Separation of Legionella pneumophila proteases and purification of a protease which produces lesions like those of Legionnaires' disease in guinea pig lung

Six discrete protease activities were recovered from the supernatant broth of Legionella pneumophila cultures by ion-exchange chromatography. One of these demonstrated in vitro activity against collagen, casein and gelatin. When administered into the lungs of guinea-pigs this protease elicited lesions which were pathologically similar to those seen in clinical and experimentally induced Legionnaires' disease.     

Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila

The mechanisms by which Legionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained. Several earlier studies indicated that L. pneumophila does not elaborate siderophores. However, we now present evidence that supernatants from L. pneumophila cultures can contain a nonproteinaceous, high-affinity iron chelator. More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM), L. pneumophila secretes a substance that is reactive in the chrome azurol S (CAS) assay. Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract. Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity. The Legionella CAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and other Legionella species. The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction. As would also be expected for a siderophore, the addition of 0.5 or 2.0 microM iron to the cultures repressed the expression of the CAS-reactive substance. Interestingly, the supernatants were negative in the Arnow, Csáky, and Rioux assays, indicating that the Legionella siderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure. We have designated the L. pneumophila siderophore legiobactin.     

A rapid method for purification of homogenous Legionella pneumophila cytotoxic protease using fast protein liquid chromatography

A purification method was developed to isolate Legionella pneumophila cytotoxic protease in a form suitable for biological assays. Culture supernatant of a clinical isolate of L. pneumophila, Knoxville 1 strain, was used as the starting material. The protease was purified by FPLC on a Mono Q column followed by ultrafiltration. The isolated proteolytic enzyme has a specific activity of 90 azocasein units/mg protein and is a 42 kDa monomeric protein as determined by SDS-PAGE and gel filtration chromatography. It is heat-labile and toxic to a variety of cells e.g. McCoy, SIRC, HeLa, and rhabdomyosarcoma cells, baby hamster and green monkey kidney cells, and human embryonic lung fibroblasts.     

Analysis of a cloned sequence of Legionella pneumophila encoding a 38 kD metalloprotease possessing haemolytic and cytotoxic activities

The DNA encoding the zinc metalloprotease of Legionella pneumophila Philadelphia 1 has been isolated and expressed in Escherichia coli. This protein, which is 38,000 Daltons in size, possesses immunological and biochemical properties identical to those previously described for the purified L. pneumophila protease. Periplasmic extracts of E. coli clones expressing the recombinant protease are also capable of causing the haemolysis of canine erythrocytes and the cytotoxic destruction of CHO cells. Using transposon mutagenesis, it was determined that a maximum of 1.2 kb of DNA encoded all three biological activities. Inactivation of proteolytic activity by transposon insertion occurred concomitantly with losses of the haemolytic and cytotoxic phenotypes. A putative regulatory sequence approximately 200-500 bp upstream of the gene's coding region was identified. A 4.0 kb fragment encoding these activities hybridized to the chromosomal DNA of the parent strain of L. pneumophila Philadelphia 1 as well as clinical isolates of L. pneumophila.     

Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB)

The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.     

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.     

Inactivation of human alpha-1-antitrypsin by a tissue-destructive protease of Legionella pneumophila

Three extracellular proteases produced by Legionella pneumophila during growth in liquid medium were examined for their effects on human alpha-1-antitrypsin (alpha-1-AT). One of these proteases, tissue-destructive protease (TDP) destroyed completely the trypsin-inhibitory capacity of alpha-1-AT at protease: inhibitor molar ratios down to 0.002:1. After inactivation by TDP, the Mr of alpha-1-AT was reduced by 5000 in SDS-PAGE. This suggested that inactivation entailed only limited cleavage.     

