incubation temperature. The results obtained using short (micromethod) and long (macromethod) term methods showed that L. pneumophila Lp-1 dies rapidly at 4 degrees C in the "macromethod" assay. When the same temperature (4 degrees C) was applied to the "micromethod" assay, L. pneumophila Lp-1 survived for three weeks, although it progressively decreased. At an incubation temperature of 30 degrees C, the aquatic environment was more favourable and better survival emerged in the "macromethod"; in contrast, this favourable temperature condition did not improve the survival of L. pneumophila Lp-1 cultured with the "micromethod". The role of the protozoa Acanthamoeba polyphaga proved to be indispensable for legionella survival only when environmental conditions become unfavourable.     

An oxaloacetate decarboxylase homologue protein influences the intracellular survival of Legionella pneumophila

Abstract Legionella pneumophila is a facultative intracellular parasite which is able to survive in various eukaryotic cells. We characterised a Tn5-mutant of the L. pneumophila Corby strain and were able to identify the insertion site of the transposon. It is localised within an open reading frame which shows high homology to the α-subunit of the oxaloacetate decarboxylase (OadA) of Klebsiella pneumoniae . The OadA homologous protein of L. pneumophila was detected in the wild-type strain by Western blotting. Since the intracellular multiplication of the oad A⁻ mutant strain is reduced in guinea pig alveolar macrophages and human monocytes, it is concluded that the oad A gene product has an effect on the intracellular survival of L. pneumophila .     

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.     

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.     

广州地区嗜肺军团菌环境分离株的基因序列分型分析

摘要:【目的】研究广州市嗜肺军团菌的基因特征,对来自不同水域环境的嗜肺军团菌进行分子分型研究。【方法】选择嗜肺军团菌的7个基因flaA、asd、mip、pilE、mompS、proA和neuA 作为目的基因, 对在2006-2009年间广州地区分离的44株嗜肺军团菌进行PCR扩增和测序,并将核苷酸序列上传至欧洲军团菌病感染工作组（EWGLI）数据库进行比对,得到基因型别（Sequence type, ST）,对结果进行基因序列分型（Sequence-Based Typing, SBT）和系统进化分析。【结     

Ultra-structure and localisation of formazan formed by human neutrophils and amoebae phagocytosing virulent and avirulent Legionella pneumophila

Abstract Legionella pneumophila (LP) strains of differing virulence were incubated with a solution of nitroblue-tetrazolium (NBT) at a concentration of 1 mg · ml⁻ in the presence of Acanthamoeba polyphaga or human polymorphonuclear neutrophils (PMN). Reduction of NBT to formazan occurred at a faster rate in the presence of virulent strains. Reduction appeared to be temperature dependent; at 37°C the reaction rate was higher than at 20°C. On microscopic examination, deposits of formazan around Legionella cells were observed inside amoebae similar to those deposited in human neutrophils. Electron microscopy revealed electron-dense particles surrounding virulent legionellae, which appeared to be associated with formazan foundation. Formazan formation inside amoebae may suggest the presence of a respiratory burst against LP, which is more intense with virulent strains.     

Legionella pneumophila restrains autophagy by modulating the host's sphingolipid metabolism

Sphingolipids are bioactive molecules playing a key role as membrane components, but they are also central regulators of many intracellular processes including macroautophagy/autophagy. In particular, sphingosine-1-phosphate (S1P) is a critical mediator that controls the balance between sphingolipid-induced autophagy and cell death. S1P levels are adjusted via S1P synthesis, dephosphorylation or degradation, catalyzed by SGPL1 (sphingosine-1-phosphate lyase 1). Intracellular pathogens are able to modulate many different host cell pathways to allow their replication. We have found that infection of eukaryotic cells with the human pathogen Legionella pneumophila triggers a change in the host cell sphingolipid metabolism and specifically affects the levels of sphingosine. Indeed, L. pneumophila secretes a protein highly homologous to eukaryotic SGPL1 (named LpSPL). We solved the crystal structure of LpSPL and showed that it encodes lyase activity, targets the host's sphingolipid metabolism, and plays a role in starvation-induced autophagy during L. pneumophila infection to promote intracellular survival.     

带GFP标记嗜肺军团菌的构建和在细胞感染中的应用

【目的】为能实时直观了解嗜肺军团菌感染细胞的过程,研究细菌在细胞内的变化及其与宿主细胞间的相互作用关系。【方法】通过基因敲除、克隆回补等重组构建绿色荧光蛋白(GFP)稳定高表达的嗜肺军团菌株,利用该菌株建立小鼠巨噬细胞Raw264.7的感染模型。【结果】通过荧光显微镜可实时观察细菌感染细胞的全过程,包括细菌在细胞内的形态变化、增殖和裂解宿主细胞等。【结论】重组菌可替代野生菌株在细胞感染中应用,为直观研究嗜肺军团菌与被感染细胞之间的相互作用关系,以及进行相关药物模型的制备、药物筛选、耐药机制研究等提供了新的手段。     

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。