Identification and characterization of a new conjugation/type IVA secretion system (trb/tra) of Legionella pneumophila Corby localized on two mobile genomic islands

Horizontal gene transfer probably contributes to evolution of Legionella pneumophila and its adaptation to different environments. Although horizontal gene transfer was observed in Legionella, the mechanism is still not specified. In this study we identified and analysed a new type of conjugation/type IVA secretion system (trb/tra) of L. pneumophila Corby, a virulent human isolate. Two similar versions of this conjugation system were identified, localized on two different genomic islands (Trb-1, 42,710 bp and Trb-2, 34,434 bp). Trb-1 and Trb-2 are integrated within the tRNA^Pro gene (lpc2778) and the tmRNA gene (lpc0164), respectively. Both islands exhibit an oriT region and both can be excised from the chromosome forming episomal circles. Trb-1 was analysed in more detail. It is active and can be horizontally transferred to other Legionella strains by conjugation and then integrated into the genome in a site-specific manner within the tRNA^Pro gene. We characterized the sequence of the excision and integration sites of Trb-1 in three different L. pneumophila strains. Here we demonstrate that L. pneumophila exhibits a functional oriT region and that genomic islands in Legionella can be mobilized and conjugated to other species of Legionella. Thus, we describe for the first time a mechanism that may explain the observed horizontal transfer of chromosomal DNA in Legionella.     

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.     

LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway

Legionella pneumophila uses a type IV secretion system to deliver effector molecules into the host cell and establish its replication vacuole. In this study, we investigated the role of LidA, a translocated substrate associated with the surface of the L. pneumophila-containing vacuole. LidA is secreted into the host cell throughout the replication cycle of the bacteria and associates with compartments of the early secretory pathway. When overexpressed in mammalian cells or yeast, LidA interferes with the early secretory pathway, probably via a domain predicted to be rich in coiled-coil structure. Finally, during intracellular replication, the replication vacuoles are in close contact with the endoplasmic reticulum-Golgi intermediate compartment and the Golgi apparatus, suggesting a positive correlation between intracellular growth and association of the vacuole with compartments of the early secretory pathway. We propose that LidA is involved in the recruitment of early secretory vesicles to the L. pneumophila-containing vacuole and that the vacuole associates with the secretory pathway to facilitate this process.     

Differential 2-D protein gel electrophoresis analysis of Legionella pneumophila wild type and Tat secretion mutants

The twin-arginine translocation (Tat) pathway is a secretory pathway for translocation of folded proteins with two arginines in their signal peptide across the cytoplasmic membrane. Recently, we showed the presence of the Tat secretion pathway in Legionella pneumophila Philadelphia-1 and its role in intracellular replication and biofilm formation. To analyse the importance of the Tat pathway in protein export and its role in L. pneumophila virulence, a comparative 2-D protein gel electrophoresis analysis was performed on supernatants of the wild type and two Tat secretion mutants in order to identify possible Tat substrates. Twenty proteins were identified as differential proteins, eight of which were present in a lower quantity in the supernatant of the tat mutants. Among these, one protein with a typical twin-arginine motif in its signal peptide was identified as the 3′,5′-cyclic nucleotide phosphodiesterase. Two other proteins that resulted as differential proteins from this study were flagellin and LvrE, which were studied in more detail and their Tat-dependence was further confirmed with specific antibodies. LvrE was shown to play a role in intracellular growth in differentiated U937 cells.     

The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages

We have previously shown that Legionella pneumophila induces caspase 3-dependent apoptosis in mammalian cells during early stages of infection. In this report, we show that nine L. pneumophila strains with mutations in the dotA, dotDCB, icmT, icmGCD, and icmJB loci are completely defective in the induction of apoptosis, in addition to their severe defects in intracellular replication and pore formation-mediated cytotoxicity. Importantly, all nine dot/icm mutants were complemented for all their defective phenotypes with the respective wild-type loci. We show that the role of the Dot/Icm type IV secretion system in the induction of apoptosis is independent of the RtxA toxin, the dot/icm-regulated pore-forming toxin, and the type II secretion system. However, the pore-forming toxin, which is triggered upon entry into the postexponential growth phase, enhances the ability of L. pneumophila to induce apoptosis. Our data provide the first example of the role of a type IV secretion system of a bacterial pathogen in the induction of apoptosis in the host cell.     

