Expression of Multiple Pili by Legionella pneumophila: Identification and Characterization of a Type IV Pilin Gene and Its Role in Adherence to Mammalian and Protozoan Cells

Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 μm) or short (0.1 to 0.6 μm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilE_L). An insertion mutation was constructed in pilE_L and introduced into the L. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilE_L mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that the pilE_L locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires’ disease and invasion of amoebas in the environment.     

Legionella pneumophila NudA Is a Nudix hydrolase and virulence factor

We studied the identity and function of the 528-bp gene immediately upstream of Legionella pneumophila F2310 ptsP (enzyme I(Ntr)). This gene, nudA, encoded for a Nudix hydrolase based on the inferred protein sequence. NudA had hydrolytic activity typical of other Nudix hydrolases, such as Escherichia coli YgdP, in that Ap(n)A's, in particular diadenosine pentaphosphate (Ap(5)A), were the preferred substrates. NudA hydrolyzed Ap(5)A to ATP plus ADP. Both ptsP and nudA were cotranscribed. Bacterial two-hybrid analysis showed no PtsP-NudA interactions. Gene nudA was present in 19 of 20 different L. pneumophila strains tested and in 5 of 10 different Legionella spp. other than L. pneumophila. An in-frame nudA mutation was made in L. pneumophila F2310 to determine the phenotype. The nudA mutant was an auxotroph that grew slowly in liquid and on solid media and had a smaller colony size than its parent. In addition, the mutant was more salt resistant than its parent and grew very poorly at 25 degrees C; all of these characteristics, as well as auxotrophy and slow-growth rate, were reversed by transcomplementation with nudA. The nudA mutant was outcompeted by about fourfold by the parent in competition studies in macrophages; transcomplementation almost completely restored this defect. Competition studies in guinea pigs with L. pneumophila pneumonia showed that the nudA mutant was outcompeted by its parent in both lung and spleen. NudA is of major importance for resisting stress in L. pneumophila and is a virulence factor.     

Outer membrane proteins from Legionella pneumophila serogroups and other Legionella species.

Outer membranes were isolated from eight serogroups of L. pneumophila and five other Legionella species. The protein composition of the membranes was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single, disulfide stabilized protein with a molecular size of 29,000 to 30,000 daltons was found to be the major outer membrane protein (MOMP) of all the serogroups. The equivalent of the L. pneumophila MOMP was not observed in any of the other Legionella species examined. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels revealed distinctive patterns for each serogroup and other Legionella species that were not observed by staining with Coomassie blue and may result from the presence of lipopolysaccharide in the membrane preparations. The MOMP from serogroup 1 was isolated by exposing crude peptidoglycan to detergent in the presence of heat and reducing agent and was found to be tightly associated with lipopolysaccharide. Antibodies to this complex were used to probe the outer membranes of the remaining, L. pneumophila serogroups and other Legionella species by Western blotting. Serogroup 1 anti-MOMP antibodies were found to react with the MOMP from the remaining seven serogroups examined, whereas antibodies directed against the lipopolysaccharide of serogroup 1 only reacted with lipopolysaccharide from two of the remaining seven serogroups.     

Pleomorphism of Legionella pneumophila

Legionella pneumophila (Lp), serogroups 1-6, was grown in vitro on a variety of media, in embryonated hens' eggs, and in guinea pigs. The morphology of the microbe was examined by light, immunofluorescent, and electron microscopy (transmission, scanning, negative staining). The configuration of all serogroups examined differed somewhat on agar media, in liquid media, and in vivo. Each serogroup of Lp showed pleomorphic features indistinguishable from the others. Except for filamentous forms, pleomorphism was least conspicuous on agar. By contrast, pleomorphism was most apparent in yeast extract broth, and it was detected by all of the morphologic techniques employed. Bacilli were seen most commonly, but the spectrum of forms was as follows: cocci, coccobacilli (short bacilli), medium bacilli, bacilli with terminal cocci, filamentous forms, and branches. Diplococci, branches, and stalks were only rarely seen, and the latter form was never visualized by immunofluorescence. In tissue samples from infected guinea pigs and embryonated hens' eggs, Lp was typically a short bacillus, but coccoid and coccobacillary forms were seen. Lp is clearly a pleomorphic bacterium, particularly when grown in yeast extract broth. The variety of forms described herein might provide clues to taxonomy, ecologic niche, and physiology of Lp.     

