Phenotype versus genotype of the 19 kD peptido-glycan associated protein of Legionella (PpIA), among Legionellae and other gram-negative bacteria.

The protein PpIA (19 kD) cloned from a genomic library of Legionella pneumophila, Philadelphia 1, represents a peptido-glycan associated outer membrane protein in recombinant E. coli K-12 and L. pneumophila. It exhibits distinct sequence homology to lipoproteins of Haemophilus influenzae and E. coli. A ppIA specific DNA probe generated by PCR was used in Southern hybridizations of chromosomal DNA of Legionella strains and other Gram-negative pathogens. Under conditions of high stringency, hybridization could only be observed in L. pneumophila isolates, but all other Legionella strains tested displayed hybridization under lower stringency. No signals appeared after hybridization of chromosomal DNA from a variety of other bacteria. Using anti-PpIA monospecific polyclonal antibodies in Western blots, it was demonstrated that PpIA related proteins of nearly the same size are found in all L. pneumophila isolates and in a variety of, but not all, the Legionella species analysed here.     

Distribution, expression, and long-range mapping of legiolysin gene (lly)-specific DNA sequences in legionellae.

The legiolysin gene (lly) cloned from Legionella pneumophila Philadelphia 1 confers the phenotypes of hemolysis and browning of the culture medium. An internal lly-specific DNA probe was used in Southern hybridizations for the detection of lly-specific DNA in the genomes of legionellae and other gram-negative pathogenic bacteria. Under conditions of high stringency, the lly DNA probe specifically reacted with DNA fragments from L. pneumophila isolates; by reducing stringency, hybridization was also observed for all other Legionella strains tested. No hybridization occurred with DNAs isolated from bacteria of other genera. The lly gene was mapped by pulsed-field gel electrophoresis to the respective genomic NotI fragments of Legionella isolates. By using antilegiolysin monospecific polyclonal antibodies in Western blots (immunoblots), Lly proteins could be detected only in L. pneumophila isolates.     

Identification of mip-like genes in the genus Legionella.

The mip gene of Legionella pneumophila serogroup 1 strain AA100 encodes a 24-kilodalton surface protein (Mip) and enhances the abilities of L. pneumophila to parasitize human macrophages and to cause pneumonia in experimental animals. To determine whether this virulence factor is conserved in the genus Legionella, a large panel of Legionella strains was examined by Southern hybridization and immunoblot analyses for the presence and expression of mip-related sequences. Strains representing all 14 serogroups of L. pneumophila contained a mip gene and expressed a 24-kilodalton Mip protein. Although the isolates of the 29 other Legionella species did not hybridize with mip DNA probes under high-stringency conditions, they did so at reduced stringency. In support of the notion that these strains possess mip-like genes, these species each expressed a protein (24 to 31 kilodaltons in size) that reacted with specific Mip antisera. Moreover, the cloned mip analog from Legionella micdadei encoded the cross-reactive protein. Thus, mip is conserved and specific to L. pneumophila, but mip-like genes are present throughout the genus, perhaps potentiating the intracellular infectivity of all Legionella species.     

Characterization of a Legionella pneumophila extracellular protease exhibiting hemolytic and cytotoxic activities.

A preliminary screening of selected Legionella species for proteolytic and hemolytic phenotypes suggested a correlation between these activities. To investigate the relationship of these phenotypes, a mutant strain of Legionella pneumophila deficient in the expression of a 38-kilodalton (kDa) exoprotease was isolated and characterized. This strain, designated PRT8, was also found to be nonhemolytic when screened on blood agar composed of 5% canine or guinea pig erythrocytes. Strain PRT8 was serologically and biochemically identical to the parental strain with the exception of the expression of the exoprotease. Immunoblot analysis of concentrated culture filtrates from PRT8 probed with polyclonal anti-38-kDa exoprotease serum revealed no cross-reactive peptides. To resolve the role of the exoprotease in the hemolytic phenotype, the exoprotease was purified from the culture supernatant of the parental strain by a combination of ion-exchange and hydrophobic interaction chromatography steps. The hemolytic activity was found to copurify with the proteolytic activity, and analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot revealed a single protein species exhibiting an apparent molecular mass of 38 kDa. Finally, the purified exoprotease and concentrated culture supernatant from the parental strain, but not from PRT8, exhibited cytotoxicity (minimum cytotoxic activity of 0.17 U of protease activity) in a Chinese hamster ovary cell assay. These data suggest that the exoprotease is responsible for the hemolytic and cytotoxic phenotypes described for this species and therefore may be one of several determinants associated with virulence.     

