Isoprenoid quinones of the genus Legionella.

Representative strains of each of the named species of Legionella were examined for isoprenoid quinones by reverse-phase thin-layer chromatography. All strains contained three or more ubiquinones (Q9, Q10, Q11, Q12, Q13) which were useful for placing the species into one of three distinct groups. Group 1 contained L. longbeachae, L. bozemanii, L. dumoffi, and L. gormanii; group 2 contained only L. micdadei; and group 3 contained only L. pneumophila. The identities of the quinones were established by UV spectroscopy and mass spectrometry.     

Disulfide-bonded outer membrane proteins in the genus Legionella.

Staphylococcus aureus has been classified into at least eight different capsular types by using polyclonal rabbit antisera specific for their associated capsular polysaccharides. We produced and characterized monoclonal antibodies reactive with two serologically distinct capsular types, types 5 and 8, which account for more than 70% of all S. aureus bacteremias. These type-specific, monoclonal antibodies reacted with S. aureus clinical isolates possessing the homologous capsular type and exhibited no cross-reactivity against S. aureus clinical isolates possessing the heterologous capsular type, nontypeable S. aureus clinical isolates, Staphylococcus epidermidis clinical isolates, or a variety of gram-negative organisms. The anti-type 8 monoclonal antibodies also reacted with purified capsular polysaccharide derived from the prototype type 8 S. aureus strain.     

Identification of Legionella pneumophila genes required for growth within and killing of human macrophages.

Legionella pneumophila was mutagenized with Tn903dIIlacZ, and a collection of mutants was screened for defects in macrophage killing (Mak-). Of 4,564 independently derived mutants, 55 (1.2%) showed a reduced or complete lack in the ability to kill HL-60-derived human macrophages. Forty-nine of the Mak- mutants could be assigned to one of 16 DNA hybridization groups. Only one group (9 of the 10 members) could be complemented for macrophage killing by a DNA fragment containing icm and dot, two recently described L. pneumophila loci that are required for macrophage killing. Phenotypic analysis showed that none of the mutants were any more sensitive than the wild type to human serum, oxidants, iron chelators, or lipophilic reagents nor did they require additional nutrients for growth. The only obvious difference between the Mak-mutants and wild-type L. pneumophila was that almost all of the Mak- mutants were resistant to NaCl. The effects of LiCl paralleled the effects of NaCl but were less pronounced. Resistance to salt and the inability to kill human macrophages are linked since both phenotypes appeared when Tn903dIIlacZ mutations from two Mak- strains were transferred to wild-type backgrounds. However, salt sensitivity is not a requisite for killing macrophages since a group of Mak- mutants containing a plasmid that restored macrophage killing remained resistant to NaCl. Mak- mutants from groups I through IX associated with HL-60 cells similarly to wild-type L. pneumophila. However, like the intracellular-multiplication-defective (icm) mutant 25D, the Mak- mutants were unable to multiply within macrophages. Thus, the ability of L. pneumophila to kill macrophages seems to be determined by many genetic loci, almost all of which are associated with sensitivity to NaCl.     

Identification of Legionella pneumophila by latex agglutination.

A latex agglutination test for the identification of Legionella pneumophila serogroups 1 through 6 is described. The reagent is specific for L. pneumophila and enables the ready identification of L. pneumophila colonies on agar plates. Preliminary evidence suggests that latex agglutination enables the detection of soluble L. pneumophila antigens in respiratory secretions of patients suspected of having legionellosis.     

Determination of catalase, peroxidase, and superoxide dismutase within the genus Legionella.

We examined 40 strains of Legionella for reduced-oxygen scavenging enzymes. Using a simple reaction chamber with a Swinney filter for the Beers and Sizer assay, we determined the catalase activity of live cells grown on buffered charcoal-yeast extract agar. For 29 strains of Legionella pneumophila, the apparent first-order rate constants for catalase ranged from 0.000 to 0.005. Similarly, low values ranging from 0.001 to 0.005 were observed for Legionella wadsworthii, Legionella oakridgensis, and Legionella gormanii. High catalase activities were found for Legionella jordanis, Legionella longbeachae, Legionella micdadei, and Legionella bozemanii, with first-order rate constant values of 0.010 to 0.035. Cell-free extracts were analyzed for catalase, peroxidase, and superoxide dismutase. Cell-free extracts of all strains had superoxide dismutase levels ranging from 8.2 to 30.5 U per mg of protein. The species could be characterized by their catalase and peroxidase since L. pneumophila and L. gormanii had only peroxidase (relative molecular weight [Mr], 150,000); L. dumoffii had a peroxidase (Mr, 150,000) plus a catalase (Mr, 174,000); and all remaining species had catalase only (Mr, 300,000, 220,000, or 150,000).     

