Cross-reactive Legionella antigens and the antibody response during infection

In order to define cross-reactive Legionella antigens suitable for diagnostic purposes, we investigated sonicate antigens from two Legionella species, including two serogroups of L. pneumophila. The antigens were reacted with heterologous and homologous rabbit antisera in Western blot. Sera from seven patients with culture-verified L. pneumophila infection and nine patients with serologically confirmed L. micdadei infection were also investigated for reactivity with the corresponding antigens. Among the cross-reactive Legionella antigens defined, non-specific reactivity in patients' sera with the 58-kDa common antigen (CA) was noted. Specific reactions were observed with the Legionella flagellum antigen and with the macrophage infectivity potentiator (Mip) protein; with both antigens, however, the reactive sera were too few to suggest the use of a single antigen in a diagnostic test.     

Cross-reactions between Legionella pneumophila (serogroup 1) and twenty-eight other bacterial species, including other members of the family Legionellaceae.

Cross-reactions between Legionella pneumophila serogroup 1 and 28 other bacterial species were studied by various quantitative immunoelectrophoretic techniques. A sonicated L. pneumophila antigen and purified homologous rabbit antibody were used as a reference system. Few antigens (0 to 6) cross-reacted with non-Legionellaceae, but two were found in nearly all gram-negative bacteria tested (antigens no. 1 and 66). Antigen no. 66 of the L. pneumophila reference system was shown to be antigenically similar to the "common antigen" of Pseudomonas aeruginosa reported in many gram-negative bacteria. Greater than 85% of the antigens from L. pneumophila serogroup 1 cross-reacted with the other six serogroups of L. pneumophila. By contrast, Fluoribacter (Legionella) bozemanae, F. (L.) dumoffii, F. (L.) gormanii, and Tatlockia (Legionella) micdadei cross-reacted with only 45, 53, 39, and 43% of the reference system antigens, respectively. The antigenic relatedness of members of the Legionellaceae, expressed as a matching coefficient, is discussed in terms of its taxonomic significance. Serogroup-, genus-, and family-specific antigens are identified in the L. pneumophila reference system.     

Antigenic analysis of Legionella pneumophila and Tatlockia micdadei (Legionella micdadei) by two-dimensional (crossed) immunoelectrophoresis.

The antigens of the six serogroups of Legionella pneumophila were compared by two-dimensional (crossed) immunoelectrophoresis by using rabbit antisera to serogroups 1, 2, 3 and 4. The close relationship among the serogroups was shown by the fact that 27 of the 31 antigens demonstrated so far were common. However, distinctive group-specific antigens with slow electrophoretic mobility were observed for serogroups 1, 2, 3, and 4. When intact serogroup 1 organisms were extracted with EDTA, the group-specific antigen was recovered in a virtually pure form. The group-specific antigen was pronase resistant, heat stable, and amphiphilic and had a surface location, all of which are properties suggestive of lipopolysaccharide. L. pneumophila shared four to five antigens with Tatlockia micdadei (Legionella micdadei). The large number of common antigens in the serogroups of L. pneumophila has important implications for the specific detection of antigens and antibodies by fluorescent and other tagged antibody methods.     

Agglutinating antibody titers to members of the family Legionellaceae in cystic fibrosis patients as a result of cross-reacting antibodies to Pseudomonas aeruginosa.

The objective of this study was to evaluate the prevalence and significance of antibody titers to organisms in the family Legionellaceae in 128 serum samples collected from cystic fibrosis patients at routine examinations. Antibody titers were determined for 10 antigenic types of Legionellaceae; Legionella pneumophila serogroups 1 to 6, Fluoribacter (Legionella) bozemanae, Fluoribacter (Legionella) dumoffii, Fluoribacter (Legionella) gormanii, and Tatlockia (Legionella) micdadei. The method of antibody titer determination was the microagglutination test. Elevated titers (greater than or equal to 1:64) to one or more antigens were found in 41.3% of cystic fibrosis patients but in only 9.7% of 103 normal control subjects (P less than 0.01). Titers to 8 of the 10 antigens were directly correlated with the number of Pseudomonas aeruginosa precipitating antibodies in patient sera, as determined by crossed immunoelectrophoresis (correlation coefficients, greater than or equal to 0.74). Cross-reactions between P. aeruginosa and L. pneumophila were substantiated by crossed immunoelectrophoresis of hyperimmune rabbit serum as well as patient sera against P. aeruginosa and Legionellaceae antigens. Monospecific antibody to the "common antigen" of P. aeruginosa was used to demonstrate the presence of this antigen in L. pneumophila. The presence of cross-reacting antibodies in cystic fibrosis patients chronically infected with P. aeruginosa emphasizes the need for cautious interpretation of antibody titers to members of the family Legionellaceae.     

