Type II protein secretion is critical for Legionella pneumophila infection of amoebae, macrophages, and mice. Previously, we found several enzymes to be secreted by this (Lsp) secretory pathway. To better define the L. pneumophila type II secretome, a 2D electrophoresis proteomic approach was used to compare proteins in wild-type and type II mutant supernatants. We identified 20 proteins that are type II-dependent, including aminopeptidases, an RNase, and chitinase, as well as proteins with no homology to known proteins. Because a chitinase had not been previously reported in Legionella, we determined that wild type secretes activity against both p-nitrophenyl triacetyl chitotriose and glycol chitin. An lsp mutant had a 70 -75% reduction in activity, confirming the type II dependency of the secreted chitinase. Newly constructed chitinase (chiA) mutants also had Ϸ75% less activity, and reintroduction of chiA restored the mutants to normal levels of activity. Although chiA mutants were not impaired for in vitro intracellular infection, they were defective upon intratracheal inoculation into the lungs of A/J mice, and antibodies against ChiA were detectable in infected animals. In contrast, mutants lacking a secreted phosphatase, protease, or one of several lipolytic enzymes were not defective in vivo. In sum, this study shows that the output of type II secretion is greater in magnitude than previously appreciated and includes previously undescribed proteins. Our data also indicate that an enzyme with chitinase activity can promote infection of a mammalian host.bacterial protein secretion ͉ bacterial virulence ͉ Legionnaires' disease

The gram-negative bacterium Legionella pneumophila grows in both natural and man-made water systems and in the mammalian lung as a facultative intracellular parasite. The PilD prepilin peptidase of L. pneumophila promotes type IV pilus biogenesis and type II protein secretion. Whereas pili enhance adherence, Legionella type II secretion is critical for intracellular growth and virulence. Previously, we observed that pilD transcript levels are greater in legionellae grown at 30 versus 37°C. Using a new pilD::lacZ fusion strain, we now show that pilD transcriptional initiation increases progressively as L. pneumophila is grown at 30, 25, and 17°C. Legionella pilD mutants also had a dramatically reduced ability to grow in broth and to form colonies on agar at the lower temperatures. Whereas strains specifically lacking type IV pili were not defective for lowtemperature growth, mutations in type II secretion (lsp) genes greatly impaired the capacity of L. pneumophila to form colonies at 25, 17, and 12°C. Indeed, the lsp mutants were completely unable to grow at 12°C. The growth defect of the pilD and lsp mutants was complemented by reintroduction of the corresponding intact gene. Interestingly, the lsp mutants displayed improved growth at 25°C when plated next to a streak of wild-type but not mutant bacteria, implying that a secreted, diffusible factor promotes low-temperature growth. Mutants lacking either the known secreted acid phosphatases, lipases, phospholipase C, lysophospholipase A, or protease grew normally at 25°C, suggesting the existence of a critical, yet-to-be-defined exoprotein(s). In summary, these data document, for the first time, that L. pneumophila replicates at temperatures below 20°C and that a bacterial type II protein secretion system facilitates growth at low temperatures.

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...

Several Legionella pneumophila proteins were highly expressed in low-temperature supernatants. One of these proteins was the peptidyl-prolyl isomerase PpiB. Mutants lacking ppiB exhibited reduced growth at 17°C. Since PpiB lacked a signal sequence and was present in 17°C supernatants of type II and type IV secretion mutants, this protein may be secreted by a novel mechanism.

Legionella pneumophila is an aquatic bacterium that is also the agent of Legionnaires' disease pneumonia. Since L. pneumophila is transmitted directly from the environment to the lung, it is important to understand how legionellae survive at low temperatures. To identify genes that are needed for L. pneumophila growth at low temperature, we screened a population of mutagenized legionellae for strains that are specifically impaired for growth at 17°C. From the 7400 mutants tested, eleven displayed defects ranging from ca. 10-fold to a complete inability to grow at the low temperature. PCR and sequence analysis were then utilized to identify the genes whose loss had compromised growth. The proteins thereby implicated in low-temperature growth included components of the type II secretion system (LspE, LspG, LspH), a lipid A biosynthetic enzyme (LpxP), a ribonuclease (RNAse R), an RNA helicase (CsdA/DeaD), TCA cycle enzymes (citrate synthase), enzymes linked to fatty acid (FadB) or amino acid (aspartate aminotransferase) catabolism, and two putative membrane proteins that were, based upon their sequences, unlike previously characterized proteins. Given the magnitude of their mutant's defect, the aspartate aminotransferase, RNA helicase, and one of the putative membrane proteins were the factors most critical for L. pneumophila low-temperature growth. Thus, L. pneumophila not only employs some of the same processes and factors as other bacteria do in order to survive at low temperatures (e.g., LpxP, CsdA) but it also appears to possess novel modes of cold adaptation.Legionella pneumophila is a gram-negative γ-proteobacterium that is ubiquitous in natural and man-made water systems [17,29,45,60]. In its aquatic habitats, it exists planktonically, as an intracellular parasite of protozoa, and as a component of biofilms [35,39,45]. However, the ubiquity of L. pneumophila is also due to its capacity to survive at many temperatures, including those between 63°C and 4°C [17,29,56,60]. Beyond its natural niche, L. pneumophila is also a human pathogen, whereby the inhalation of contaminated water droplets from aerosolgenerating devices results in Legionnaires' disease [15]. Given that L. pneumophila is transmitted to humans directly from water sources, it is important to understand how the legionellae survive in water, including how they adapt to high and low temperature conditions in order to survive in both natural habitats throughout the year, from the warmer to colder months, and man-made systems that provide both warm and cold water. Since L. pneumophila grows best in the laboratory at 32 to 37°C, there are many studies devoted to understanding its physiology at the higher temperatures [56]. In contrast, studies on L. pneumophila at low temperatures are sparse, even though a large number of studies have identified factors that facilitate the low-temperature growth of other bacteria [7,9,43] [12,14,48,54]. Finally, a secreted peptidyl-prolyl isomerase (PpiB), whose secretion is not dependent upon T2S or the known...