Identification of a cytotoxin produced by Legionella pneumophila.

Culture filtrates of Legionella pneumophila were cytotoxic for Chinese hamster ovary cells. The cytotoxin was found to be methanol soluble, heat stable, and stable from pH 5 through 8.     

Identification of Legionella species by lipopolysaccharide antigen pattern.

Electrophoretic analysis of lipopolysaccharide (LPS) extracts from 430 previously serotyped Legionella isolates and 28 American Type Culture Collection (ATCC) non-Legionella pneumophila Legionella reference strains representing different Legionella species and serogroups has been performed. LPS was prepared from Legionella suspensions by sonication and proteinase K digestion. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, LPS bands were either stained with silver nitrate or transferred onto a nitrocellulose membrane and detected with rabbit antibodies raised against L. pneumophila serogroup 5, which was known to cross-react with L. pneumophila serogroups 1 to 14. Silver staining revealed that each of the 28 ATCC non-L. pneumophila Legionella strains possessed an individual and characteristic LPS banding pattern. The LPS profile was defined by the molecular weight of the visualized bands and/or the individual ladder-like LPS pattern. It was demonstrated by immunoblotting that non-L. pneumophila Legionella strains did not react with the serogroup 5 antiserum, thus allowing for the differentiation between L. pneumophila and non-L. pneumophila species.     

Identification of protein antigens of Legionella pneumophila serogroup 1.

Growth of Legionella pneumophila serogroup 1 in yeast extract broth was standardized, and protein profiles of detergent-solubilized cells were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Over 30 protein bands were identified, 6 of which were more prominent both in Coomassie brilliant blue-stained profiles and in fluorograms with intrinsically radiolabeled bacteria. These major proteins were 22,000 dalton (22K), 24K, 43K, 63K, 76K, and 78K. We found that the 24K and 63K major proteins were antigenic, as demonstrated both by radioimmunoprecipitation (RIP) of [35S]methionine-labeled organisms and by immunoblotting with rabbit hyperimmune sera. In addition, both techniques detected antigens migrating at 58K, 72K, 76K, and 78K. The major 22K and 43K major proteins and antigens migrating at 25.5K, 29K, and 46K were only detected by radioimmunoprecipitation, whereas antigens of 19K, 48K, 53K, and 68K were detected only by immunoblotting. Cell-surface localization of the proteins was determined by a modified radioimmunoprecipitation assay designed to react specifically with surface antigens and by the use of hyperimmune sera that had been extensively preabsorbed with intact cells to deplete the sera of antibodies directed against surface components. The 22K, 24K, 43K, 63K, and 78K major proteins and several minor proteins were found to be located on the surface of L. pneumophila cells.     

Identification of Legionella pneumophila-specific genes by genomic subtractive hybridization with Legionella micdadei and identification of lpnE, a gene required for efficient host cell entry

Legionella pneumophila is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of L. pneumophila, we performed genomic subtractive hybridization between L. pneumophila serogroup 1 strain 02/41 and L. micdadei strain 02/42. A total of 144 L. pneumophila-specific clones were sequenced, revealing 151 genes that were absent in L. micdadei strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among L. pneumophila isolates of various serogroups as well as strains of Legionella longbeachae, L. micdadei, Legionella gormanii, and Legionella jordanis. Twelve predicted ORFs were L. pneumophila specific, including the gene encoding the dot/icm effector, lepB, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all L. pneumophila serotypes tested and an isolate of L. gormanii. These included icmD, the 5' end of a pilMNOPQ locus, and two genes known to be upregulated during growth within macrophages, cadA2 and ceaA. Disruption of an L. pneumophila-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed lpnE, for "L. pneumophila entry." In summary, this investigation has revealed important genetic differences between L. pneumophila and other Legionella species that may contribute to the phenotypic and clinical differences observed within this genus.     

Identification of a species-specific antigen in Legionella pneumophila by a monoclonal antibody.

A species-specific antigen in Legionella pneumophila was identified by a monoclonal antibody in enzyme-linked immunosorbent and immunofluorescence assays of serogroups 1 through 8. The species-specific antigen was heat stable, and the molecular weight of the major band was 29,000 by immunoblot analysis. In direct immunofluorescence assays, the antigen was cryptic or only partly exposed on the surface of the cells but was effectively exposed by treating the cells with detergent and EDTA. The monoclonal antibody was utilized in direct immunofluorescence assays to specifically identify multiple cultures of L. pneumophila serogroups.     

Identification of two new antigenic subgroups within the genus Mobiluncus.