Cross-reactivity and sensitivity of two Legionella urinary antigen kits, Biotest EIA and Binax NOW, to extracted antigens from various serogroups of L. pneumophila and other Legionella species

Legionella antigen detection kits for diagnosing legionellosis from urine have become widely used, but basic information about reactivity of the kits to non-serogroup (SG) 1 L. pneumophila and other Legionella species remains incomplete. We evaluated Biotest EIA and the most recently developed Binax NOW by using in-vitro extracted antigens of 22 L. pneumophila SG 1 to 15 strains and of 27 other Legionella species. Both kits showed excellent sensitivity to L pneumophila SG 1 antigens, but reacted to different sets of non-SG I L. pneumophila with different sensitivity. No cross-reactivity was observed to Legionella species other than L. pneumophila.     

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。     

Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses

Legionella pneumophila is the causative pathogen of Legionnaires' disease, which is characterized by severe pneumonia. In regard to the pathophysiology of Legionella infection, the role of inflammatory phagocytes such as macrophages has been well documented, but the involvement of dendritic cells (DCs) has not been clarified. In this study, we have investigated the immune responses that DCs generate in vitro and in vivo after contact with L. pneumophila. Heat- and formalin-killed L. pneumophila, but not live L. pneumophila, induced immature DCs to undergo similar phenotypic maturation, but the secreted proinflammatory cytokines showed different patterns. The mechanisms of the DC maturation by heat- or formalin-killed L. pneumophila depended, at least in part, on Toll-like receptor 4 signaling or on Legionella LPS, respectively. After transfer to naive mice, DCs pulsed with dead Legionella produced serum Ig isotype responses specific for Legionella, leading to protective immunity against an otherwise lethal respiratory challenge with L. pneumophila. The in vivo immune responses required the Ag presentation of DCs, especially that on MHC class II molecules, and the immunity yielded cross-protection between clinical and environmental strains of L. pneumophila. Although the DC maturation was impaired by live Legionella, macrophages were activated by live as well as dead L. pneumophila, as evidenced by the up-regulation of MHC class II. Finally, DCs, but not macrophages, exhibited a proliferative response to live L. pneumophila that was consistent with their cell cycle progression. These findings provide a better understanding of the role of DCs in adaptive immunity to Legionella infection.     

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.     

Specific DNA probe for detecting Legionella pneumophila

A fragment of the gene for cytolysin has been cloned. The product of the gene has been earlier identified as an immunoserological marker of Legionella pneumophila. Clones were selected by immunodetection of cytolysin gene product expression. An EcoRI 3.8 kb fragment of the genomic DNA from Legionella pneumophila strain Philadelphia-1 was used as a DNA probe that hybridized with the bacterial colonies of 20 Legionella pneumophila strains but not with the colonies of 10 other Legionella species or 8 other bacterial genera. The cloned fragment has been shown to be unique for Legionella pneumophila. The region of homology is suggested to be longer than a possible dimension of the cytolysin gene.     

Use of the dnaJ gene for the detection and identification of all Legionella pneumophila serogroups and description of the primers used to detect 16S rDNA gene sequences of major members of the genus Legionella

We sequenced about 930 bp of the dnaJ gene from 15 Legionella pneumophila serogroups and some other members of the genus Legionella. As L. pneumophila 16S rDNA sequences could not discriminate between all subspecies and serogroups, we assessed the use of dnaJ gene sequences to differentiate between Legionella subspecies as well as between L. pneumophila serogroups. A phylogenetic analysis revealed that dnaJ gene sequences were more variable between the L. pneumophila serogroups than mip gene and 16S rDNA sequences. By studying 61 strains from 41 species of the genus Legionella, as well as other genera, we established a PCR method that could amplify 285 bp of dnaJ gene from all L. pneumophila serogroups. This primer set was more sensitive than mip gene primers and was able to detect 0.25 ng of purified DNA. We also describe the 16S rDNA primers that were used to detect most Legionella genus members.     

