SummaryAdaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii . The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner-Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro , may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA ( s 28 ) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival.

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Bacterial flagella are highly complex molecular machines. They are surface organelles assembled from over 40 different protein components that mediate bacterial motility. To ensure maximal efficiency and accuracy during flagellar biogenesis, bacteria use hierarchical regulatory networks involving transcriptional and posttranscriptional mechanisms to control the ordered expression of the individual components of the flagellar organelle. Although significant differences exist between the regulatory mechanisms used by different bacteria, a salient feature in all cases is that the flagellar genes can be classified based upon their temporal gene expression and on their dependence on various nested transcriptional regulators (for a recent review, see reference 33).The bacterial pathogen Legionella pneumophila lives in natural and manmade water systems and replicates intracellularly within aquatic protozoa (41). When inhaled by humans, L. pneumophila is able to survive and replicate within alveolar macrophages (28). After entry into host cells, L. pneumophila inhibits phagolysosomal fusion (26, 27) and establishes a specialized Legionella-containing vacuole (LCV) surrounded by endoplasmic reticulum in which L. pneumophila represses transmissive traits and starts to replicate (15,37,43). During the bacterial late replicative phase, the LCV merges with lysosomes (44). Finally, induced by a nutrient decline the bacteria enter the transmissive phase, which is reflected by a major shift in gene expression (2,8,14,19,37,51). In the transmissive phase, L. pneumophila expresses many virulence-associated traits promoting the release of the bacteria and infection of a new host (2,3,23,36,42,45,46,51). One striking feature of transmissive L. pneumophila is the expression of a single monopolar flagellum composed of the flagellin subunit FlaA. The flagellum mediates invasivness of L. pneumophila for human macrophage-like cell lines and cytotoxicity to macrophages (13,20). Furthermore, it was shown that flagellin sensed by nonpermissive mouse macrophages mediates cell death by activating the cytosolic Naip5 (Birc1e) receptor (35,40). Expression of the flagellum is dependent on the regulatory circuit controlling phase transition (for a review, see reference 1) and different environmental factors (21,22).Several studies have been undertaken to understand the regulatory mechanisms governing this life cycle switch, including the regulation of flagellar gene expression. The two-component system LetA/LetS, a system homologous to BarA/UvrY of Escherichia coli and RsmA/RsmS of Pseudomonas aeruginosa, was shown to have an important role in the regulation of the life cycle switch and in flagellar gene expression (17,20,32,36,42). It is suggested that LetA/LetS responds to the alarmone molecule (p)ppGpp, synthesized by RelA and SpoT (8,

SummaryThe haploid social soil amoeba Dictyostelium discoideum has been established as a host model for several pathogens including Pseudomonas aeruginosa , Cryptococcus neoformans , Mycobacterium spp. and Legionella pneumophila . The research areas presently pursued include (i) the use of Dictyostelium wild-type cells as screening system for virulence of extracellular and intracellular pathogens and their corresponding mutants, (ii) the use of Dictyostelium mutant cells to identify genetic host determinants of susceptibility and resistance to infection and (iii) the use of reporter systems in Dictyostelium cells which allow the dissection of the complex host-pathogen cross-talk. The body of information presented in this review demonstrates that the availability of host cell markers, the knowledge of cell signalling pathways, the completion of the genome sequencing project and the tractability for genetic studies qualifies Dictyostelium for the study of fundamental cellular processes of pathogenesis.

BackgroundLegionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells.ResultsWild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter.ConclusionsTaken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease.

Legionella pneumophila (Lp) is commonly found in freshwa-13 C]glucose showed that the carbohydrate is also used as a substrate to feed the central metabolism. The specific labeling patterns due to [1,2-13 C 2 ]glucose identified the Entner-Doudoroff pathway as the predominant route for glucose utilization. In line with these observations, a mutant lacking glucose-6-phosphate dehydrogenase (⌬zwf) did not incorporate label from glucose at significant levels and was slowly outcompeted by the wild type strain in successive rounds of infection in Acanthamoeba castellanii, indicating the importance of this enzyme and of carbohydrate usage in general for the life cycle of Lp.

