Importance of Type II Secretion for Survival of Legionella pneumophila in Tap Water and in Amoebae at Low Temperatures

Abstract

Legionella pneumophila type II secretion mutants showed reduced survival in both tap water at 4 to 17°C and aquatic amoebae at 22 to 25°C. Wild-type supernatants stimulated the growth of these mutants, indicating that secreted factors promote low-temperature survival. There was a correlation between low-temperature survival and secretion function when 12 additional Legionella species were examined.Legionella pneumophila is widespread in natural and manmade water systems (8,21,29,39,41,47,53,64). In these habitats, L. pneumophila exists planktonically, within protozoa, and in biofilms (14,35,36,41,45). The ubiquity of L. pneumophila is also a result of the organism's ability to survive at many temperatures, including ones as low as 4°C (21,29,62,64). L. pneumophila is an important pathogen of humans, with the inhalation of contaminated water droplets originating from aerosol-generating devices resulting in Legionnaires' disease (16). Given the manner in which infection occurs, it is important to better understand how legionellae survive in water, in protozoa, and at low temperatures. Recently, we found that L. pneumophila type II protein secretion is critical for growth in rich broth or agar at 12 to 25°C but not in medium at 30 to 37°C (56). Operative in many gram-negatives (9), type II secretion is a multistep process in which proteins are translocated across the inner membrane in a Sec-or Tat-dependent manner, recognized in the periplasm, and then delivered to the T2S apparatus, whereupon a pilus-like structure "pushes" proteins through a dedicated outer membrane pore or secretin (28).To investigate the connection between type II secretion and low-temperature survival under conditions that more closely mimic natural habitats, we compared wild-type serogroup 1 strain 130b (Table 1) and its type II secretion mutants for persistence in tap water incubated at 37°C, 25°C, and 17°C. We used three mutants: NU258, containing a mutation in the genes encoding the type II outer membrane secretin (lspD) and the inner membrane ATPase (lspE); NU275, containing a mutation in the gene for the inner membrane platform protein (lspF); and NU272 mutated in the gene encoding the pseudopilin peptidase (pilD) (51). Tap water was obtained from laboratory sinks and filter sterilized. Following growth at 37°C in buffered yeast extract (BYE) broth to late log phase (56), wild types and mutants were inoculated into flasks containing 50 ml of the tap water, and then the cultures were incubated with shaking. As with other wild-type L. pneumophila (30,41,42,54,57), 130b persisted in low-temperature tap water for extended times (Fig. 1). Also similar to previous work (27), the recovery of CFU was maintained for a longer period at low temperatures below 37°C. But across the 17 to 37°C range, the secretion mutants behaved differently than their parent (Fig. 1). At 37°C, the mutants displayed a greater recoverability than 130b between days 7 and 20 (P Ͻ 0.05). In a similar vein, at 25°C, the mutants were recovered more than the wild type was be...

Surface Translocation byLegionella pneumophila: a Form of Sliding Motility That Is Dependent upon Type II Protein Secretion

“…One Legionella species that stands out as being unusual is L. micdadei. Indeed, all six strains of this species tested clearly failed to display surface translocation and surfactant production, and such a finding is compatible with previous work showing that L. micdadei strains do not express other T2S-associated phenotypes despite harboring lsp genes (110). Based on our latest results in Legionella, L. micdadei not withstanding, it is quite likely that the T2S systems of other types of gram-negative bacteria also promote surfactant secretion and surface translocation.…”

“…This might have been due to either some form of suppression of the pilD and lsp mutations or some T2S mutant lysis that released a stimulatory substance whose secretion had been blocked by the mutation. In past studies of L. pneumophila T2S mutants, we observed examples of both suppression and lysis (30,110). Because T2S mutants grow comparably to the wild type at 30°C (111), their reduced ability to spread was not an indirect effect of a generalized growth defect.…”

“…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 epithelia (27,106,113).…”

Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis inVibrio cholerae

“…In addition, our studies showed that V. cholerae T2S mutants are unable to survive the passage through the infant mouse gastrointestinal tract (65). Growth defects at low temperatures under laboratory conditions as well as in tap water and amoebae were also observed for T2S mutants of Legionella pneumophila (68).…”

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

“…In the aquatic environment, T2S promotes L. pneumophila survival at low temperatures and is critical for infection of at least four genera of amoebae (13)(14)(15). In mammalian hosts, T2S contributes to both intracellular infection of macrophages and the destruction of lung tissue.…”