Immunologic response of patients with legionellosis against major protein-containing antigens of Legionella pneumophila serogroup 1 as shown by immunoblot analysis.

Major protein-containing antigens of Legionella pneumophila serogroup 1 were were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis with rabbit antisera to 14 different Legionella species or serogroups. Fourteen bands were observed in immunoelectropherograms of whole-cell, sonicated cell, and heated cell preparations, seven of which appeared in the supernatant fluid from the heated cells and three of which were shown in an outer membrane fraction. Immunoblots of whole-cell antigen preparations of 14 Legionella species or serogroups revealed seven major Legionella proteins: antigens with molecular weights of 58,000, 79,000, and 154,000 were present in all Legionella sp. strains, antigens with molecular weights of 44,000 and 97,000 occurred in multiple species, and antigens with molecular weights of 14,000 and 25,000 were present only in L. pneumophila strains. All sera from 15 patients with culture-confirmed L. pneumophila serogroup 1 disease and 14 of 18 (78%) sera from serologically diagnosed patients reacted with the 58-kilodalton (kDa) common antigen. In contrast, less than one-half of the sera reacted with the L. pneumophila-specific proteins (14 and 25 kDa). Absorption of sera with Escherichia coli cells had no effect on their reactivity with the 58-kDa antigen, whereas absorption with L. pneumophila serogroup 1 cells removed reactivity. These data suggest that the 58-kDa antigen may prove useful in serodiagnostic tests for legionellosis.     

Rapid identification of Legionella pneumophila zinc metalloprotease using chromogenic detection

Strains of Legionella spp. produce extracellular proteases than can be detected using synthetic chromogenic peptides. Chromogenic tri- and tetrapeptides show a high degree of sensitivity, specificity and reagent stability when linked to para-nitroaniline (pNA). For example, SucOMe-Arg-Pro-Tyr.pNa (S-2586) is specifically hydrolysed by proteases of Legionella pneumophila and some other Legionella species. A paper disc method to sample protease directly from agar plates has been used to evaluate chromogenic peptides as reagents for diagnostic purposes. Strains of Legionella spp., Pseudomonas spp. and Enterobacteriaceae were examined, together with a recombinant Escherichia coli strain containing the cloned 38 kDa zinc metalloprotease from L. pneumophila, S-2586 was hydrolysed by 282 out of 283 L. pneumophila strains, and by the recombinant E. coli. Two of the six strains representing other Legionella species, and 22 of the 50 strains from the Pseudomonas group were also positive. No reaction was seen with any of the Enterobacteriaceae strains. Although there was functional homology between proteases from several bacterial groups, the high prevalence of S 2586-hydrolysing proteases within L. pneumophila indicates a potential usefulness for phenotypic identification.     

Cloning and temperature-dependent expression in Escherichia coli of a Legionella pneumophila gene coding for a genus-common 60-kilodalton antigen.

