Legionella pneumophila (Lp) is the primary agent of Legionnaires'disease, a common and potentially fatal form of pneumonia. The bacterium is ubiquitous in natural and man-made water systems and infects the lungs after the inhalation of contaminated aerosols. In water and the lungs, Lp grows as an intracellular parasite, infecting either aquatic protozoa, macrophages, or epithelia. Previously, we determined that Lp possesses a type II secretion (T2S) system and hypothesized that proteins secreted by T2S are mediators of environmental persistence, intracellular infection, and virulence. During the last grant period, we documented that T2S is required for intracellular infection of amoebae, alveolar macrophages, and lung epithelia as well as bacterial persistence in the lungs of experimentally-infected mice. Proteomic analysis of Lp supernatants and in silico analysis of the Lp genome were then combined to reveal the extensive nature of the T2S output. In addition to predicted activities, which we confirmed, "novel" exoproteins were identified that had no similarity to known proteins. Using mutants lacking one or more T2S-dependent exoproteins (effectors), we determined that a metalloprotease (ProA) and ribonuclease (SrnA) promote amoeba infection, phospholipases C (PlcA/B) and a chitinase (ChiA) facilitate infection of mammalian cells, and a novel protein (Lpg0264) promotes both amoebal and macrophage infection. In vivo competition assays then identified four T2S effectors that appear to promote Lp survival in lungs;i.e., ChiA, PlcA/B, Lpg0264, and an astacin-like protease (LegP). Additionally, we discovered that Lp exhibits a swarming phenotype on agar surfaces that is dependent upon T2S. In sum, Lp T2S is uniquely critical for intracellular infection, swarming, virulence, as well as low-temperature survival in water, elaborating more effectors and encoding a wider variety of activities than is appreciated for any other T2S system. In the current proposal, we will use genetics and various infection models and cell biological tools to i) determine the importance of a select number of new "novel" effectors for intracellular infection, ii) determine the importance of T2S-dependent swarming for intracellular infection, iii) determine the intracellular location of and trafficking patterns influenced by ProA, SrnA, PlcA/B, and Lpg0264, and iv) confirm the importance of PlcA/B, Lpg0264, and LegP, the new novel effectors, and swarming in a murine model of pneumonia. The proposed studies will i) increase significantly our understanding of the pathogenesis of Lp, which is an important public health concern within the US and throughout the world, ii) expand our molecular understanding of the both bacterial protein secretion and intracellular infection, iii) have implications for other important, environmental pathogens that utilize or are predicted to use T2S, and iv) offer potential new targets for disease diagnosis, treatment, or prevention.

Public Health Relevance

We have discovered that the so-called type II secretion system of Legionella pneumophila promotes bacterial growth in mammalian host cells (e.g., macrophages and epithelial cells) and in the lungs of experimentally infected mice. We therefore hypothesize that the proteins (effectors) secreted by the bacterium through this system are virulence factors and therefore are potential targets for disease diagnosis or prevention. To test this hypothesis, we will i) characterize bacterial mutants that are lacking specific effectors using in vitro models of intracellular infection and an animal model of pneumonia in order to identify those effectors that promote infection, and ii) monitor the intracellular expression patterns of those proteins that are shown to be important for infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI043987-14
Application #
8197087
Study Section
Special Emphasis Panel (ZRG1-IDM-H (02))
Program Officer
Korpela, Jukka K
Project Start
1998-12-01
Project End
2013-11-30
Budget Start
2011-12-01
Budget End
2012-11-30
Support Year
14
Fiscal Year
2012
Total Cost
$298,931
Indirect Cost
$102,911
Name
Northwestern University at Chicago
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
005436803
City
Chicago
State
IL
Country
United States
Zip Code
60611
Gunderson, Felizza F; Cianciotto, Nicholas P (2013) The CRISPR-associated gene cas2 of Legionella pneumophila is required for intracellular infection of amoebae. MBio 4:e00074-13
Karaba, Sara M; White, Richard C; Cianciotto, Nicholas P (2013) Stenotrophomonas maltophilia encodes a type II protein secretion system that promotes detrimental effects on lung epithelial cells. Infect Immun 81:3210-9
Tyson, Jessica Y; Pearce, Meghan M; Vargas, Paloma et al. (2013) 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. Infect Immun 81:1399-410
Pearce, Meghan M; Theodoropoulos, Nicole; Noskin, Gary A et al. (2011) Native valve endocarditis due to a novel strain of Legionella. J Clin Microbiol 49:3340-2
Stewart, Catherine R; Burnside, Denise M; Cianciotto, Nicholas P (2011) The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species. J Bacteriol 193:5971-84
McCoy-Simandle, Kessler; Stewart, Catherine R; Dao, Jenny et al. (2011) Legionella pneumophila type II secretion dampens the cytokine response of infected macrophages and epithelia. Infect Immun 79:1984-97
Soderberg, Maria A; Cianciotto, Nicholas P (2010) Mediators of lipid A modification, RNA degradation, and central intermediary metabolism facilitate the growth of Legionella pneumophila at low temperatures. Curr Microbiol 60:59-65
Cianciotto, Nicholas P (2009) Many substrates and functions of type II secretion: lessons learned from Legionella pneumophila. Future Microbiol 4:797-805
Rossier, Ombeline; Dao, Jenny; Cianciotto, Nicholas P (2009) A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis. Microbiology 155:882-90
Pearce, Meghan M; Cianciotto, Nicholas P (2009) Legionella pneumophila secretes an endoglucanase that belongs to the family-5 of glycosyl hydrolases and is dependent upon type II secretion. FEMS Microbiol Lett 300:256-64

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