Interaction of Legionella micdadei with human monocytes.

We have recently shown that Legionella micdadei is ingested, but not killed, by human neutrophils. Herein we investigate the role of human monocytes in defense against this organism. Serum and monocytes from normal donors having no detectable antibody to L. micdadei were used. Egg-passaged L. micdadei organisms multiplied inside these monocytes with a peak growth of 2 log units within 12 h. No growth occurred when monocytes were omitted or when sonicated monocytes were used. Electron microscopy 18 h after infection revealed these organisms to be intracellular in normal-appearing phagosomes. When the input multiplicity of L. micdadei was greater than 1 CFU per monocyte, no intracellular growth occurred. When egg-passaged Legionella pneumophila organisms were used, intracellular organisms were found in phagosomes studded with ribosomes at the same time period. Peak intracellular growth of L. pneumophilia occurred by 48 h. L. micdadei activated the complement system and was opsonized by C3. However the use of complement-depleted (heat-inactivated) serum as the opsonic source had no effect on the bacterium's ingestion or growth in the monocyte. Thus, L. micdadei multiples in human monocytes. This entry and growth is independent of antibody or complement. The intracellular locations of L. micdadei and L. pneumophila differ, suggesting different mechanisms for the survival of these two organisms in the monocyte.     

An unusual strain of Legionella micdadei.

A microorganism antigenically identified as Legionella micdadei but showing a cellular fatty acid profile distinct from that described previously for this species and more similar to that of L. bozemanii has been studied by phenotypic characterization, crossed immunoelectrophoresis, gas-liquid chromatography, and transmission electron microscopy. Although the phenotypic characters, the crossed immunoelectrophoresis, and the ultrastructural features were similar to those of L. micdadei, the fatty acid composition differed significantly from this species; moreover it differed also from L. bozemanii, even though it was quantitatively more similar.     

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.     

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.     

Effect of Legionella pneumophila cytotoxic protease on human neutrophil and monocyte function.

The extracellular metalloprotease of Legionella pneumophila, also called tissue-destructive protease or major secretory protein, has been proposed as one of the virulence factors of this organism. Considering the decisive role played by the phagocytic cells in host defense against Legionella infection, we investigated the effect of this protease on the function of human neutrophils and monocytes. L. pneumophila protease inhibited the chemotactic response of neutrophils to F-Met-Leu-Phe and zymosan-activated serum in a concentration-dependent and heat-labile manner. A direct effect of the protease on the chemotactic activity of neutrophils was demonstrated by the continued inhibition of neutrophil chemotaxis when the protease was removed following pre-incubation of the cells. In contrast, the enzyme had no effect on monocyte chemotaxis. The protease inhibited, also in a concentration-dependent and heat-labile manner, the binding of F-Met-Leu-Phe to both cell types. Neutrophil and monocyte oxidative burst response, as measured by superoxide release and chemiluminescence response, was not significantly affected by the enzyme. A slight enhancement of PMA-stimulated superoxide release was induced by the protease in both cell types. Lastly, the protease inhibited the killing of Listeria monocytogenes by neutrophils or monocytes. Inhibition of Listeria killing was concentration-dependent, heat-labile, and did not require the presence of the enzyme in the bactericidal assay. The inhibitory activity of L. pneumophila protease on neutrophil chemotaxis and on the listericidal activity of human neutrophils and monocytes demonstrated in this study provides evidence for a role of this enzyme in the pathogenesis of Legionnaires' disease.     

Characterization of a Legionella micdadei mip mutant.

The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human macrophages and freshwater protozoa. Southern hybridization and immunoblot analyses demonstrated that mip sequences were present and expressed within a panel of virulent L. micdadei strains. Using allelic exchange mutagenesis, we then constructed an L. micdadei strain that completely and specifically lacked Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip- organism displayed a 24-fold reduction in survivability immediately after its entry into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei.     

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.     

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.     

Legionella micdadei lung abscess in a patient with HIV-associated nephropathy.

A patient with end-stage renal disease due to human immunodeficiency-associated nephropathy developed fever, cough and chest pain over a week's duration. He was diagnosed with lung abscess and started on antibiotic coverage. He underwent bronchoscopy because of progression of his illness and persistent fever and bronchoalveolar lavage culture isolated Legionella micdadei. In spite of appropriate antibiotic therapy, the patient remained febrile for 10 days, necessitating chest tube drainage. After a 6-week course of antibiotics and drainage, the patient made an uneventful recovery. Infections due to L. micdadei may be hard to diagnose because of difficulties in isolating this bacteria.     

Acid-fast bacilli in sputum: a case of Legionella micdadei pneumonia.

