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.     

Incomplete activation of macrophage apoptosis during intracellular replication of Legionella pneumophila

The ability of the intracellular bacterium Legionella pneumophila to cause disease is totally dependent on its ability to modulate the biogenesis of its phagosome and to replicate within alveolar cells. Upon invasion, L. pneumophila activates caspase-3 in macrophages, monocytes, and alveolar epithelial cells in a Dot/Icm-dependent manner that is independent of the extrinsic or intrinsic pathway of apoptosis, suggesting a novel mechanism of caspase-3 activation by this intracellular pathogen. We have shown that the inhibition of caspase-3 prior to infection results in altered biogenesis of the L. pneumophila-containing phagosome and in an inhibition of intracellular replication. In this report, we show that the preactivation of caspase-3 prior to infection does not rescue the intracellular replication of L. pneumophila icmS, icmR, and icmQ mutant strains. Interestingly, preactivation of caspase-3 through the intrinsic and extrinsic pathways of apoptosis in both human and mouse macrophages inhibits intracellular replication of the parental stain of L. pneumophila. Using single-cell analysis, we show that intracellular L. pneumophila induces a robust activation of caspase-3 during exponential replication. Surprisingly, despite this robust activation of caspase-3 in the infected cell, the host cell does not undergo apoptosis until late stages of infection. In sharp contrast, the activation of caspase-3 by apoptosis-inducing agents occurs concomitantly with the apoptotic death of all cells that exhibit caspase-3 activation. It is only at a later stage of infection, and concomitant with the termination of intracellular replication, that the L. pneumophila-infected cells undergo apoptotic death. We conclude that although a robust activation of caspase-3 is exhibited throughout the exponential intracellular replication of L. pneumophila, apoptotic cell death is not executed until late stages of the infection, concomitant with the termination of intracellular replication.     

Evidence for apoptosis of human macrophage-like HL-60 cells by Legionella pneumophila infection.

Legionella pneumophila, the causative agent of Legionnaires' disease and Pontiac fever, replicates within and eventually kills human macrophages. In this study, we show that L. pneumophila is cytotoxic to HL-60 cells, a macrophage-like cell line. We demonstrate that cell death mediated by L. pneumophila occurred at least in part through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented host cell DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether potential virulence factors like the metalloprotease and the macrophage infectivity potentiator of L. pneumophila are involved in the induction of apoptosis. None of these factors are essential for the induction of apoptosis in HL-60 cells but may be involved in other cytotoxic mechanisms that lead to accidental cell death (necrosis). The ability of L. pneumophila to promote cell death may be important for the initiation of infection, bacterial survival, and escape from the host immune response. Alternatively, the triggering of apoptosis in response to bacterial infection may have evolved as a means of the host immune system to reduce or inhibit bacterial replication.     

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.     

Molecular Pathogenesis of Infections Caused by Legionella pneumophila

Summary: The genus Legionella contains more than 50 species, of which at least 24 have been associated with human infection. The best-characterized member of the genus, Legionella pneumophila, is the major causative agent of Legionnaires'' disease, a severe form of acute pneumonia. L. pneumophila is an intracellular pathogen, and as part of its pathogenesis, the bacteria avoid phagolysosome fusion and replicate within alveolar macrophages and epithelial cells in a vacuole that exhibits many characteristics of the endoplasmic reticulum (ER). The formation of the unusual L. pneumophila vacuole is a feature of its interaction with the host, yet the mechanisms by which the bacteria avoid classical endosome fusion and recruit markers of the ER are incompletely understood. Here we review the factors that contribute to the ability of L. pneumophila to infect and replicate in human cells and amoebae with an emphasis on proteins that are secreted by the bacteria into the Legionella vacuole and/or the host cell. Many of these factors undermine eukaryotic trafficking and signaling pathways by acting as functional and, in some cases, structural mimics of eukaryotic proteins. We discuss the consequences of this mimicry for the biology of the infected cell and also for immune responses to L. pneumophila infection.     

Legionella anisa, a possible indicator of water contamination by Legionella pneumophila

Legionella anisa is one of the most frequent species of Legionella other than Legionella pneumophila in the environment and may be hospital acquired in rare cases. We found that L. anisa may mask water contamination by L. pneumophila, suggesting that there is a risk of L. pneumophila infection in immunocompromised patients if water is found to be contaminated with Legionella species other than L. pneumophila.     

Identification of Legionella pneumophila by latex agglutination.

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

Pathogenicity of Legionella pneumophila.