All Legionella species express a 60-kilodalton (kDa) protein which contains a genus-specific epitope recognized by murine monoclonal antibody GW2X4B8B2H6. A genomic cosmid library of Legionella pneumophila chromosomal DNA was constructed in pHC79 and screened for 60-kDa antigen-expressing clones with the monoclonal antibody. A 3.2-kilobase EcoRI fragment from cosmid 14B11 expressing a 60-kDa protein was subcloned into pUC19 (pSH16), and deletion of a 1.2-kilobase HindIII fragment (pSH16A) generated a 33-kDa truncated polypeptide no longer reactive with the monoclonal antibody. Southern blot analysis of chromosomal DNA from selected Legionella species restricted with EcoRI and probed with the 1.2-kilobase fragment coding for the carboxyl region of the protein revealed DNA homology which was not observed with DNA from Escherichia coli. Maxicell analysis of pSH16 identified a second polypeptide of approximately 15 kDa expressed from a gene (htpA) upstream of the gene coding the 60-kDa protein (htpB). Both proteins were preferentially synthesized by L. pneumophila following heat shock (temperature shift from 25 to 42 degrees C), and under steady-state growth conditions the relative level of 60-kDa protein was unaffected by temperature. In E. coli, expression of a 60-kDa protein from pSH16 also increased following heat shock (25 to 42 degrees C), but under steady-state conditions expression was temperature dependent. Temperature-dependent expression from pSH16 was not observed in an rpoH (htpR) mutant strain of E. coli. The Legionella 60-kDa protein appears to be a heat shock protein which shares cross-reactive epitopes with the GroEL homolog of E. coli. In addition, a region of htpB encoding the 27-kDa carboxyl portion of the protein containing the monoclonal antibody-reactive epitope also contains DNA sequences unique to and conserved within the genus.     

Evaluation of the Gen-Probe DNA probe for the detection of legionellae in culture.

A commercial DNA probe kit designed to detect rRNA from legionellae was evaluated for its ability to correctly discriminate between legionellae and non-legionellae taken from culture plates. The probe kit, made by the Gen-Probe Corp. (San Diego, Calif.), was radiolabeled with 125I, and probe bacterial RNA hybridization, detected in a simple one-tube system hybridization assay, was quantitated with a gamma counter. A total of 156 Legionella sp. strains were tested, of which 125 were Legionella pneumophila and the remainder were strains from 21 other Legionella spp. A total of 106 gram-negative non-legionellae, isolated from human respiratory tract (81%) and other body site (19%) specimens, were also tested; 14 genera and 28 species were represented. The probe easily distinguished all of the legionellae from the non-legionellae. The average legionellae/non-legionellae hybridization ratio was 42:1, and the lowest ratio was 2:1; a minor modification in the procedure increased the lowest ratio to 5:1. In addition to correctly identifying all Legionella species, the probe was able to separate some of the various species of Legionella. L. pneumophila strains hybridized more completely to the probe than did the other Legionella spp.; L. wadsworthii and L. oakridgensis hybridized only about 25% of the probe relative to L. pneumophila. Some strains of phenotypically identified L. pneumophila had much lower hybridization to the probe than other members of the species and may represent a new Legionella species. The simplicity of the technique and specificity of the probe make it a good candidate for confirming the identity of legionellae in culture.     

Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein.

After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L. pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern. Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L. micdadei has extensive homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).     

Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody.

Serogroup-specificity of Legionella pneumophila is related to lipopolysaccharide (LPS), and few cross-reactions between serogroups have been observed with rabbit or monkey antisera. C57BL/6 mice were sequentially immunized with crude outer membrane fractions of L. pneumophila serogroups 1, 5, and 7, Legionella bozemanii, and Legionella micdadei. Spleen cells from these mice were then fused with the Sp2-0/Ag14 mouse myeloma cell line. Outer membrane-rich fractions and LPS were prepared from L. pneumophila serogroups 1 to 8 and other Legionella and non-Legionella species. Immunoblots of these extracts were performed with monoclonal antibody obtained from these fusions. One of these monoclonal antibodies recognized an epitope common to all tested serogroups of L. pneumophila and attached to the major constituent of the outer membrane, LPS. This antibody did not react with other Legionella species and numerous gram-negative rods other than Pseudomonas fluorescens CDC93. This monoclonal antibody may be useful in preliminary identification of L. pneumophila as an alternative to direct fluorescent-antibody testing.     

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).     

DNA probe specific for Legionella pneumophila.