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.     

Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed which detected soluble antigens from culture extracts of Legionella pneumophila serogroups 1 to 8, L. micdadei, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. longbeachae serogroups 1 and 2, L. wadsworthii, L. oakridgensis, L. anisa, L. feeleii serogroup 1, and L. jordanis. The assay was approximately 10-fold more sensitive for the eight L. pneumophila serogroups than for the other Legionella species tested. The ELISA detected Legionella antigens in the urine specimens of 25 of 35 patients with L. pneumophila serogroup 1, 3, 4, 6, and 8; L. micdadei; and L. longbeachae serogroup 1 infections. None of the 334 urine specimens from patients with either non-Legionella pneumonia or urinary tract infections was positive. For 10 patients from whom sequential urine specimens were available, Legionella antigens were not detectable from 7 to 19 days after laboratory diagnosis. Test sensitivity was not affected by heavy bacterial contamination. This ELISA offers the detection of a broad spectrum of Legionella antigens by a single test.     

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).     

Genetic, immunological, and cytotoxic comparisons of Legionella proteolytic activities.

Several strains of Legionella pneumophila and other species of Legionella with proteolytic activities were compared by assays, including Southern hybridizations and Western immunoblots, to determine their proteolytic, hemolytic, and cytotoxic activities. Only proteases from strains of L. pneumophila were both hemolytic and cytotoxic, and proteolytic activities extracted from other species of Legionella possessed only hemolytic activity. A 4.0-kilobase DNA sequence encoding the 38-kilodalton metalloprotease from L. pneumophila Philadelphia 1 that we showed previously was responsible for the observed hemolytic and cytotoxic phenotypes (F. D. Quinn and L. S. Tompkins, Mol. Microbiol., 3:797-805, 1989) was used in Southern hybridizations to probe chromosomal DNA from several strains of L. pneumophila and other Legionella species. The probe hybridized to the chromosomal DNA of all serogroups of L. pneumophila but not to any strains of L. dumoffii, L. micdadei, L. feeleii, or L. jordanis that we examined. Additionally, Western immunoblots done with rabbit antisera made to the cloned L. pneumophila protease demonstrated cross-reactions among 38-kilodalton proteins from strains of L. pneumophila, but no reactions were observed with proteins from other species of Legionella. Similarly, the cloned protease from L. pneumophila reacted with convalescent-phase sera from patients infected with L. pneumophila, but not with antisera isolated from patients infected with other Legionella species. Thus, despite some similarities among the proteolytic activities of members of the genus Legionella, including proteolytic and hemolytic phenotypes, metal requirements for zinc or iron, sensitivity to EDTA, and temperature and pH optima, we documented distinct genetic, immunological, and cytotoxicity differences among the proteolytic activities produced by Legionella species.     

Serological cross-reactions between Coxiella burnetii and Legionella micdadei.

Coxiella burnetii and Legionella micdadei are both gram-negative bacteria potentially responsible for identical clinical syndromes resembling upper respiratory infections. These infections, quite common in immunocompromised patients, are usually diagnosed by serology with a microimmunofluorescence assay. We found that 34.5% of Q fever patients had a significant titer of antibodies against L. micdadei. Cross-reactions involved immunoglobulin G antibodies and were demonstrated by a cross-adsorption study and protein immunoblotting. Western blot analysis performed after treatment with proteinase K indicated that cross-reactions were probably due to both protein and lipopolysaccharide antigens. It is critical that the existence of this cross-reaction be recognized, as misdiagnosis of either condition may lead to incorrect and ineffective treatment.     

Comparative Analysis of Legionella pneumophila and Legionella micdadei Virulence Traits

While the majority of Legionnaire's disease has been attributed to Legionella pneumophila, Legionella micdadei can cause a similar infection in immunocompromised people. Consistent with its epidemiological profile, the growth of L. micdadei in cultured macrophages is less robust than that of L. pneumophila. To identify those features of the Legionella spp. which are correlated to efficient growth in macrophages, two approaches were taken. First, a phenotypic analysis compared four clinical isolates of L. micdadei to one well-characterized strain of L. pneumophila. Seven traits previously correlated with the virulence of L. pneumophila were evaluated: infection and replication in cultured macrophages, evasion of phagosome-lysosome fusion, contact-dependent cytotoxicity, sodium sensitivity, osmotic resistance, and conjugal DNA transfer. By nearly every measure, L. micdadei appeared less virulent than L. pneumophila. The surprising exception was L. micdadei 31B, which evaded lysosomes and replicated in macrophages as efficiently as L. pneumophila, despite lacking both contact-dependent cytopathicity and regulated sodium sensitivity. Second, in an attempt to identify virulence factors genetically, an L. pneumophila genomic library was screened for clones which conferred robust intracellular growth on L. micdadei. No such loci were isolated, consistent with the multiple phenotypic differences observed for the two species. Apparently, L. pneumophila and L. micdadei use distinct strategies to colonize alveolar macrophages, causing Legionnaire's disease.     

