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.     

Long-term survival of Legionella pneumophila serogroup 1 under low-nutrient conditions and associated morphological changes

Abstract Extended survival of Legionella pneumophila , using both a clinical and an environmental isolate, was studied in drinking water, creek water, and estuarine water microcosms. Legionella populations were monitored by acridine orange direct counts (AODC) and viable count on buffered charcoal yeast extract agar amended with alpha-ketoglutarate (BCYEα). Initial colony counts of the clinical isolate in drinking and creek water microcosms were 2 × 10⁸ cfu/ml and, after incubation for 1.5 years, the plate counts decreased to 3 × 10⁶ cfu/ml. The AODC counts, however, did not change significantly. The clinical isolate in estuarine water decreased in plate counts to 10² (cfu/ml) over the same period. After incubation for 1.5 years at 15°C in the microcosms, Legionella plate counts of creek and drinking water decreased by two logs. Direct microscopic examination of aliquots removed from all microcosms revealed the presence of small bacilli, large bacilli and rare filamentous cells. The environmental isolate demonstrated only one colony morphology upon culture on BCYEα. Interestingly, after four months incubation in the microcosm, upon plating the clinical isolate on BCYEα, two distinct colony types were evident. Examination by immunofluorescent staining employing a monoclonal antibody against L. pneumophila revealed both bacillus and filamentous forms. The total cellular proteins of both morphotypes were examined by sodium dodecyl sulfate polyacrylyamide gel electrophoresis (SDS-PAGE), demonstrating identical protein patterns. Those Legionella cells remaining culturable during 1.5 years of incubation grew rapidly when transferred to BCYEα. Incubation was continued and it was found that some strains of L. pneumophila serogroup 1 can remain viable for longer than 2.4 years under low-nutrient conditions.     

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。     

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.     

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.     

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     

Genetic approaches to study Legionella pneumophila pathogenicity

Abstract: Legionella pneumophila is an intracellular pathogen replicating in human macrophages during the course of infection of the lungs, infection by legionellae often leads to severe pneumonia, termed Legionnaires' disease. Genetic approaches to identify the factors responsible for L. pneumophila pathogenicity started with the construction of genomic libraries in Escherichia coli. Various L. pneumophila -specific genes were cloned in E. coli K-12 by identifaction using functional assays, antibody screening and hybridization ('reverse genetics'). By disrupting the genes via allelic exchange, mutants have been created to assess the influence of the factors on pathogenicity. Among the cloned genes, only for the gene product of the mip gene, encoding a 24-kDa surface-associated protein (macrophage infectivity potentiator) unequivocal evidence for its contribution to pathogenicity could be provided. Two hemolytic factors that have been cloned do not seem to play a role in L. pneumophila pathogenicity. Genetic systems for transposon mutagenesis of the L. pneumophila genome (Tn5, Tn903dlIlacZ, MudphoA), including TnphoA shuttle mutagenesis, have been established and specifically adapted to identify mutants which displayed an impaired capability to multiply inside macrophages and with a reduced in vivo virulence. Furthermore, by complementation of avirulent mutants, genetic loci could be identified which restored the virulence.     

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。     

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.     

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.     

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。     

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 .     

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。     

Study of nonculturable Legionella pneumophila cells during multiple-nutrient starvation

Abstract: The nonculturable form of Legionella pneumophila in multiple-nutrient starvation culture was studied. During extended starvation, the total direct counts (TDC) of L. pneumophila did not change significantly, but no colonies were detected on day 50. Quantitative PCR detection of L. pneumophila DNA demonstrated that nonculturable cells retained PCR-detectable DNA even after starvation for 300 days, and part of the nonculturable population possessed nonspecific esterase activity. However, resuscitation trials of nonculturable L. pneumophila on day 100 of starvation recovered no colony-forming units, and electrophoresis of nucleic acids extracted from nonculturable cells revealed rRNA subunit (23S, 16S and 5S) degradation. When legionellae have lost the ability to multiply, at least some DNA and enzyme functions may be retained for prolonged periods.     

Legionella pneumophila in commercial bottled mineral water

Sixty-eight commercial bottled mineral waters (64 brands, 68 different 'best-before dates') were tested for the presence of bacteria and fungi. Six samples were Legionella antigen positive and six were Legionella pneumophila PCR positive. Two samples were both Legionella antigen and L. pneumophila PCR positive. Legionella cultures were negative. Although the PCR might have detected only dead Legionella cells, the PCR has been described to detect specifically viable but not culturable (VBNC) L. pneumophila cells as well. Whether VBNC bacteria may be present in bottled mineral waters and the risk for infection this may pose for severely immunocompromised patients should be investigated.     

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.     

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

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。