聚合酶链反应-酶切分型鉴定广州地区环境水源军团菌

【目的】探讨聚合酶链反应-酶切分型在快速鉴定环境水源军团菌方面的应用价值,并了解广州地区环境水源军团菌的分布状况。【方法】对广州地区采集的44份环境水样,作军团菌分离培养,再对分离菌株进行16Sr DNA PCR-酶切分型鉴定、16S rDNA基因测序和mip基因测序鉴定。【结果】在广州地区环境水源分离的112株军团菌,经聚合酶链反应-酶切分型鉴定、16S rDNA基因测序和mip基因测序鉴定,检出嗜肺军团菌66株,非嗜肺军团菌46株,其中菲氏军团菌20株,戈氏军团菌17株,橡树岭军团菌7株,长滩军团菌2株。【结论】聚合酶链反应-酶切分型检测环境水源军团菌是一种简便、快速、特异的鉴定方法;在广州地区环境水源中普遍存在军团菌,主要是嗜肺军团菌,其次是菲氏军团菌,戈氏军团菌,橡树岭军团菌和长滩军团菌。     

Evaluation of the Duopath Legionella lateral flow assay for identification of Legionella pneumophila and Legionella species culture isolates

Duopath Legionella (Merck KGaA, Darmstadt, Germany) is a new immunochromatographic assay for the simultaneous identification of cultured L. pneumophila and Legionella species other than L. pneumophila. In tests of 89 L. pneumophila strains and 87 Legionella strains other than L. pneumophila representing 41 different species, Duopath and a widely used latex agglutination assay detected L. pneumophila with 100% and 98% accuracy, respectively, whereas the percentages differed significantly for other Legionella spp. (93% versus 37% [P < 0.001]). Since many countries' regulations require the identification of Legionella spp. in water and environmental samples, the use of Duopath Legionella to comply with those regulations could contribute to significantly fewer false-negative results.     

Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method.

The presence of Legionella spp. in cooling tower water was investigated by using the polymerase chain reaction. Total Legionella spp. detection was performed with 20-mer 5S rRNA complementary DNA sequence primers, and specific Legionella pneumophila detection was performed with 20-mer and then 21-mer macrophage infectivity potentiator gene sequence primers. Of 27 cooling tower water samples, 25 were positive for Legionella spp., and 14 of these contained L. pneumophila.     

Highly sensitive quenched fluorescent substrate of Legionella major secretory protein (msp) based on its structural analysis

Legionella pneumophila has been shown to secrete a protease termed major secretory protein (Msp). This protease belongs to the M4 family of metalloproteases and shares 62.9% sequence similarity with pseudolysin (EC 3.4.24.26). With the aim of developing a specific enzymatic assay for the detection and quantification of Msp, the Fluofast substrate library was screened using both enzymes in parallel. Moreover, based on the crystal structure of pseudolysin, a model of the Msp structure was built. Screening of the peptide library identified a lead substrate specifically cleaved by Msp that was subsequently optimized by rational design. The proposed model for Msp is consistent with the enzymatic characteristics of the studied peptide substrates and provides new structural information useful for the characterization of the protease. This study leads to the identification of the first selective and high affinity substrate for Msp that is able to detect picomolar concentrations of the purified enzyme. The identified substrate could be useful for the development of a novel method for the rapid detection of Legionella.     

Isolation of Legionella species from drinking water

Three different species of Legionella were recovered from samples of water taken from chlorinated public water supplies where no coliform bacteria were simultaneously detected. Five of 856 samples yielded Legionella isolates. Three isolates were identified as Legionella pneumophila serogroup 1, the fourth was identified as Legionella dumoffii, and the fifth was identified as Legionella jordanis. Studies to determine the survival of L. pneumophila Flint 1 serogroup 1 in tap water at various temperatures and in tap water with added sodium hypochlorite were done. These organisms were found to survive for 299 days in tap water at 24 and 5 degrees C but not at 35 degrees C. A concentration of at least 0.2 mg of residual chlorine per ml was required to eliminate at least 90% of L. pneumophila and Escherichia coli inocula in 2 h.