Detection of cell-associated or soluble antigens of Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, Legionella gormanii, and Legionella micdadei by staphylococcal coagglutination tests.

Current methods used for the detection of whole-cell isolates of Legionella or for the detection of Legionella soluble antigens are technically impractical for many clinical laboratories. The purpose of this study was to explore practical alternatives. The results showed that whole cell isolates of Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, Legionella gormanii, and Legionella micdadei were identified specifically by a simple slide agglutination test or slide coagglutination test in which the reagent antisera are first bound to staphylococcal protein A. Soluble antigens were also identified specifically by the slide coagglutination test and by a sandwich immunofluorescence assay. The latter test may be useful in detecting antigen in body fluids of patients with legionellosis or in environmental samples.     

Legionella micdadei (Pittsburgh pneumonia agent): direct fluoresent-antibody examination of infected human lung tissue and characterization of clinical isolates.

Legionella micdadei (Pittsburgh pneumonia agent) was identified by direct fluorescent-antibody (DFA) examination of lung tissue in six of seven persons diagnosed previously as having L. micdadei pneumonia only by histopathology and in four persons who also had positive cultures of the organism. No cross-reactions occurred with monospecific DFA conjugates prepared against Legionella pneumophila serogroups 1 to 6, Legionella bozemanii, Legionella dumoffii, and Legionella gormanii. One person had L. pneumophila serogroup 6 identified by DFA examination of lung tissue and subsequent culture of stored pulmonary secretions. Characterization of the four strains of L. micdadei revealed specific DFA reactions, bacteriological behavior, and cellular fatty acid composition that allow identification of the organism. DFA testing appears to be a sensitive method for identifying L. micdadei prescent in human lung tissue or cultured on artificial media.     

Role of the alveolar macrophage in host defense and immunity to Legionella micdadei pneumonia in the guinea pig

Guinea pigs develop a lethal pneumonia after intratracheal infection with Legionella micdadei, and the lung displays pathological changes similar to those observed in humans. To investigate the role of the resident alveolar macrophage in the pathogenesis of L. micdadei pneumonia, guinea pig alveolar macrophages obtained by bronchoalveolar lavage were cultured in vitro and infected with L. micdadei. In the absence of opsonins L. micdadei was phagocytized by, and multiplied within, alveolar macrophages with greater than a 100-fold increase in cell-associated colony forming units over 20 h. L. micdadei opsonized with complement or antibody multiplied within alveolar macrophages at the same rate as unopsonized bacteria. Guinea pigs which were treated with antimicrobials after infection with L. micdadei and recovered from the pneumonia were immune to challenge with an otherwise lethal inoculum of L. micdadei. However, the growth curve of both unopsonized and opsonized L. micdadei in the alveolar macrophages from immune animals was essentially identical to that in macrophages from susceptible animals. Thus, the resident alveolar macrophage is not capable of limiting the growth of Legionella. Rather, the alveolar macrophages appear to be the primary site of Legionella multiplication within the lung. Although alveolar macrophages may participate in other aspects of pulmonary immunity to the legionellae, these data indicate that the alveolar macrophage alone does not act as an effector cell in cell-mediated immunity to Legionella.     

Abscess and empyema caused by Legionella micdadei.

Legionella micdadei is the second most common species implicated in the occurrence of Legionella pneumonia (D. J. Bremer, Semin. Respir. Infect. 4:190-205, 1987). Although there has been a reported lung abscess caused by dual infection (L. micdadei and L. pneumophila), there are no known cases of L. micdadei as the only causative organism. We report a case of a patient with a lung abscess from which L. micdadei was the sole organism isolated.     

Monoclonal antibodies to Legionella Mip proteins recognize genus- and species-specific epitopes.

Monoclonal antibodies (MAbs) against the virulence-associated Mip protein of Legionella spp. were raised by immunizing BALB/c mice with (i) Legionella pneumophila, (ii) Legionella micdadei, and (iii) purified recombinant native Mip protein cloned from L. pneumophila Philadelphia 1. Following screening of seeded wells by immunoblot analysis with homologous antigens, eight Mip-specific MAbs were found. These MAbs were chosen to investigate the antigenic diversity of Mip proteins in the genus Legionella. Mip was detected in 82 Legionella strains representing all 34 species tested. One of these MAbs, obtained from immunization with L. micdadei, recognized an epitope common to all Legionella species tested by immunoblot analysis. Another MAb was discovered to be specific for the Mip protein of L. pneumophila. The remaining six MAbs recognized 18 to 79% of Legionella species included in this study. By making use of the MAbs introduced in this study, it could be shown that, based on Mip protein epitope expression, Legionella species can be divided into at least six antigenetically distinct groups. As demonstrated by 43 L. pneumophila strains representing all serogroups, no antigenic diversity of Mip proteins was found for this species. In addition, 18 non-Legionella species, including Chlamydia trachomatis, Neisseria meningitidis, Pseudomonas aeruginosa, and Saccharomyces cerevisiae, all of which are known to carry genes homologous to the Legionella mip genes, were reacted against all eight MAbs. No cross-reactivity was detectable in any of those strains.     