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of amoebae, macrophages, and epithelial cells. The pathology of Legionella infections involves alveolar cell destruction, and several proteins of L. pneumophila are known to contribute to this ability. By screening a genomic library of L. pneumophila, we found an additional L. pneumophila gene, plaB, which coded for a hemolytic activity and contained a lipase consensus motif in its deduced protein sequence. Moreover, Escherichia coli harboring the L. pneumophila plaB gene showed increased activity in releasing fatty acids predominantly from diacylphosphoand lysophospholipids, demonstrating that it encodes a phospholipase A. It has been reported that culture supernatants and cell lysates of L. pneumophila possess phospholipase A activity; however, only the major secreted lysophospholipase A PlaA has been investigated on the molecular level. We therefore generated isogenic L. pneumophila plaB mutants and tested those for hemolysis, lipolytic activities, and intracellular survival in amoebae and macrophages. Compared to wild-type L. pneumophila, the plaB mutant showed reduced hemolysis of human red blood cells and almost completely lost its cell-associated lipolytic activity. We conclude that L. pneumophila plaB is the gene encoding the major cell-associated phospholipase A, possibly contributing to bacterial cytotoxicity due to its hemolytic activity. On the other hand, in view of the fact that the plaB mutant multiplied like the wild type both in U937 macrophages and in Acanthamoeba castellanii amoebae, plaB is not essential for intracellular survival of the pathogen.Legionella pneumophila is an inhabitant of fresh water, where it intracellularly colonizes protozoa (20). When bacteria-laden aerosols are inhaled by humans, L. pneumophila exploits alveolar macrophages and epithelial cells for its multiplication, leading to a severe pneumonia characterized by destruction of alveolar cells (60). The cytopathology of Legionnaires' disease involves several cytotoxic or hemolytic factors produced by L. pneumophila, for example, the zinc metalloprotease ProA, the legiolysin Lly, and several pore-forming toxins, one of which is an RTX (repeats in structural toxin) protein (12,26,31,35,36,47,61). The zinc metalloprotease ProA is the major extracellular protease of L. pneumophila and its export depends on the L. pneumophila type II protein secretion system (28, 37). The enzyme hydrolyzes a broad spectrum of protein substrates and confers hemolytic as well as cytolytic activities (47, 53). Additionally, ProA has been shown to contribute to bacterial pathogenesis in a guinea pig model of pneumonia (38). Another hemolytic, but not cytotoxic, protein is the L. pneumophila legiolysin Lly, which is also responsible for color production and fluorescence of the bacterium (61). Pore-forming activities of L. pneumophila confer contact-dependent hemolytic and cytotoxic activities toward a variety of cells, especially at high bacterial nu...

Legionella pneumophila, the etiologic agent of Legionnaires' disease, contains a single, monopolar flagellum which is composed of one major subunit, the FlaA protein. To evaluate the role of the flagellum in the pathogenesis and ecology of Legionella, the flaA gene of L. pneumophila Corby was mutagenized by introduction of a kanamycin resistance cassette. Immunoblots with antiflagellin-specific polyclonal antiserum, electron microscopy, and motility assays confirmed that the specific flagellar mutant L. pneumophila Corby KH3 was nonflagellated. The redelivery of the intact flaA gene into the chromosome (L. pneumophila Corby CD10) completely restored flagellation and motility. Coculture studies showed that the invasion efficiency of the flaA mutant was moderately reduced in amoebae and severely reduced in HL-60 cells. In contrast, adhesion and the intracellular rate of replication remained unaffected. Taking these results together, we have demonstrated that the flagellum of L. pneumophila positively affects the establishment of infection by facilitating the encounter of the host cell as well as by enhancing the invasion capacity.Legionella pneumophila, the etiologic agent of Legionnaires' disease, is a ubiquitous microorganism inhabiting natural and man-made freshwater biotopes (5). In these environments, the gram-negative, rod-shaped bacteria survive as intracellular pathogens of protozoan organisms such as Acanthamoeba castellanii, Hartmannella vermiformis, and Naegleria spp. (15). Upon transmission to individuals via L. pneumophila-containing aerosols generated by showerheads and air-conditioning systems, the bacteria invade and multiply within alveolar macrophages (1, 2, 7) and nonphagocytic cells (17). The infection which mainly affects immunocompromised patients results in a life-threatening atypical pneumonia (7).Detailed ultrastructural and molecular studies of the intracellular fate of the bacterium revealed that human macrophages and protozoan cells infected with L. pneumophila exhibit remarkable similarities concerning the establishment of a replicative phagosome (3,16,22,42,45). However, significant differences were observed during early stages of infection (21). Uptake by Hartmannella is accomplished by a microfilamentindependent mechanism that is sensitive to methylamine, which is an inhibitor of receptor-mediated endocytosis (28). So far, one receptor of Hartmanella vermiformis, a Gal/GalNAc lectin, could be identified (46). Attachment of L. pneumophila to this lectin results in tyrosine dephosphorylation of multiple host cell proteins. However, depending on the type of amoeba, different receptors might be involved (22). In contrast, the uptake by human macrophages occurs following binding of complement receptors CR1 and CR3 via microfilament-dependent phagocytosis (26). In addition to this cytochalasin D-sensitive mechanism, complement-independent mechanisms for uptake by nonphagocytic cells have been described (39).The influence of bacterial motility on infection processes or on survival of legio...