Rapid and complete fusion of macrophage lysosomes with phagosomes containing Salmonella typhimurium.

The virulence of Salmonella typhimurium for mice results, in part, from its ability to survive after phagocytosis by macrophages. Although it is generally agreed that intracellular bacteria persist in membrane-bound phagosomes, there remains some question as to whether these phagosomes fuse with macrophage lysosomes. This report describes the maturation of phagosomes containing S. typhimurium inside mouse bone marrow-derived macrophages. Macrophages were infected briefly and incubated for various intervals; then they were examined by fluorescence microscopy for colocalization of bacteria with lysosomal markers. These markers included LAMP-1, cathepsin L, and fluorescent proteins or dextrans preloaded into lysosomes by endocytosis. By all measures, phagosomes containing S. typhimurium merged completely with the lysosomal compartment within 20 min of phagocytosis. The rate of phagosome-lysosome fusion was similar to the rate for phagocytosed latex beads. Phagolysosomes remained accessible to fluid-phase probes and contained lysosomal markers for many hours. Moreover, a large percentage of the wild-type bacteria that were viable 20 min after infection survived longer incubations inside macrophages, indicating that the survivors were not a minor subpopulation that avoided phagosome-lysosome fusion. Therefore, we conclude that S. typhimurium survives within the lysosomal compartments of macrophages.     

Caspase-11 promotes the fusion of phagosomes harboring pathogenic bacteria with lysosomes by modulating actin polymerization

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L.?pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L.?pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L.?pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.     

Surfactant protein D increases fusion of Mycobacterium tuberculosis-containing phagosomes with lysosomes in human macrophages

Lung surfactant protein D (SP-D) binds to Mycobacterium tuberculosis surface lipoarabinomannan and results in bacterial agglutination, reduced uptake, and inhibition of growth in human macrophages. Here we show that SP-D limits the intracellular growth of bacilli in macrophages by increasing phagosome-lysosome fusion but not by generating a respiratory burst.     

Mycobacterium tuberculosis and Legionella pneumophila phagosomes exhibit arrested maturation despite acquisition of Rab7

Rab7 is a small GTPase that regulates vesicular traffic from early to late endosomal stages of the endocytic pathway. Phagosomes containing inert particles have also been shown to transiently acquire Rab7 as they mature. Disruption in the pathway prior to the acquisition of Rab7 has been suggested as playing a role in the altered maturation of Mycobacterium bovis BCG phagosomes. As a first step to determine whether disruption in the delivery or function of Rab7 could play a role in the altered maturation of Legionella pneumophila and M. tuberculosis phagosomes, we have examined the distribution of wild-type Rab7 and the GTPase-deficient, constitutively active mutant form of Rab7 in HeLa cells infected with L. pneumophila or M. tuberculosis. We have found that the majority of L. pneumophila and M. tuberculosis phagosomes acquire relatively abundant staining for Rab7 and for the constitutively active mutant Rab7 in HeLa cells that overexpress these proteins. Nevertheless, despite acquisition of wild-type or constitutively active Rab7, both the L. pneumophila and the M. tuberculosis phagosomes continue to exhibit altered maturation as manifested by a failure to acquire lysosome-associated membrane glycoprotein 1. These results demonstrate that L. pneumophila and M. tuberculosis phagosomes have receptors for Rab7 and that the altered maturation of these phagosomes is not due to a failure to acquire Rab7.     

