Legionella pneumophila, the agent of Legionnaires' disease pneumonia, is transmitted to humans following the inhalation of contaminated water droplets. In aquatic systems, L. pneumophila survives much of time within multi-organismal biofilms. Therefore, we examined the ability of L. pneumophila (clinical isolate 130b) to persist within biofilms formed by various types of aquatic bacteria, using a bioreactor with flow, steel surfaces, and low-nutrient conditions. L. pneumophila was able to intercalate into and persist within a biofilm formed by Klebsiella pneumoniae, Flavobacterium sp. or Pseudomonas fluorescens. The levels of L. pneumophila within these biofilms were as much as 4×104 CFU per cm2 of steel coupon and lasted for at least 12 days. These data document that K. pneumoniae, Flavobacterium sp., and P. fluorescens can promote the presence of L. pneumophila in dynamic biofilms. In contrast to these results, L. pneumophila 130b did not persist within a biofilm formed by Pseudomonas aeruginosa, confirming that some bacteria are permissive for Legionella colonization whereas others are antagonistic. In addition to colonizing certain mono-species biofilms, L. pneumophila 130b persisted within a two-species biofilm formed by K. pneumoniae and Flavobacterium sp. Interestingly, the legionellae were also able to colonize a two-species biofilm formed by K. pneumoniae and P. aeruginosa, demonstrating that a species that is permissive for L. pneumophila can override the inhibitory effect(s) of a non-permissive species.

The type II secretion (T2S) system of Legionella pneumophila is required for the ability of the bacterium to grow within the lungs of A/J mice. By utilizing mutants lacking T2S (lsp), we now document that T2S promotes the intracellular infection of both multiple types of macrophages and lung epithelia. Following infection of macrophages, lsp mutants (but not a complemented mutant) elicited significantly higher levels of interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-␣), IL-10, IL-8, IL-1␤, and MCP-1 within tissue culture supernatants. A similar result was obtained with infected lung epithelial cell lines and the lungs of infected A/J mice. Infection with a mutant specifically lacking the T2S-dependent ProA protease (but not a complemented proA mutant) resulted in partial elevation of cytokine levels. These data demonstrate that the T2S system of L. pneumophila dampens the cytokine/chemokine output of infected host cells. Upon quantitative reverse transcription (RT)-PCR analysis of infected host cells, an lspF mutant, but not the proA mutant, produced significantly higher levels of cytokine transcripts, implying that some T2S-dependent effectors dampen signal transduction and transcription but that others, such as ProA, act at a posttranscriptional step in cytokine expression. In summary, the impact of T2S on lung infection is a combination of at least three factors: the promotion of growth in macrophages, the facilitation of growth in epithelia, and the dampening of the chemokine and cytokine output from infected host cells. To our knowledge, these data are the first to identify a link between a T2S system and the modulation of immune factors following intracellular infection.The aquatic bacterium Legionella pneumophila is the primary agent of Legionnaires' disease, a potentially fatal form of pneumonia (38). L. pneumophila is especially pathogenic for the immunocompromised, the elderly, and smokers. Recent studies highlight the significance of travel-associated Legionnaires' disease and an increasing incidence of legionellosis (79). In natural and man-made waters, Gram-negative L. pneumophila survives planktonically, in biofilms, and as an intracellular parasite of protozoa (115). Infection occurs after the inhalation of contaminated droplets that originate from a variety of aerosol-generating devices as well as potable waters (33, 86). In the lung, L. pneumophila multiplies in resident alveolar macrophages, although persistence might also involve growth in alveolar epithelia and extracellular survival (82). Much of the pathogenesis and ecology of L. pneumophila is mediated by secreted factors, including protein and nonprotein molecules (2,25,27,44,57). For secreting proteins into the extracellular milieu and/or target host cells, L. pneumophila uses both the type II secretion (T2S) and the type IV secretion (T4S) system, large membrane-spanning apparatuses that are each composed of at least 10 component proteins (27,82).T2S promotes the physiology and ecology of many bacteria as well as the virulence o...

Legionella pneumophila exhibits surface translocation when it is grown on a buffered charcoal yeast extract (BCYE) containing 0.5 to 1.0% agar. After 7 to 22 days of incubation, spreading legionellae appear in an amorphous, lobed pattern that is most manifest at 25 to 30°C. All nine L. pneumophila strains examined displayed the phenotype. Surface translocation was also exhibited by some, but not all, other Legionella species examined. L. pneumophila mutants that were lacking flagella and/or type IV pili behaved as the wild type did when plated on low-percentage agar, indicating that the surface translocation is not swarming or twitching motility. A translucent film was visible atop the BCYE agar, advancing ahead of the spreading legionellae. Based on its abilities to disperse water droplets and to promote the spreading of heterologous bacteria, the film appeared to manipulate surface tension and, as such, acted like a surfactant. Indeed, a sample obtained from the film rapidly dispersed when it was spotted onto a plastic surface. L. pneumophila type II secretion (Lsp) mutants, but not their complemented derivatives, were defective for both surface translocation and film production. In contrast, mutants defective for type IV secretion exhibited normal surface translocation. When lsp mutants were spotted onto film produced by the wild type, they were able to spread, suggesting that type II secretion promotes the elaboration of the Legionella surfactant. Together, these data indicate that L. pneumophila exhibits a form of surface translocation that is most akin to "sliding motility" and uniquely dependent upon type II secretion.The genus Legionella was established in 1977, following the isolation of Legionella pneumophila from patients with a form of pneumonia now known as Legionnaires' disease (33). Today, L. pneumophila is recognized as a common cause of both community-acquired and nosocomial pneumonia (84). Legionellosis occurs sporadically and in large outbreaks, with the largest outbreak occurring as recently as 2003 and encompassing 800 suspected and 449 confirmed cases (43). L. pneumophila is especially pathogenic for the elderly and the immunocompromised, large and growing segments of the population (39, 84), and recent studies have been highlighting the growing significance of travel-associated Legionnaires' disease (107). L. pneumophila is a gram-negative, gammaproteobacterium that is widespread in natural and manufactured water systems (22,39,93). Infection occurs after the inhalation of Legionellacontaminated water droplets originating from a wide variety of aerosol-generating devices (39). Alarmingly, outbreaks can occur following the airborne spread of L. pneumophila over distances of Ͼ10 km from cooling towers or scrubbers (86). Within its aquatic habitats, L. pneumophila survives over a wide temperature range and grows on surfaces, in biofilms, and as an intracellular parasite of protozoa (9,39,110). Within the mammalian lung, the organism has the ability to attach to and invade macrophages and epit...

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum-and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various nonLegionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.