The bacterium Legionella pneumophila is the principal etiologic agent of Legionnaires' disease, a form of lobar pneumonia. Ubiquitous in aquatic environments, the gram-negative Legionella organism is a facultative, intracellular parasite of protozoa. The pathogenesis of legionellosis is largely due to the ability of L. pneumophila to invade and grow within alveolar macrophages, and it is widely believed that this ability results from a prior adaptation to intracellular niches in nature. Indeed, intracellular legionellae display a remarkable capacity to avoid endosomal and lysosomal bactericidal activities and to establish a unique replicative phagosome. In recent years, much progress has been made toward identifying the bacterial factors that promote intracellular infection and virulence. Surface structures that enhance infection include LPS, flagella, type IV pili, an outer membrane porin, and the Mip propyl-proline isomerase. Both type II and type IV protein secretion systems are critical for L. pneumophila pathogenesis. Whereas the type II (Lsp) system secretes a collection of degradative enzymes, the type IV (Dot/Icm) system likely exports effector proteins that are especially critical for trafficking of the Legionella phagosome. In addition to facilitating pilus formation and type II secretion, the inner membrane prepilin peptidase (PilD) of L. pneumophila appears to mediate a third, potentially novel pathway that is operative in the mammalian host. Periplasmic and cytosolic infectivity determinants include a catalase-peroxidase and the HtrA and Hsp60 stress-response proteins. The stationary phase response and the iron acquisition functions of L. pneumophila also play key roles in pathogenesis, as do a number of other loci, including the pts, mil and enh genes.     

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.     

Legionella pneumophila suppresses interleukin-12 production by macrophages

In vitro infection of macrophages with Legionella pneumophila induced interleukin-1alpha (IL-1alpha), IL-10, monocyte chemotactic protein 1 (MCP-1), and MCP-3 but not IL-12. The lipopolysaccharide (LPS)-induced production of IL-12 was down-regulated by infection with virulent L. pneumophila, but other cytokines were not affected. In contrast, avirulent L. pneumophila or UV-killed, virulent L. pneumophila did not induce any suppression of IL-12. The IL-12 suppression occurred at the level of mRNA accumulation for IL-12 genes in response to LPS stimulation, but the infection induced a marked accumulation of mRNA for both MCP-1 and MCP-3, which are known to suppress IL-12 production in LPS-stimulated macrophages. However, pretreatment of macrophages with MCP-1 did not suppress LPS-induced IL-12 production at the concentrations induced by L. pneumophila infection. These results suggest that L. pneumophila selectively suppresses IL-12 production induced by LPS from macrophages in vitro by an MCP-independent mechanism.     

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.     

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.     

Identification of a cytotoxin produced by Legionella pneumophila.

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

Purification and characterization of a UDP-glucosyltransferase produced by Legionella pneumophila

Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.     

Induction of tumor necrosis factor by Legionella pneumophila.

Mice were inoculated with Legionella pneumophila via an intratracheal route to establish an experimental model of infection. Lung lavage fluid obtained from infected mice contained a cytolytic factor identified as tumor necrosis factor (TNF). Peak levels of TNF were produced at about 24 h postinfection and rapidly declined thereafter. Treatment of the mice with dextran sulfate before inoculation with the bacteria resulted in lowered amounts of TNF in the lung lavage fluid, suggesting that macrophages were responsible for production of the cytokine. Furthermore, cultures of adherent lung leukocytes and a macrophage cell line, PU 5-1.8, were stimulated to produce TNF by exposure to Legionella antigens. In addition, adherent lung leukocytes from Legionella-infected mice spontaneously released TNF into the culture supernatant. Inoculation of mice with saline or latex particles failed to induce TNF in vivo, indicating that bacterial antigens or products were the stimulating signals. Since there was no detectable TNF activity in sera at any time after intratracheal inoculation, TNF production appeared to be confined to the site of infection. Pretreatment of PU 5-1.8 cultures with gamma interferon, which was detected in the lung lavage fluid before TNF, resulted in augmented TNF production, suggesting cooperativity may exist between the two cytokines, either in the pathogenicity of the bacterium or in a possible immunomodulatory function of TNF and interferon during infection.     

Virulence associated ingestion of Legionella pneumophila by HeLa cells

Invasion of HeLa cell monolayers by Legionella pneumophila was studied. Virulent L. pneumophila strains efficiently entered cultured HeLa cells whereas isogenic avirulent isolates were nearly 1000-fold less efficient at cellular entry. Animal passage of avirulent strains, which restored bacterial virulence, coincided with a return to the virulent parental level of HeLa cell invasion. Diminished HeLa cell invasion by avirulent strains was not a function of reduced bacterial association with the cell monolayer. These data suggest that ingestion of L. pneumophila by non-professional phagocytes is a virulence directed property.     

Extracellular enzymes of Legionella pneumophila.

All strains of Legionella pneumophila tested produced detectable levels of extracellular protease, phosphatase, lipase, deoxyribonuclease, ribonuclease, and beta-lactamase activity. Weak starch hydrolysis was also demonstrated for all strains. Elastase, collagenase, phospholipase C, hyaluronidase, chondroitinase, neuraminidase, or coagulase were not detected in any of these laboratory-maintained strains.