Membrane vesicles released by intestinal epithelial cells infected with rotavirus inhibit T-cell function

Rotavirus (RV) predominantly replicates in intestinal epithelial cells (IEC), and "danger signals" released by these cells may modulate viral immunity. We have recently shown that human model IEC (Caco-2 cells) infected with rhesus-RV release a non-inflammatory group of immunomodulators that includes heat shock proteins (HSPs) and TGF-β1. Here we show that both proteins are released in part in association with membrane vesicles (MV) obtained from filtrated Caco-2 supernatants concentrated by ultracentrifugation. These MV express markers of exosomes (CD63 and others), but not of the endoplasmic reticulum (ER) or nuclei. Larger quantities of proteins associated with MV were released by RV-infected cells than by non-infected cells. VP6 co-immunoprecipitated with CD63 present in these MV, and VP6 co-localized with CD63 in RV-infected cells, suggesting that this viral protein is associated with the MV, and that this association occurs intracellularly. CD63 present in MV preparations from stool samples from 36 children with gastroenteritis due or not due to RV were analyzed. VP6 co-immunoprecipitated with CD63 in 3/8 stool samples from RV-infected children, suggesting that these MV are released by RV-infected cells in vivo. Moreover, fractions that contained MV from RV-infected cells induced death and inhibited proliferation of CD4(+) T cells to a greater extent than fractions from non-infected cells. These effects were in part due to TGF-β, because they were reversed by treatment of the T cells with the TGF-β-receptor inhibitor ALK5i. MV from RV-infected and non-infected cells were heterogeneous, with morphologies and typical flotation densities described for exosomes (between 1.10 and 1.18?g/mL), and denser vesicles (>1.24?g/mL). Both types of MV from RV-infected cells were more efficient at inhibiting T-cell function than were those from non-infected cells. We propose that RV infection of IEC releases MV that modulate viral immunity.     

Oligosaccharide receptor mimics inhibit Legionella pneumophila attachment to human respiratory epithelial cells

Legionnaire's disease is caused by the intracellular pathogen Legionella pneumophila, presenting as an acute pneumonia. Attachment is the key step during infection, often relying on an interaction between host cell oligosaccharides and bacterial adhesins. Inhibition of this interaction by receptor mimics offers possible novel therapeutic treatments. L. pneumophila attachment to the A549 cell line was significantly reduced by treatment with tunicamycin (73.6%) and sodium metaperiodate (63.7%). This indicates the importance of cell surface oligosaccharide chains in adhesion. A number of putative anti-adhesion compounds inhibited attachment to the A549 and U937 cell lines. The most inhibitory compounds were polymeric saccharides, GalNAcbeta1-4Gal, Galbeta1-4GlcNAc and para-nitrophenol. These compounds inhibited adhesion to a range of human respiratory cell lines, including nasal epithelial, bronchial epithelial and alveolar epithelial cell lines and the human monocytic cell line, U937. Some eukaryotic receptors for L. pneumophila were determined to be the glycolipids, asialo-GM1 and asialo-GM2 that contain the inhibitory saccharide moiety, GalNAcbeta1-4Gal. The identified compounds have the potential to be used as novel treatments for Legionnaire's disease.     

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.     

Oropharyngeal colonization with Legionella pneumophila.

A total of 186 volunteers, including 40 hospital patients, participated in a cross-sectional survey of oropharyngeal colonization with Legionella pneumophila. Colonization was defined as the appearance of any L. pneumophila organisms on culture or a positive direct fluorescent-antibody (FA) test or both in the absence of signs or symptoms of pneumonia. The direct FA tests were performed on throat swabs, using a polyvalent conjugate directed against L. pneumophila serogroups I through IV. Throat swabs were cultured for L. pneumophila on a selective medium. Blood specimens were tested for antibody, using an indirect FA test and heat-killed polyvalent antigen for L. pneumophila serogroups I through IV. Eight people, none of whom had pneumonia or fever, had positive direct FA tests; no subject had a positive culture for L. pneumophila. Whether the positive direct FA results represent colonization cannot be stated with assurance. In any case, the results suggest that colonization occurs infrequently.     

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.     

Interaction of Legionella pneumophila with Acanthamoeba castellanii: uptake by coiling phagocytosis and inhibition of phagosome-lysosome fusion.

