Pseudomonas aeruginosa is an important opportunistic pathogen both in terms of the morbidity and mortality of infections it causes. Most patients with cystic fibrosis (CF), are colonized at an early age with this organism and most CF patients ultimately succumb to a chronic lung infection from P. aeruginosa. The reason for the extraordinary pathogenicity of P. aeruginosa in these patients, as compared to other Pseudomonads for example, is not clear. It is highly probable that the myriad of virulence determinants P. aeruginosa produces contributes to its pathogenic potential. Unfortunately, the exact contribution of these factors, alone or in combination, to even the simplest kind of P. aeruginosa infection has not yet been elucidated. In the past few years studies using molecular, biochemical and genetic approaches have begun to elucidate the structure-function relationships and mechanisms of regulation of virulence determinants. This research is directed at understanding the role of phospholipase C (PLC) production in the pathogenesis of P. aeruginosa infections. PLC has become recognized in recent years as a critical enzyme in both eukaryotic and prokaryotic biology. In eukaryotic organisms it is a critical second messenger in cellular processes, particularly in the function of specific and nonspecific immune mechanisms. In prokaryotic organisms there has been a resurgence of interest in PLC as a critical virulence determinant, both in gram negative and gram positive infections. P. aeruginosa produces two distinct PLCs that could play a significant role in the pathogenesis of lung, as well a other kinds of infections. One PLC is cytolytic (PLC-H) on human erythrocytes and neutrophils, while the other is not (PLC-N) lytic to these kind of cells. These and other features suggest structure-functions relationships between PLC activity and cytolytic activity that will be investigated in this research project. A more complete understanding of the structure-function relationships of both PLCs will lead to better understanding of their role in the pathogenesis of P. aeruginosa, and could result in therapeutic interventions for P. aeruginosa lung infections that were not previously considered. We also propose that derivatives of the substrate products produced by the action of both PLCs on phosphatidylcholine, the major essential lipid in lung surfactant, significantly contribute to the pathogenesis of P. aeruginosa infections. We hypothesize that some of these derivatives are especially relevant to the survival of this organism in the lungs of CF patients. We will investigate how a class of compounds, known as osmoprotectants including, glycine betaine, are able to induce the synthesis of both PLCs in P. aeruginosa. This compound, derived from the one of substrate products of both PLCs, can provide for the survival of this organism in a high osmotic environment, such as found in the lungs of CF patients or in the urinary tract. We propose that understanding this unusual regulatory process could lead to the discovery of novel agents which might at least temper the pathogenic potential of P. aeruginosa, if not directly affect its ability to persist in the lungs of CF patients, or survive in the high osmotic environment of the urinary tract.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL062608-09
Application #
6390366
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Peavy, Hannah H
Project Start
1993-06-01
Project End
2003-07-31
Budget Start
2001-09-01
Budget End
2002-07-31
Support Year
9
Fiscal Year
2001
Total Cost
$352,492
Indirect Cost
Name
University of Colorado Denver
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Ibarguren, Maitane; Sot, Jesús; Montes, L Ruth et al. (2013) Recruitment of a phospholipase C/sphingomyelinase into non-lamellar lipid droplets during hydrolysis of lipid bilayers. Chem Phys Lipids 166:12-7
Truan, Daphné; Vasil, Adriana; Stonehouse, Martin et al. (2013) High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa. Protein Expr Purif 90:40-6
Ibarguren, Maitane; López, David J; Montes, L-Ruth et al. (2011) Imaging the early stages of phospholipase C/sphingomyelinase activity on vesicles containing coexisting ordered-disordered and gel-fluid domains. J Lipid Res 52:635-45
Wargo, Matthew J; Gross, Maegan J; Rajamani, Sathish et al. (2011) Hemolytic phospholipase C inhibition protects lung function during Pseudomonas aeruginosa infection. Am J Respir Crit Care Med 184:345-54
Lopez, David J; Collado, M Isabel; Ibarguren, Maitane et al. (2011) Multiple phospholipid substrates of phospholipase C/sphingomyelinase HR2 from Pseudomonas aeruginosa. Chem Phys Lipids 164:78-82
Ibarguren, Maitane; Bomans, Paul H H; Frederik, Peter M et al. (2010) End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa. Biochim Biophys Acta 1798:59-64
Vasil, Michael L; Stonehouse, Martin J; Vasil, Adriana I et al. (2009) A complex extracellular sphingomyelinase of Pseudomonas aeruginosa inhibits angiogenesis by selective cytotoxicity to endothelial cells. PLoS Pathog 5:e1000420
Parker, Sarah K; Barkley, Robert M; Rino, John G et al. (2009) Mycobacterium tuberculosis Rv3802c encodes a phospholipase/thioesterase and is inhibited by the antimycobacterial agent tetrahydrolipstatin. PLoS One 4:e4281
Montes, L-Ruth; Lopez, David J; Sot, Jesus et al. (2008) Ceramide-enriched membrane domains in red blood cells and the mechanism of sphingomyelinase-induced hot-cold hemolysis. Biochemistry 47:11222-30
Parker, Sarah K; Curtin, Kathryn M; Vasil, Michael L (2007) Purification and characterization of mycobacterial phospholipase A: an activity associated with mycobacterial cutinase. J Bacteriol 189:4153-60

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