The Surfactant of Legionella pneumophila Is Secreted in a TolC-Dependent Manner and Is Antagonistic toward Other Legionella Species

Abstract

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.

Multiple Legionella pneumophila Type II Secretion Substrates, Including a Novel Protein, Contribute to Differential Infection of the Amoebae Acanthamoeba castellanii, Hartmannella vermiformis, and Naegleria lovaniensis

“…Strains representing other Legionella species that were also examined are listed in Table S2 in the supplemental material. Legionellae were routinely grown at 37°C on buffered charcoal-yeast extract (BCYE) agar, which, when appropriate, was supplemented with chloramphenicol at 3 g/ml, kanamycin at 25 g/ml, or gentamicin at 2.5 g/ml (27). Escherichia coli strain DH5␣ was the host for recombinant plasmids (Life Technologies, Carlsbad, CA).…”

“…To look for the presence of astacin-like activity, culture supernatants were assayed for their ability to trigger the release of Congo red from elastin-Congo red, as previously described (38,39). Swimming motility was determined by wet mount, and sliding motility was assessed by examining bacteria spotted onto BCYE medium containing only 0.5% agar (27).…”

“…DNA was isolated from L. pneumophila as described previously (27). Primers used for sequencing or PCR were obtained from Integrated DNA Technologies (Coralville, IA).…”

“…Others of our studies have shown that T2S also promotes the persistence of L. pneumophila in the environment by facilitating extracellular survival in low-temperature water samples (18,(22)(23)(24)(25). Furthermore, L. pneumophila T2S helps to mediate the secretion of a surfactant that confers both sliding motility and antibacterial activity (26,27). In the mammalian host, L. pneumophila T2S also has a multifactorial role by fostering intracellular multiplication in both macrophages and lung epithelial cells, dampening the chemokine and cytokine output from infected host cells, and elaborating tissue-destructive enzymes (7,16,28).…”

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

“…Among the secreted proteins that promote intracellular infection of amoebae are the novel proteins NttA and NttC as well as the PlaC acyltransferase, ProA metalloprotease, and SrnA RNase (26,(28)(29)(30). L. pneumophila T2S also promotes biofilm formation and sliding motility (31,32). In the lungs, T2S promotes infection of macrophages, suppression of innate immunity, and destruction of tissue (23,33,34).…”