Classifications of 48 atypical clinical isolates of Mobiluncus spp. were determined on the basis of biochemical reactions, morphology, antigenic profiles, and DNA hybridization studies. Two new subgroups with unique antigenic profiles are described. Like typical Mobiluncus species, the antigenic variant of M. mulieris is associated with bacterial vaginosis. The atypical isolates of M. curtisii were frequently recovered from women with normal vaginal flora and were also recovered from sterile body sites. These isolates may be incorrectly identified if current biochemical and morphological criteria are used for identification. Gram stain morphology, however, correctly identifies these isolates to the species level. The characterization of these atypical isolates has important implications for future investigations in which serological methods are used for diagnosis, epidemiology, and determination of pathogenicity of Mobiluncus spp.     

Identification of 22 Legionella species and 33 serogroups with the slide agglutination test.

We used the slide agglutination test to determine the serologic relationships of 22 Legionella spp. representing 33 serogroups. Antisera prepared against 14 of the Legionella spp. contained cross-reactive antibodies (1+ or greater) at their working dilutions. Numerous cross-reactions were observed for the blue-white fluorescing Legionella spp. With only three exceptions in the latter group, cross-reactive antibodies were removed by absorption, thereby producing serogroup-specific antisera. For screening tests or for identification only to the genus level, nine polyvalent antiserum pools were prepared. Routine use of slide agglutination test reagents should expand the number of Legionella spp. that can be identified in the clinical laboratory and, at the same time, provide a simpler, less costly test procedure.     

嗜肺军团菌生物膜的形成与毒力基因表达量分析

目的 初步探讨生物膜相关军团菌独特的毒力基因表达特征.方法 在富营养条件下建立静止状态的单细胞嗜肺军团菌生物膜模型.运用反转录real-time PCR技术,比较和分析浮游和生物膜状态下嗜肺军团菌部分毒力基因的表达,毒力基因包括mip、flaA和fliA基因.结果 对数生长后期与对数生长期浮游菌的mip、flaA和fliA基因表达量比值分别为0.53、4.45、3.67,对数生长期浮游菌显示复制态军团菌的毒力基凶表达特征,对数生长后期表达转移态军团菌特点.生物膜相关军团菌与对数生长期浮游菌的mip、flaA和fliA基因表达量比值为4.42、5.24、16.21;与对数生长后期浮游菌的比值为8.39、1.18、4.43,生物膜相关军团菌同时表达复制态和转移态军团菌毒力基因特征.结论 生物膜相关军团菌毒力基因的表达具有其特殊性,有待进一步研究.

Abstract：

Objective To investigate the physiological state of L. pneumophila in biofilm. Methods Genes previously identified as good markers for the transmissive and replicative phases of the L. pneumophila life cycle during growth in Acanthamoeba castellanii were examined for their expression fold change in the sessile cells as compared to planktonic cells using real-time RT PCR. Results Mature L. pneumophila biofilms were formed at 37t in 75 cm2 cell culture treated flasks for 18 days. The ratio of gene (mip, flaA and fliA) expression in post-exponential cells compared to exponential cells is 0. 53, 4. 45 and 3. 67. The exponential phase cultures display replicative traits and post-exponential bacteria express transmissive factors. The ratio of gene (mip, flaA and fliA) expression in sessile cells compared to exponential cells is 4.42, 5.24 and 16.21, while the sessile cells compared to exponential cells is 8.39, 1. 18 and 4. 43, respectively. Conclusion The violence gene expression of L pneumophila in biofilm is unique.     

Identification of genes differentially expressed in osteoclast-like cells.

Homeostasis of the skeletal system is maintained by a balance between bone formation and resorption. The receptor activator of NF-kappaB ligand (RANKL) induces the differentiation of bone-resorbing cells, osteoclasts. To identify genes regulated during osteoclast differentiation, we constructed a subtraction cDNA library using a mouse RAW264 macrophage cell line that differentiates into osteoclast-like multinucleated cells after treatment with RANKL. Northern blot analysis showed that RANKL treatment upregulated expression of 17 genes. Among these were the genes for five H(+)-ATPase subunits, two chemokines, and the osteoclast marker cathepsin K. In addition, a mouse homolog of human dendritic cell (DC)-specific transmembrane protein (DCSTAMP), whose function in osteoclastogenesis was recently revealed, was also included in the induced genes. Characterization of these inducible genes will provide an insight into the biology of osteoclasts and the mechanism of bone-related diseases.     

Identification of an antigen common to different species of the genus Campylobacter.

Affinity-purified antibody specific for a determinant on flagellin from Campylobacter jejuni was used to screen by immunoblotting strains of C. coli, C. laridis, C. fetus, "C. upsaliensis," C. pylori, and C. sputorum biovar fecalis. The antigen was detected in each of these species, but the molecular weights of the proteins bearing the common antigen varied considerably.     

Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila

Legionella pneumophila is an intracellular bacterium, and its successful parasitism in host cells involves two reciprocal phases: transmission and intracellular replication. In this study, we sought genes that are involved in virulence by screening a genomic DNA library of an L. pneumophila strain, 80-045, with convalescent-phase sera of Legionnaires' disease patients. Three antigens that reacted exclusively with the convalescent-phase sera were isolated. One of them, which shared homology with an integrin analogue of Saccharomyces cerevisiae, was named L. pneumophila adhesion molecule homologous with integrin analogue of S. cerevisiae (LaiA). The laiA gene product was involved in L. pneumophila adhesion to and invasion of the human lung alveolar epithelial cell line A549 during in vitro coculture. However, its presence did not affect multiplication of L. pneumophila within a U937 human macrophage cell line. Furthermore, after intranasal infection of A/J mice, the laiA mutant was eliminated from lungs and caused reduced mortality compared to the wild isolate. Thus, we conclude that the laiA gene encodes a virulence factor that is involved in transmission of L. pneumophila 80-045 and may play a role in Legionnaires' disease in humans.     

Legionella micdadei and Legionella dumoffii monoclonal antibodies for laboratory diagnosis of Legionella infections.

Two different monoclonal antibodies directed against Legionella micdadei and L. dumoffii (Genetic Systems Corp., Seattle, Wash.) were evaluated for their specificity and ability to detect L. micdadei and L. dumoffii in human and animal clinical samples and bacterial isolates in an indirect immunofluorescence assay. All three frozen sputum samples and all three Formalin-fixed sputum and liver samples from patients with culture-documented L. micdadei pneumonia were positive when tested with the L. micdadei monoclonal antibody. A Formalin-preserved lung sample from a patient with culture-documented L. dumoffii pneumonia was positive with its homologous monoclonal antibody. No cross-staining reactions were found with either monoclonal antibody on any of 25 human sputum samples tested from patients without Legionella infections. A total of 66 Legionella strains and 56 non-Legionella strains including 22 Pseudomonas strains and 34 other bacterial strains were studied. No cross-staining reactions were found except in Staphylococcus aureus Cowan 1 ATCC 12598. The lower limit of detection in seeded sputum samples was about 7 X 10(4) cells per ml for both monoclonal antibodies. Lung and tracheal lavage specimens from L. micdadei- or L. dumoffii-infected guinea pigs showed specific staining only with their respective monoclonal antibodies. The monoclonal antibodies stained homologous bacteria slightly less intensely than did the polyclonal antisera, but the signal-to-noise ratio was considerably higher for the monoclonal antibodies. No differences in sensitivity of staining of clinical specimens or bacterial isolates were noted between the monoclonal antibodies and the polyclonal reagents for L. micdadei and L. dumoffii (Centers for Disease Control, Atlanta, Ga., and BioDx, Denville, N.J. These monoclonal antibodies ae sensitive and specific, making them good candidates for laboratory diagnostic purposes.     

Identification of in vivo induced genes in Actinobacillus pleuropneumoniae.

We have developed an in vivo expression technology (IVET) system to identify Actinobacillus pleuropneumoniae gene promoters that are specifically induced in vivo during infection. This system is based upon an avirulent riboflavin-requiring A. pleuropneumoniae mutant and a promoter-trap vector (pTF86) that contains, in sequence, the T4 terminator, a unique Bam HI site, a promoterless copy of the V. harveyi luxAB genes, and a promoterless copy of the B. subtilis ribBAH genes in the E. coli - A. pleuropneumoniae shuttle vector pGZRS19. Sau 3A fragments of A. pleuropneumoniae genomic DNA were cloned into the Bam HI site in pTF86 and transformed into the A. pleuropneumoniae Rib- mutant. Pigs were infected with pools of 300-600 transformants by endobronchial inoculation and surviving bacteria were isolated from the pigs' lungs at 12-16 h post-infection. Infection strongly selected for transformants containing cloned promoters which drove expression of the vector ribBAH genes and allowed survival of the Rib- mutant in vivo. Strains that survived in vivo, but which minimally expressed luciferase activity in vitro, should contain cloned promoters that are specifically induced in vivo. Ten clones, designated iviA-J, were isolated which contain promoters that are induced in vivo during infection. These ivi clones were shown to be induced in the animal by luminescence of infected tissue and by direct assay of bacteria recovered from bronchoalveolar lavage. Four of these clones were putatively identified by amino acid sequence similarity as ilvI, the ilvDA operon, the secE-nusG operon, and the mrp gene. This is the first report of an IVET system for use in the family Pasteurellaceae, as well as the first report of an IVET system utilizing an infection model of pneumonia in the natural host.