Identification of Linked Legionella pneumophila Genes Essential for Intracellular Growth and Evasion of the Endocytic Pathway

Legionella pneumophila replicates within a specialized phagosome in cultured cells, a function necessary for its pathogenicity. The replicative phagosome lacks membrane marker proteins, such as the glycoprotein LAMP-1, that are indicators of the normal endocytic pathway. We describe the isolation of several Legionella genes essential for intracellular growth and evasion of the endocytic pathway, using a genetic and cell biological approach. We screened 4,960 ethyl methanesulfonate-mutagenized colonies for defects in intracellular growth and trafficking to the replicative phagosome. Six mutant strains of L. pneumophila that had severe intracellular growth defects in mouse bone marrow-derived macrophages were identified. All six mutants were found in phagosomes that colocalized with LAMP-1, indicating defects in intracellular trafficking. The growth defects of two of these strains were complemented by molecular clones from a bank constructed from a wild-type L. pneumophila strain. The inserts from these clones are located in a region of the chromosome contiguous with several other genes essential for intracellular growth. Three mutants could be complemented by single open reading frames placed in trans, one mutant by a gene termed dotH and two additional mutants by a gene termed dotO. A deletion mutation was created in a third gene, dotI, which is located directly upstream of dotH. The ΔdotI strain was also defective for intracellular growth in macrophages, and this defect was complemented by a single open reading frame in trans. Based on sequence analysis and structural predictions, possible roles of dotH, dotI, and dotO in intracellular growth are discussed.     

Essential role for the Legionella pneumophila rep helicase homologue in intracellular infection of mammalian cells

We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar to rep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the rep helicase homologue mutant, rendering it defective in intracellular replication.     

Legionella pneumophila Invasion of MRC-5 Cells Induces Tyrosine Protein Phosphorylation

After uptake and intracellular multiplication of Legionella pneumophila in MRC-5 lung fibroblasts, important cytoskeletal filament structures, like actin, tubulin, or vimentin, and a cell membrane-associated fibronectin were rearranged during early infection, resulting in a loss of cell adhesion and collapse of the cytoskeleton. Dysregulation of the cellular phosphorylation and dephosphorylation cascade may contribute to the observed changes and may support intracellular survival and multiplication of L. pneumophila. We therefore studied expression of phosphoproteins during intracellular growth of L. pneumophila. By using an anti-tyrosine phosphoprotein antibody we showed that proteins phosphorylated on tyrosine residues accumulated progressively during late infection exclusively around or in phagosomes filled with bacteria. In contrast, expression of serine/threonine phosphoproteins did not change. To discern the origin of phosphorylated proteins, the host cells were treated with cycloheximide, an inhibitor of eukaryotic protein synthesis. The newly synthesized proteins were labeled metabolically with [(35)S]methionine-cysteine and immunoprecipitated with a phosphotyrosine-specific antibody. Sodium dodecyl sulfate gel electrophoresis gave evidence for synthesis of at least three protein clusters (160 to 200, 35 to 60, and 19 to 28 kDa) of Legionella origin that were phosphorylated on tyrosine residues 24 h after infection. Treatment of infected host cells with genistein, a tyrosine kinase inhibitor, revealed that tyrosine protein phosphorylation was not important for bacterial uptake but contributed to intracellular growth of L. pneumophila. Bacterial tyrosine phosphoproteins and the observed intracellular structural changes may be important to understanding the process involved in intracellular growth of L. pneumophila.     