A procedure for preparing a DNA probe to be used in the specific detection of Legionella pneumophila by dot or colony hybridization has been devised. When total DNA from L. pneumophila was used as a radioactive probe, cross-hybridization occurred with DNA from many other species belonging to various families (including Legionellaceae, Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae). Cross-hybridizing restriction fragments in L. pneumophila ATCC 33152 DNA were identified on Southern blots. When unlabeled DNA from strain ATCC 33152 was cleaved by endonuclease BamHI, the DNA fragments cross-hybridizing with the labeled DNA from all of the other species and genera tested (or with Escherichia coli 16 + 23 S RNA) had a size of 21.4 and 16.2 kilobase pairs (major bands) and 28.0, 12.8, and 10.1 kilobase pairs (minor bands). BamHI restriction fragments of L. pneumophila DNA deprived of the cross-hybridizing fragments were pooled and used as a probe for the detection of L. pneumophila. This probe proved to be specific for L. pneumophila in colony and dot hybridization. It can potentially be used for the detection of L. pneumophila in clinical and water samples. The procedure described can be readily applied to the preparation of probes specific for phylogenetically isolated bacterial species other than L. pneumophila.     

Chromosomal DNA, iron-transport systems, outer membrane proteins, and enterotoxin (heat labile) production in Salmonella typhi strains.

We examined a representative collection of Salmonella typhi strains from Chile, Peru, Mexico, India, and England for the presence of several properties. All strains had a conserved pattern of outer membrane proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The electrophoresis profiles of chromosomal DNA digested with EcoRI and PstI restriction enzymes were similar for all the strains. A conserved pattern of hybridization was observed when digested chromosomal DNA was hybridized with DNA probes for the 36-kilodalton porin, enterobactin synthesis, and enterobactin receptor genes. All the strains produced enterobactin but not aerobactin in bioassays. None of the strains produced heat-labile toxin, as measured by an enzyme-linked immunosorbent assay. Colony and Southern hybridizations with DNA probes for aerobactin synthesis and its receptor and heat-labile toxin genes were negative. These results indicate that S. typhi strains from different origins have similar phenotypic and genetic properties and, as has been suggested, constitute a clone.     

Cross-reactions in Legionella antisera with Bordetella pertussis strains.

While preparing slide agglutination test antisera and immunofluorescence conjugates for the identification of Legionella species and serogroups, we found that several of the reagents cross-reacted with Bordetella pertussis strains. To determine the extent of this problem and to estimate the specificity of Legionella reagents, we tested slide agglutination test antisera against 22 species and 35 serogroups with 92 bacterial strains representing 19 genera. The only cross-reactions observed were with Legionella pneumophila serogroup 10, L. maceachernii, L. gormanii, and L. feeleii serogroup 1 antisera and 4 of 10 B. pertussis strains. Nineteen conjugates, previously available from the Centers for Disease Control but no longer distributed as reference reagents, were tested with the four cross-reactive B. pertussis strains. Two conjugates, L. micdadei and L. wadsworthii, stained three of the B. pertussis strains at a fluorescence intensity of greater than or equal to 3+. All cross-reactions were removed from the antisera and conjugates by absorption with the cross-reacting strain without diminishing the homologous reaction. Special emphasis should be placed on the identification and removal of cross-reactions in Legionella reagents with strains that have similar morphologic and growth characteristics.     

Spectrum of Legionella species whose intracellular multiplication in murine macrophages is genetically controlled by Lgn1.

We examined the intracellular growth of 20 strains within six species of Legionella in thioglycolate-elicited peritoneal macrophages from A/J and C57BL/6 mice and a congenic strain derived from them (A.B Lgn1). With the exception of Legionella pneumophila Togus-1 and Bloomington-2, the intracellular growth of the 15 L. pneumophila strains tested was controlled by Lgn1. However, the intracellular growth of five Legionella species other than L. pneumophila was not under Lgn1's control.     

Molecular fingerprinting of Legionella species by repetitive element PCR.

