Legionella pneumophila, the causative agent of Legionnaires' disease and related pneumonias, infects, replicates within and eventually kills human macrophages. A key feature of the intracellular life-style is the ability of the organism to replicate within a specialized phagosome which does not fuse with lysosomes or acidify. Avirulent mutants that are defective in intracellular multiplication and host-cell killing are unable to prevent phagosome-lysosome fusion. In a previous study, a 12 kb fragment of the L. pneumophila genome containing the icm locus (intracellular multiplication) was found to enable the mutant bacteria to prevent phagosome-lysosome fusion, to multiply intracellularly and to kill human macrophages. The complemented mutant also regained the ability to produce lethal pneumonia in guinea-pigs. In order to gain information about how L. pneumophila prevents phagosome-lysosome fusion and alters other intracellular events, we have studied the region containing the icm locus. This locus contains four genes, icmWXYZ, which appear to be transcribed from a single promoter to produce a 2.1-2.4 kb mRNA. The deduced amino acid sequences of the Icm proteins do not exhibit significant similarity to other proteins of known sequence, suggesting that they may carry out novel functions. The icmX gene encodes a product with an apparent signal sequence suggesting that it is a secreted protein. The icmWXYZ genes are located adjacent to and on the opposite strand from the dot gene, which is also required for intracellular multiplication and the ability of L. pneumophila to modify organelle traffic in human macrophages. Five L. pneumophila Icm mutants that had been generated with transposon Tn903dIIlacZ were found to have inserted the transposon within the icmX, icmY, icmZ and dot genes, confirming their role in the ability of the organism to multiply intracellularly.

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...

The maltose system in Escherichia coli consists of cell envelope-associated proteins and enzymes that catalyze the uptake and utilization of maltose and a,1-4-linked maltodextrins. The presence of these sugars in the growth medium induces the maltose system (exogenous induction), even though only maltotriose has been identified in vitro as an inducer (0. Raibaud and E. Richet, J. Bacteriol., 169:3059-3061, 1987). Induction is dependent on MalT, the positive regulator protein of the system. In the presence of exogenous glucose, the maltose system is normally repressed because of catabolite repression and inducer exclusion brought about by the phosphotransferase-mediated vectorial phosphorylation of glucose. In contrast, the increase of free, unphosphorylated glucose in the cell induces the maltose system. A ptsG ptsM glk mutant which cannot grow on glucose can accumulate ['4CJglucose via galactose permeases. In this strain, internal glucose is polymerized to maltose, maltotriose, and maltodextrins in which only the reducing glucose residue is labeled. This polymerization does not require maltose enzymes, since it still occurs in malT mutants. Formation of maltodextrins from external glucose as well as induction of the maltose system is absent in a mutant lacking phosphoglucomutase, and induction by external glucose could be regained by the addition of glucose-lphosphate entering the cells via a constitutive glucose phosphate transport system. malQ mutants, which lack amylomaltase, are constitutive for the expression of the maltose genes. This constitutive nature is due to the formation of maltose and maltodextrins from the degradation of glycogen.The Escherichia coli maltose system consists of a maltodextrin-specific pore (encoded by lamB) (17, 30) in the outer membrane and a binding-protein-dependent transport system in the cell envelope (encoded by malE malF malG malK) (38), as well as one periplasmic enzyme (encoded by malS) (35) and three cytoplasmic enzymes (encoded by malQ, malP, and malZ) (27,34,41). Expression of all mal genes depends on the positive regulator MalT (33).The transport system (11,12,20,24,40) can recognize and accumulate maltose and linear a,1-4-linked maltodextrins up to a chain length of seven glucose units (15). The major enzymes of the system (see Fig. 1) are the cytoplasmic amylomaltase (MalQ) (42) and maltodextrin phosphorylase (MalP) (34). Amylomaltase recognizes maltotriose and larger maltodextrins (donors), cleaving off the reducing glucose residue and transferring the remaining dextrinyl residue onto the nonreducing end of maltodextrin (acceptors), including maltose and glucose. With maltotriose, the smallest donor substrate, as well as with longer linear maltodextrins, amylomaltase thus produces glucose and longer maltodextrins (26). Maltodextrin phosphorylase subsequently releases glucose-i-phosphate from the nonreducing end of maltodextrins with a minimal chain length of five glucose residues (37). The glucose and glucose-l-phosphate are both transformed into glucose-6-phosphat...

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.

Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct factors. To understand the regulation of L. pneumophila flagellin expression, we cloned the factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA. FliA is a member of the 28 class of alternative factors identified in several bacterial genera. The gene fliA has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12. This library was transformed into a fliA mutant of E. coli K-12 containing a plasmid carrying the L. pneumophila-specific flaA promoter fused to the reporter gene luxAB. Screening the obtained transformants for luciferase activity, we isolated the major part of the fliA gene on a 1.64-kb fragment. This fragment was sequenced and used for reverse PCR in order to recover the complete fliA gene. The resulting 1.03-kb fragment was shown to contain the entire fliA gene. L. pneumophila FliA has 55 and 43% amino acid identity with the homologous sequences of Pseudomonas aeruginosa and E. coli. Furthermore, the L. pneumophila fliA gene was able to restore the flagellation and the motility defect of an E. coli fliA mutant. This result suggests that the L. pneumophila 28 protein can bind to the E. coli core RNA polymerase to direct transcription initiation from the flaA-specific promoter.Legionella pneumophila, a ubiquitous microorganism inhabiting freshwater biotopes and man-made water systems, is also a pathogen of humans causing severe pneumonia termed Legionnaires' disease. In the environment, the bacteria are able to replicate intracellularly in amoebae and other protozoa (14,39,46). Legionella infection occurs after inhalation of aerosolized bacteria. The bacteria enter the human lungs, where they are capable of invading and proliferating in alveolar macrophages and blood monocytes.A growing number of virulence factors elaborated by L. pneumophila contribute to the pathogenicity of the organism. These include a number of genetic loci that have been implicated in being important for intracellular growth (for a review, see reference 6). The icm locus is essential for the intracellular multiplication of L. pneumophila within human macrophages and is also required for virulence in guinea pigs (5, 30). Adjacent to icm is dot, a gene essential for intracellular replication and proper trafficking of host organelles during macrophage infection (3-5). Several surface-associated cellular components have been implicated in virulence. The Mip (macrophage infectivity potentiator) protein contributes to intracellular survival of L. pneumophila, and invasion experiments using a null mutation in mip revealed that Mip is required for early survival processes of L. pneumophila in eukaryotic cells (9, 10, 47). Convincing evidence suggests that the major outer membrane protein (encoded by ompS) is involved in the uptake of bacteria by the host cell (22). The major outer membr...