Protein kinase LegK2 is a type IV secretion system effector involved in endoplasmic reticulum recruitment and intracellular replication of Legionella pneumophila

Legionella pneumophila is the etiological agent of Legionnaires' disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting the Legionella phagosome. Many Legionella effectors display distinctive eukaryotic domains, among which are protein kinase domains. In silico analysis and in vitro phosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemic L. pneumophila Lens strain. Except for LegK5, the Legionella protein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant of legK2 exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed by L. pneumophila to establish a replicative niche and evade host cell defenses.     

Components of the Legionella pneumophila flagellar regulon contribute to multiple virulence traits, including lysosome avoidance and macrophage death

Legionella pneumophila is a motile intracellular pathogen of macrophages and amoebae. When nutrients become scarce, the bacterium induces expression of transmission traits, some of which are dependent on the flagellar sigma factor FliA (sigma(28)). To test how particular components of the L. pneumophila flagellar regulon contribute to virulence, we compared a fliA mutant with strains whose flagellar construction is disrupted at various stages. We find that L. pneumophila requires FliA to avoid lysosomal degradation in murine bone marrow-derived macrophages (BMM), to regulate production of a melanin-like pigment, and to regulate binding to the dye crystal violet, whereas motility, flagellar secretion, and external flagella or flagellin are dispensable for these activities. Thus, in addition to flagellar genes, the FliA sigma factor regulates an effector(s) or regulator(s) that contributes to other transmissive traits, notably inhibition of phagosome maturation. Whether or not the microbes produced flagellin, all nonmotile L. pneumophila mutants bound BMM less efficiently than the wild type, resulting in poor infectivity and a loss of contact-dependent death of BMM. Therefore, bacterial motility increases contact with host cells during infection, but flagellin is not an adhesin. When BMM contact by each nonmotile strain was promoted by centrifugation, all the mutants bound BMM similarly, but only those microbes that synthesized flagellin induced BMM death. Thus, the flagellar regulon equips the aquatic pathogen L. pneumophila to coordinate motility with multiple traits vital to virulence.     

Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires' disease pneumonia

Legionella pneumophila, the gram-negative agent of Legionnaires' disease, possesses type IV pili and a type II protein secretion (Lsp) system, both of which are dependent upon the PilD prepilin peptidase. By analyzing multiple pilD mutants and various types of Lsp mutants as well as performing trans-complementation of these mutants, we have confirmed that PilD and type II secretion genes are required for L. pneumophila infection of both amoebae and human macrophages. Based upon a complete analysis of lspDE, lspF, and lspG mutants, we found that the type II system controls the secretion of protease, RNase, lipase, phospholipase A, phospholipase C, lysophospholipase A, and tartrate-sensitive and tartrate-resistant acid phosphatase activities and influences the appearance of colonies. Examination of the developing L. pneumophila genome database indicated that the organism has two other loci (lspC and lspLM) that are predicted to promote secretion and thus a set of genes that is comparable to the type II secretion genes in other gram-negative bacteria. In contrast to lsp mutants, L. pneumophila pilus mutants lacking either the PilQ secretin, the PspA pseudopilin, or pilin were not defective for colonial growth, secreted activities, or intracellular replication. L. pneumophila dot/icm mutants were also not impaired for type II-dependent exoenzymes. Upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibited a reduced ability to grow in the lung, as measured by competition assays. The lspF mutant was also defective in an in vivo kinetic assay. Examination of infected mouse sera revealed that type II secreted proteins are expressed in vivo. Thus, the L. pneumophila Lsp system is a virulence factor and the only type II secretion system linked to intracellular infection.     