Repetitive element PCR (rep-PCR) uses outward-facing primers to amplify multiple segments of DNA located between conserved repeated sequences interspersed along the bacterial chromosome. Polymorphisms of rep-PCR amplification products can serve as strain-specific molecular fingerprints. Primers directed at the repetitive extragenic palindromic element were used to characterize isolates of Legionella pneumophila and other Legionella species. Substantial variation was seen among the rep-PCR fingerprints of different Legionella species and serogroups. More limited, but distinct, polymorphisms of the rep-PCR fingerprint were evident among epidemiologically unrelated isolates of L. pneumophila serogroup 1. Previously characterized Legionella isolates from nosocomial outbreaks were correctly clustered by this method. These results suggest the presence of repetitive extragenic palindromic-like elements within the genomes of members of the family Legionellaceae that can be used to discriminate between strains within a serogroup of L. pneumophila and between different Legionella species. rep-PCR appears to be a useful technique for the molecular fingerprinting of Legionella species.     

Characterization of a tandem repeat polymorphism in Legionella pneumophila and its use for genotyping

We have analyzed the variability of minisatellite sequences (also called variable-number tandem repeats [VNTRs]) in the genome of Legionella pneumophila. Based upon the genome sequence of the Philadelphia-1 strain (serogroup 1), 25 minisatellites were selected and their polymorphisms were analyzed by PCR with the DNA of serogroup 1 to 14 reference strains. For 22 markers, a PCR product of the expected size was found with the DNA of the Philadelphia-1 strain. Most of these markers did not amplify the DNA of other Legionella species or other bacteria used as controls. A polymorphism was observed for seven markers among the L. pneumophila strains tested. To check whether these markers could be used to compare strains of L. pneumophila, we analyzed two groups of isolates from clinical and environmental samples which had been independently genotyped by other methods. The results showed that, for the isolates in these two sets of samples, VNTR typing is as informative as pulsed-field gel electrophoresis for comparison of strains. Sequencing of one minisatellite from 14 reference strains was performed. Comparison of the sequences allowed a classification and confirmed the existence of subspecies of L. pneumophila. We also tested the usefulness of one very polymorphic marker as a tool for the rapid screening of colonies grown from water samples. This allowed the rapid identification of the L. pneumophila colonies and gave a first hint as to the presence of several strains in a single sample.     

Molecular analysis of hemolytic and phospholipase C activities of Pseudomonas cepacia.

By using a gene-specific fragment from the hemolytic phospholipase C (PLC) gene of Pseudomonas aeruginosa as a probe and data from Southern hybridizations under reduced stringency conditions, we cloned a 4.2-kb restriction fragment from a beta-hemolytic Pseudomonas cepacia strain which expressed hemolytic and PLC activities in Escherichia coli under the control of the lac promoter. It was found, by using a T7 phage promoter-directed expression system, that this DNA fragment carries at least two genes. One gene which shares significant DNA homology with both PLC genes from P. aeruginosa encodes a 72-kDa protein, while the other gene encodes a 22-kDa protein. When both genes on the 4.2-kb fragment were expressed from the T7 promoter in the same cell, hemolytic and PLC activities could be detected in the cell lysate. In contrast, when each individual gene was expressed in different cells or when lysates containing the translated products of each separate gene were mixed, neither hemolytic activity nor PLC activity could be detected. Clinical and environmental isolates of P. cepacia were examined for beta-hemolytic activity, PLC activity, sphingomyelinase activity, and reactivity in Southern hybridizations with a probe from P. cepacia which is specific for the larger gene which encodes the 72-kDa protein. There were considerable differences in the ability of the different strains to express hemolytic and PLC activities, and the results of Southern DNA-DNA hybridizations of the genomic DNAs of these strains revealed considerable differences in the probe-reactive fragments between high- and medium-stringency conditions as well as remarkable variation in size and number of probe-reactive fragments among different strains. Analysis of the genomic DNAs from hemolytic and nonhemolytic variants of an individual strain (PC-69) by agarose gel electrophoresis. Southern hybridization, and transverse alternating pulsed field gel electrophoresis suggests that the conversion of the hemolytic phenotype to the nonhemolytic phenotype is associated with either the loss of a large plasmid (greater than 200 kb) or a large deletion of the chromosome of P. cepacia PC-69.