Legionella pneumophila is a facultative intracellular parasite able to survive within both human monocytes and amoebae. We have demonstrated that processing of L. pneumophila by the free-living amoeba Acanthamoeba castellanii shows many similarities to the processing of L. pneumophila by monocytes. These similarities include uptake of L. pneumophila by coiling phagocytosis and the subsequent confinement of L. pneumophila in a ribosome-studded phagosome. In addition, as in monocytes, inhibition of lysosomal fusion with phagosomes containing L. pneumophila was detected in amoebae. With all clinical isolates, inhibition of phagosomes-lysosome fusion correlated with virulence. However, with one of the environmental isolates tested, no significant difference in phagosome-lysosome fusion was observed between the virulent and avirulent forms. These results indicate that the avirulent form of this isolate differed from the virulent form in some other respect critical to intracellular survival. Therefore, intracellular multiplication of L. pneumophila within A. castellanii may not be solely dependent upon the inhibition of lysosomal fusion.     

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.     

Deviant expression of Rab5 on phagosomes containing the intracellular pathogens Mycobacterium tuberculosis and Legionella pneumophila is associated with altered phagosomal fate

The intracellular human pathogens Legionella pneumophila and Mycobacterium tuberculosis reside in altered phagosomes that do not fuse with lysosomes and are only mildly acidified. The L. pneumophila phagosome exists completely outside the endolysosomal pathway, and the M. tuberculosis phagosome displays a maturational arrest at an early endosomal stage along this pathway. Rab5 plays a critical role in regulating membrane trafficking involving endosomes and phagosomes. To determine whether an alteration in the function or delivery of Rab5 could play a role in the aberrant development of L. pneumophila and M. tuberculosis phagosomes, we have examined the distribution of the small GTPase, Rab5c, in infected HeLa cells overexpressing Rab5c. Both pathogens formed phagosomes in HeLa cells with molecular characteristics similar to their phagosomes in human macrophages and multiplied in these host cells. Phagosomes containing virulent wild-type L. pneumophila never acquired immunogold staining for Rab5c, whereas phagosomes containing an avirulent mutant L. pneumophila (which ultimately fused with lysosomes) transiently acquired staining for Rab5c after phagocytosis. In contrast, M. tuberculosis phagosomes exhibited abundant staining for Rab5c throughout its life cycle. To verify that the overexpressed, recombinant Rab5c observed on the bacterial phagosomes was biologically active, we examined the phagosomes in HeLa cells expressing Rab5c Q79L, a fusion-promoting mutant. Such HeLa cells formed giant vacuoles, and after incubation with various particles, the giant vacuoles acquired large numbers of latex beads, M. tuberculosis, and avirulent L. pneumophila but not wild-type L. pneumophila, which consistently remained in tight phagosomes that did not fuse with the giant vacuoles. These results indicate that whereas Rab5 is absent from wild-type L. pneumophila phagosomes, functional Rab5 persists on M. tuberculosis phagosomes. The absence of Rab5 on the L. pneumophila phagosome may underlie its lack of interaction with endocytic compartments. The persistence of functional Rab5 on the M. tuberculosis phagosomes may enable the phagosome to retard its own maturation at an early endosomal stage.     

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.     

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.     

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.     

延安市286例呼吸科患者血清嗜肺军团菌抗体的检测

目的了解延安市嗜肺军团菌的感染情况。方法采用微量凝集法对286份呼吸科住院患者血清标本进行嗜肺军团菌Lp1型军团菌抗体测定。结果抗体阳性率为17．13％,其中男为18．56％,女为15．13％;在各年龄组均有嗜肺军团菌Lp1型抗体阳性者,其中40岁年龄组的感染率（26．09％）最高;呼吸科疾病中肺结核患者合并嗜肺军团菌Lp1感染率较高,为26．56％,各疾病类型之间感染率比较差异无统计学意义（P〉0．05）。结论延安市存在一定的嗜肺军团菌感染,需加强对军团病血清学的监测工作。