Legionella micdadei has been implicated as a cause of nosocomial pneumonia. There are no reports of L. micdadei pneumonia diagnosed by acid-fast stain of expectorated sputum. We report a case of L. micdadei pneumonia in which expectorated sputum harbored acid-fast bacteria that reacted specifically with fluorescent antiserum to L. micdadei, confirmed by culture. In a patient at risk for nosocomial infection, the differential diagnosis of a positive sputum stain for acid-fast bacilli should include L. micdadei in addition to mycobacteria. Therapy for L. micdadei infection should be considered pending confirmation of the diagnosis.     

Increased recovery of Legionella micdadei and Legionella bozemanii on buffered charcoal yeast extract agar supplemented with albumin.

The recovery of Legionella micdadei and L. bozemanii serogroups 1 and 2 from infected guinea pig spleens was evaluated by using two culture media: buffered charcoal yeast extract agar with 0.1% alpha-ketoglutarate (BCYE alpha) and the same medium supplemented with 1% bovine serum albumin (ABCYE alpha). At the lowest dilution of spleen tissue (10(-1)), recovery of all strains of L. micdadei and L. bozemanii was more efficient on ABCYE alpha than on BCYE alpha. L. micdadei strains had higher recovery rates on ABCYE alpha after another 10-fold dilution, but recoveries of L. bozemanii were similar on both media. Recovery rates for most test strains were comparable on BCYE alpha and ABCYE alpha at the highest dilution (10(-3)) of tissue tested. The presence of albumin in BCYE alpha increased the recovery rate of L. micdadei more than that of L. bozemanii. The use of ABCYE alpha medium in place of BCYE alpha may improve the recovery of L. micdadei and L. bozemanii from clinical specimens. Preliminary studies indicate that this medium also enhances recovery of certain Legionella spp. from environmental samples.     

Heterogeneity in intracellular replication and cytopathogenicity of Legionella pneumophila and Legionella micdadei in mammalian and protozoan cells.

In contrast to Legionella pneumophila, little is known about the pathogenesis of other legionellae species that are capable of causing Legionnaires' disease. In this report, we contrast L. pneumophila and L. micdadei for their cytopathogenicity and intracellular replication within mammalian and protozoan cells. We show by transmission electron microscopy that L. micdadei replicates within an endoplasmic reticulum (RER)-free phagosome within human macrophages, alveolar epithelial cells, and within the protozoan Hartmannella vermiformis. In contrast, L. pneumophila replicates within a RER-surrounded phagosome within the same host cells. In contrast to replication of L. pneumophila within Acanthamoebae polyphaga, L. micdadei does not replicate within this protozoan host. Despite the prolific intracellular replication, L. micdadei is less cytopathogenic to all host cells than L. pneumophila. Since both species replicate intracellularly to a similar level, we have examined whether the reduced cytopathogenicity of L. micdadei is due to a reduced capacity to induce apoptosis or pore formation-mediated necrosis, both of which contribute to killing of the host cell by L. pneumophila. The data show that both species induced apoptosis-mediated killing of mammalian cells to a similar level. In contrast to L. pneumophila, expression of the pore-forming toxin by L. micdadei and its necrotic effect on macrophages and alveolar epithelial cells is undetectable. This has been further confirmed showing that L. micdadei is completely defective in contact-dependent haemolysis of RBCs, an activity mediated by the pore-forming toxin. Finally, in contrast to L. pneumophila, there was no significant intrapulmonary replication of L. micdadei in the A/J mice animal model. Our data show dramatic differences between L. pneumophila and L. micdadei in intracellular replication, cytopathogenicity, and infectivity to mammalian and protozoan cells.     

Opsonic requirements for phagocytosis of Legionella micdadei by polymorphonuclear leukocytes

The roles of the classical and alternative pathways of complement activation and of antibody in the phagocytosis of Legionella micdadei by polymorphonuclear leukocytes were studied. Normal serum was treated with the appropriate chelators or with heat to inactivate the classical, alternate, or both pathways of complement activation. Normal and complement-depleted sera with or without antibody were employed as opsonins for L. micdadei in phagocytosis assays. There was no difference in the phagocytosis of L. micdadei promoted by normal serum and either C4-deficient serum or serum in which the classical pathway had been inactivated. Both normal and classical pathway-deficient sera promoted significantly greater phagocytosis than did sera in which the alternate pathway or both the alternate and classical pathways had been inactivated. Thus, polymorphonuclear leukocyte phagocytosis of L. micdadei in the absence of antibody required an intact alternate pathway. Specific antibody partially restored opsonization to sera deficient in the alternate or both complement pathways, but phagocytosis was still significantly less than that with the alternate pathway intact.     

Effects of trifluoperazine on human neutrophil function.