A specific genomic location within the icm/dot pathogenesis region of different Legionella species encodes functionally similar but nonhomologous virulence proteins

Legionella pneumophila, the major causative agent of Legionnaires' disease, is a facultative intracellular pathogen that grows within human macrophages and amoebae. Intracellular growth involves the formation of a replicative phagosome that requires the Icm/Dot type IV secretion system. Part of the icm/dot region in L. pneumophila contains the icmTSRQPO genes. The proteins encoded by the icmR and icmQ genes were shown to exhibit a chaperone-substrate relationship. Analysis of this region from other pathogenic Legionella species, i.e., L. micdadei and L. longbeachae, indicated that the overall organization of this region is highly conserved, but it was found to contain a favorable site for gene variation. In the place where the icmR gene was expected to be located, other open reading frames that are nonhomologous to each other or to any entry in the GenBank database were found (migAB in L. micdadei and ligB in L. longbeachae). Examination of these unique genes revealed an outstanding phenomenon; by use of interspecies complementation, the icmR, migB, and ligB gene products were found to be functionally similar. In addition, the function of these proteins was usually dependent on the presence of the corresponding IcmQ proteins. Moreover, each of these proteins (IcmR, LigB, and MigB) was found to interact with the corresponding IcmQ proteins, and the genes encoding these proteins were found to be regulated by CpxR. This study reveals new evidence of gene variation occurring in the same genomic location within the icm/dot locus in various Legionella species. The genes found at this site were shown to be similarly regulated and to encode species-specific, nonhomologous, but functionally similar proteins.     

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.     

Escherichia coli CS31A fimbriae: molecular cloning, expression and homology with the K88 determinant

CS31A is a plasmid-encoded K88-related fimbrial antigen. A Sau3AI library was constructed from p31A, a 180 kb CS31A encoding plasmid, in the pSUP202 vector. Bacterial recombinant clones expressing CS31A were isolated. A 8.5 kb EcoRI-HinIII DNA fragment from one of them was subcloned in pBR322 and pHSG575 vectors, leading to pAG315 and pEH524 recombinant plasmids respectively. Escherichia coli harboring pAG315 or pEH524 expressed CS31A fimbrial antigens on their cell surface. Analysis of these plasmids in minicells showed that at least seven mature polypeptides were encoded by the EcoRI-HindIII DNA fragment, with apparent molecular masses of 76,000, 54,000, 30,000, 29,000, 28,000, 15,500 and 13,500 daltons respectively. The genetic organization of the CS31A gene cluster was determined and showed to be similar to that of the K88 operon. The nucleotide sequence homology between CS31A and K88 determinants was investigated by Southern blot hybridization at high stringency. This indicated that extensive nucleotide sequence homology exists throughout both gene clusters except for the subunit structural genes.     

Crossed immunoelectrophoretic analysis of Legionella pneumophila serogroup 1 antigens.

By crossed immunoelectrophoresis, 85 different antigens were demonstrated in sonicated preparations of Legionella pneumophila serogroup 1 (Lp1). The precipitin patterns of 82 anodic-migrating antigens were numbered and were designated the Lp1 reference system. Eleven antigens were stable to boiling, and seven of these were shown to be surface antigens. One heat-stable surface antigen (antigen no. 61) was highly reactive with limulus amoebocyte lysates and formed a precipitin resembling lipopolysaccharide. Serum from an isolation confirmed case of Lp1 infection and serogroup-specific rabbit antiserum reacted specifically with antigen no. 61, which was designated the serogroup-specific antigen. Normal human and rabbit sera commonly had antibodies to antigen no. 66 of the Lp1 reference system. This antigen is antigenically related to the "common antigen" of Pseudomonas aeruginosa.     

Community-acquired lung abscess caused by Legionella micdadei in a myeloma patient receiving thalidomide treatment

Legionella infection causes 2 to 14% of community-acquired pneumonia (CAP). Legionella micdadei constitutes <1% of these infections. We describe a case of cavitary L. micdadei CAP in a myeloma patient receiving thalidomide treatment. The importance of considering pneumonia and problems in diagnosing pneumonia caused by L. micdadei in this patient population are reviewed.