Specificity of Legionella pneumophila and Coxiella burnetii vacuoles and versatility of Legionella pneumophila revealed by coinfection

Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.     

Legionella pneumophila serogroup 4 isolated from joint tissue

We report the isolation of Legionella pneumophila serogroup 4 from synovial tissue obtained from an 80-year-old female with chronic swelling of her right metacarpophalangeal joint. Synovial tissue infections caused by L. pneumophila are rare. Interestingly, this isolate was recovered from chocolate agar after 5 days of incubation.     

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.     

Ultrastructure of Legionella pneumophila.

Eleven lung samples positive for Legionnaires' disease, 12 strains of Legionella pneumophila cultured on various bacteriological media, and one strain growth in the yolk sac of fertile hens' eggs were examined by negative staining, thin sectioning, and scanning electron microscopy. All organisms studied were ultrastructurally similar irrespective of strain, source, or method of cultivation, presenting mainly as short rods, 0.6 x 1.5 micrometer, with tapered ends, though long forms and filaments were also evident. In this they resembled typical Gram-negative organisms. Division was by non-septate binary fission, and the cell wall was composed of two triple-unit membranes with morphological evidence of a peptidoglycan layer. The bacterial cytoplasm was rich in ribosomes and nuclear elements and often contained vacuoles. No acid polysaccharides or bacterial appendages were detected surrounding the organisms. In lung tissue and yolk sac membranes, the organisms replicated within the cytoplasm of infected cells and in the intercellular spaces and were specifically identified in thin sections by immunoferritin techniques.     

Clinical Application of a Multiplex Real-Time PCR Assay for Simultaneous Detection of Legionella Species,Legionella pneumophila,and Legionella pneumophila Serogroup 1

We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.     

Immunoperoxidase staining of Legionella pneumophila

Immmunoperoxidase staining has been applied to sections of pneumonic lung from a previously published case of Legionnaires' disease. Specific staining of Legionella pneumophila was accomplished with sub-group 1 antiserum, which also revealed staining of phagosomes, and in some areas diffuse background staining of 'soluble' antigen. Some organisms remained unstained with the specific antiserum, and these were revealed by progressive haemalum staining. In other sections, some organisms stained specifically with rabbit anti-μ chain serum but not with anti-γ chain serum, this result suggesting that the organisms were coated with patient's IgM specific antibody.     

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.     

Plaque assay for virulent Legionella pneumophila.

Methods of assessing virulence of Legionella pneumophila, the etiologic agent of Legionnaires disease, include the infection of guinea pigs, fertile chicken eggs, and mammalian and protozoan cell cultures. Guinea pig assays, in particular, are expensive, laborious, or unsuitable for routine screening of Legionella isolates. We have developed a virulence assay that requires the enumeration of viruslike plaques which are the result of virulent L. pneumophila infecting mouse L929 cells. Each plaque is the consequence of the initial infection of an L cell with a single bacterium. A nonvirulent mutant derived from the serial passage of virulent L. pneumophila on Mueller-Hinton agar fails to survive within L cells and consequently fails to produce plaques.     

Isolation of Legionella pneumophila from a transtracheal aspirate.

Isolation of Legionella pneumophila from a transtracheal aspirate was achieved by using simple in vitro culture methods. Clinical microbiologists should routinely culture for this organism from appropriate body fluids obtained from normally sterile areas.     

Liquid medium for growth of Legionella pneumophila.

The medium described is a simple yeast extract broth capable of growing large number of Legionella neumophila, the causative organism of Legionnaires disease. Filtration was chosen as a means of sterilization, since medium that was autoclaved did not support growth without the presence of Norite A. The filtered medium gave rapid cell growth and maintained the initial antigen production. The observed generation time was 99 min with a maximum cell population of 2 X 10(2) COLONY-FORMING UNITS PER ML IN APPROXIMATELY 40 H.