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.     

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.     

Characterization of the Legionella pneumophila gene ligA

Legionella pneumophila is a bacterial pathogen that resides and multiplies in macrophages as well as in its natural aquatic hosts, the protozoa. Different bacterial factors contribute to pathogenicity and accompanying eukaryotic intracellular events. Sequencing of mip flanking regions revealed a gene of 2610 bp, ligA, that has no significant similarity to any of the genes identified previously. Epidemiological studies indicate that this gene is present in Legionella pneumophila, the species most often associated with cases of the Legionnaires' disease, but not in Legionella species other than L. pneumophila. The isogenic ligA deletion mutant was resistant to NaCl, and showed decreased cytotoxicity to human monocytes and decreased hemolytic activity to red blood cells. However, the most prominent effect of the L. pneumophila ligA mutant strain LEPF1 was the nearly completely reduced replication within the natural host Acanthamoeba castellanii. Since this gene is L. pneumophila specific and regulates numerous bacterial properties we designated this gene ligA for Legionella pneumophila infectivity gene A.     

Serospecificity and opsonic activity of antisera to Legionella pneumophila.

A high-molecular-weight surface component (F-1 fraction) has been isolated from the four serogroups of Legionella pneumophila. Antibody raised against live organisms was found by microagglutination assay to be specific for the homologous serogroup. Agglutinating activity of antiserum was markedly diminished after absorption with the homologous, but not heterologous, F-1 fraction. In addition, it was found that L. pneumophila organisms were not interiorized by rat alveolar macrophages or mouse peritoneal macrophages in the absence of antiserum, whereas homologous antiserum effectively opsonized the organisms. The opsonizing activity of serogroup-specific antisera was eliminated by absorption of the antisera with the homologous, but not heterologous, F-1 fraction. These data indicate that the serogroup-specific antigen of L. pneumophila resides in the F-1 fraction and that antibody to the F-1 fraction is required for phagocytosis of L. pneumophila by mammalian phagocytes.     

Molecular and cell biology of Legionella pneumophila

Legionella pneumophila is a facultative intracellular pathogen that can replicate within phagocytic host cells such as protozoa and macrophages. Evasion of phagocytic killing is mediated by the type IV Dot/Icm secretion system, which exports bacterial effectors that modulate biogenesis of the phagosome to evade endocytic fusion and also to intercept vesicles derived from the endoplasmic reticulum. Bacterial replication is associated with activation of caspase-3 in infected macrophages and is culminated in apoptosis and pore formation-mediated cytolysis of the host.     

Production of monoclonal antibodies to Legionella pneumophila serogroups 1 and 6

To better define the surface antigens of Legionella pneumophila for clinical and experimental purposes, we have produced monoclonal antibodies to L. pneumophila serogroups 1 and 6. Two hybridomas were produced in serogroup 1. One antibody, LP-I-17, recognized a serogroup-common antigen. The second antibody, LP-I-81, was specific for serogroup 1. This antibody was able to agglutinate bacterial cells belonging to the serogroup 1 reference strains. Philadelphia and Knoxville. Microagglutination assays of environmental and clinical isolates revealed a subgroup of serogroup 1 environmental isolates which were not agglutinated by LP-I-81. This subset of isolates was segregated to certain buildings in the medical complex. Immunodiffusion studies showed identity between the LP-I-81 antigen and the serogroup-specific antigen of serogroup 1 organisms. This antigen could be absorbed out of the serogroup 1 organism extract with LP-I-81-coated Staphylococcus aureus, leaving the serogroup-common antigens. Three hybridomas were produced to serogroup 6. All three produced antibodies which were serogroup 6 specific and agglutinated serogroup 6 bacteria.     

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.