Type II Secretion and Secreted Ppiases Promote Low Temperature Growth and Survival in Legionella pneumophilaPublic Deposited
PilD is an enzyme that processes prepilins that are part of the type II protein secretion apparatus and the type IV piliation machinery. Using a <em>Legionella pneumophila pilDlacZ</em> fusion strain to measure transcription, we observed a 20% increase in beta-galactosidase levels at 30°C vs. 37°C. At 25°C and 17°C vs. 37°C, this difference was 3-fold and 6-fold, respectively, indicating a temperature regulation of <em>pilD</em>. <em>L. pneumophila pilD</em> mutants had a severe growth defect in BYE broth and on BCYE plates at 12-25°C, but not at 30-37°C. This growth defect was due to the loss of the type II secretion and not the type IV pilus apparatus. Since a heavy streak or a sample of supernatants from strain 130b could restore the growth of the <em>lsp</em> mutants on low-temperature BCYE plates, we hypothesize that a type II dependent exoprotein(s) promotes low temperature growth and survival. The severe growth defect of the <em>lsp</em> mutants was partially due to the presence of iron in the media since growth at low temperatures improved significantly on BCYE plates without the iron supplement. In addition, type II secretion is important for long time survival in sterile tap water at low temperatures since strain 130b shows greater survivability than the <em>lsp</em> mutants after extended incubation at 4-17°C. To isolate secreted proteins important for growth at lower temperatures, we compared supernatants from strain 130b grown in BYE broth at 17°C and 37°C on 2D gels. Two protein spots repeatedly had higher intensities on the 17°C, 2D gel. Mass Spectrometry data identified the proteins in these spots as peptidyl proline isomerases (ppiases) encoded by the <em>L. pneumophila</em> genes <em>lpg1962</em> and <em>ppiB</em>. A <em>ppiB</em> mutant had a growth defect in BYE broth and up to a two log reduction in CFUs on BCYE plates at 17°C. An <em>lpg1962:ppiB</em> double mutant, but not the individual single mutants, had a growth defect in <em>A. castellanii</em> when amoeba infections were done at 22°C but not at 35°C. Since ppiases can modify the activity of other proteins, it is possible that these ppiases act by regulating the activity of other secreted proteins, a subset of which might be secreted through the type II secretion system.