The interaction of cytochalasin B-treated human neutrophils with the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) results in a time- and concentration-dependent generation of superoxide anion (O2-) by an extracellular release of granule-associated beta-glucuronidase and lysozyme from these cells. Granule exocytosis was not accompanied by significant cytoplasmic lactate dehydrogenase extrusion. FMLP-stimulated O2- production occurs but is significantly curtailed in the absence of extracellular calcium. Nevertheless, incubation of neutrophils with EGTA in calcium-free medium had no effect on the O2- -generating system. Trifluoperazine (TFP), an inhibitor of calmodulin (a calcium-binding protein), caused a dose-related inhibition of FMLP-elicited degranulation and O2- production in the presence of absence of extracellular calcium. This effect TFP could be reversed by washing the cells before contact with FMLP. These data suggest that TFP represents a useful tool for defining the relevance of calmodulin and calcium to neutrophil function.     

Reevaluation of the cellular fatty acid composition of Legionella micdadei Bari 2/158.

The cellular fatty acid composition of Legionella micdadei Bari 2/158 was reevaluated because of its purported differences from other L. micdadei strains and its similarity to L. bozemanii. We found the fatty acid content of this strain to be consistent with that of 11 other strains of L. micdadei, including the presence of an anteiso branched-chain, monounsaturated, 17-carbon acid (Ca17:1) which is characteristic of this species. The double-bond position of Ca17:1 was established at the omega 7 (or delta 9) position by combined gas chromatographic-mass spectrometric analysis of dimethyl disulfide derivatives. The Ca17:1 omega 7 acid was absent in each of 14 strains of L. bozemanii.     

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.     

Mannose inhibits the human neutrophil oxidative burst

Stimulated human neutrophils (PMNs) increase their oxygen consumption and secrete reactive oxygen species that are involved in bactericidal activity and inflammation. While studying lectin-mediated bacterial adherence, we observed that D-mannose appeared to inhibit PMN metabolism. Further studies showed that 100 mM mannose inhibited oxygen consumption by 82%, superoxide secretion by 84%, luminol-enhanced chemiluminescence (CL) by 98%, and hexose-monophosphate shunt activity by 100% when PMN were stimulated with 1 microM phorbol myristate acetate (PMA). Inhibition was also seen with 0.1 microM formyl-methionyl-leucyl-phenylalanine (fMLP), and 0.1 microM A23187, reagents thought to stimulate the respiratory burst by different transductional mechanisms. Inhibition was dose-responsive and specific since 100 mM D-galactose, alpha-D-glucose, or alpha-L-fucose only minimally affected PMN oxidative metabolism. Inhibition of PMA-induced superoxide production was seen almost immediately upon the addition of 50 mM mannose and was reversed by washing. Neutrophils remained viable as measured by trypan blue exclusion. These data suggest that mannose inhibits the neutrophil oxidative burst at the level of the hexose monophosphate shunt. Further investigation should elucidate the specific mechanism(s) of this burst inhibition as well as define uses for it as a tool to study oxidative as well as nonoxidative killing by PMN.     

Modulation of human neutrophil function by monohydroxy-eicosatetraenoic acids.

The generation from arachidonic acid and purification of large quantities of a series of monohydroxy-eicosatetraenoic acids (HETEs) which differed only in the position of the hydroxyl group permitted an in vitro analysis of the relative effects of the HETEs on a variety of human neutrophil functions. All of the HETEs elicited maximal neutrophil chemotactic responses of comparable magnitude, but the chemotactic potencies exhibited a distinct rank order with 5-HETE greater than 8-HETE:9-HETE (85:15, w:w) greater than 11-HETE=12-L-HETE. Peak chemotactic responses were achieved at concentrations of 1 microgram/ml for 5-HETE, 5 microgram/ml for 8-HETE:9-HETE and 10 microgram/ml for 11-HETE and 12-L-HETE. In the absence of a concentration gradient, the HETEs were similar in potency with respect to the stimulation of neutrophil chemokinesis and the enhancement of the expression of neutrophil C3b receptors. At optimally chemotactic and chemokinetic concentrations, none of the HETEs stimulated the generation of superoxide by neutrophils, altered the expression of neutrophil IgG-Fc receptors, or evoked the release of lysosomal enzymes. Methyl esterification of 5-HETE and 12-L-HETE reduced the chemotactic activity to less than 12% of that of the parent compound. The HETE methyl esters competitively inhibited the chemotactic activity of the homologous free acids by approximately 50% at equimolar concentrations, without inhibiting the chemotactic activity of formyl-methionyl peptides or of chemotactic fragments of the fifth component of complement (C5fr). The stimulus specificity of the competitive inhibition of chemotaxis by HETE methyl esters and the functional selectivity of the HETEs as compared to the formyl-methionyl peptides and C5fr, which stimulate neutrophil oxidative metabolism and lysosomal enzyme release, suggest that HETEs activate human neutrophils by a unique mechanism.