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

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic amoebae and human macrophages. A key factor for L. pneumophila in intracellular infection is its type II protein secretion system (Lsp). In order to more completely define Lsp output, we recently performed a proteomic analysis of culture supernatants. Based upon the predictions of that analysis, we found that L. pneumophila secretes two distinct aminopeptidase activities encoded by the genes lapA and lapB. Whereas lapA conferred activity against leucine, phenylalanine, and tyrosine aminopeptides, lapB was linked to the cleavage of lysineand arginine-containing substrates. To assess the role of secreted aminopeptidases in intracellular infection, we examined the relative abilities of lapA and lapB mutants to infect human U937 cell macrophages as well as Hartmannella vermiformis and Acanthamoeba castellanii amoebae. Although these experiments identified a dispensable role for LapA and LapB, they uncovered a previously unrecognized role for the type II-dependent ProA (MspA) metalloprotease. Whereas proA mutants were not defective for macrophage or A. castellanii infection, they (but not their complemented derivatives) were impaired for growth upon coculture with H. vermiformis. Thus, ProA represents the first type II effector implicated in an intracellular infection event. Furthermore, proA represents an L. pneumophila gene that shows differential importance among protozoan infection models, suggesting that the legionellae might have evolved some of its factors to especially target certain of their protozoan hosts.Legionella pneumophila is the agent of Legionnaires' disease pneumonia (29). In fresh waters, this gram-negative bacterium exists in protozoan hosts and as a part of biofilms. Disease occurs when inhaled legionellae, possibly including those still associated with protozoa or protozoan vesicles (11, 12), invade alveolar macrophages. Although many factors promote the ecology and pathogenesis of L. pneumophila, protein secretion stands out for its multifaceted significance. L. pneumophila possesses type II secretion, type IVB secretion, and type IVA secretion (7, 17), and genome sequencing suggests the existence of type I and type V secretion systems (14,40). Among these pathways, the Lsp type II system is arguably implicated in the broadest array of phenotypes (17). Indeed, this pathway is required for secretion during growth in bacteriologic media at 37°C, extracellular survival in broth/water at 12 to 25°C, colony morphology, intracellular infection of protozoa (acanthamoebae and hartmannellae), optimal intracellular infection of human macrophages and monocytes, and virulence in a murine model of pneumonia (37,44,55,61,62,68,69). Although type II secretion exists in many gram-negative organisms, including various animal and plant pathogens, L. pneumophila is the only intracellular pathogen known to possess a functional type II system (17). Type II secretion is a two-step process in which nascent proteins ar...

The environmental pathogen Legionella pneumophila possesses five proteins with Sel1 repeats (SLRs) from the tetratricopeptide repeat protein family. Three of these proteins, LpnE, EnhC, and LidL, have been implicated in the ability of L. pneumophila to efficiently establish infection and/or manipulate host cell trafficking events. Previously, we showed that LpnE is important for L. pneumophila entry into macrophages and epithelial cells. In further virulence studies here, we show that LpnE is also required for efficient infection of Acanthamoeba castellanii by L. pneumophila and for replication of L. pneumophila in the lungs of A/J mice. In addition, we found that the role of LpnE in host cell invasion is dependent on the eight SLR regions of the protein. A truncated form of LpnE lacking the two C-terminal SLR domains was unable to complement the invasion defect of an lpnE mutant of L. pneumophila 130b in both the A549 and THP-1 cell lines. The lpnE mutant displayed impaired avoidance of LAMP-1 association, suggesting that LpnE influenced trafficking of the L. pneumophila vacuole, similar to the case for EnhC and LidL. We also found that LpnE was present in L. pneumophila culture supernatants and that its export was independent of both the Lsp type II secretion system and the Dot/Icm type IV secretion system. The fact that LpnE was exported suggested that the protein may interact with a eukaryotic protein. Using LpnE as bait, we screened a HeLa cell cDNA library for interacting partners, using the yeast two-hybrid system. Examination of the protein-protein interaction between LpnE and a eukaryotic protein, obscurin-like protein 1, suggested that LpnE can interact with eukaryotic proteins containing immunoglobulin-like folds via the SLR regions. This investigation has further characterized the contribution of LpnE to L. pneumophila virulence and, more specifically, the importance of the SLR regions to LpnE function.

Hawthorn (Crataegus) may play a role in the prevention and treatment of cardiovascular diseases such as hypertension, hyperlipidemia, and in particular, congestive heart failure. Evidence is accumulating that hawthorn may induce anti-ischemia/reperfusion-injury, anti-arrhythmic, hypolipidemic and hypotensive effects. These beneficial effects may in part be due to the presence of antioxidant flavonoid components. While a number of studies have been performed to evaluate the clinical efficacy of hawthorn, an international, multicenter, prospective clinical study including a large number of New York Heart Association (NYHA) class II/III heart failure patients is ongoing to test hawthorn's long-term therapeutic effects. Further clinical trials as well as pharmacokinetic and mechanistic studies are needed to explore and confirm its effectiveness, safety and pharmacological mechanism.

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...