The diversity of roles that phospholipases C (PLCs) play in biology and medicine is extraordinary. In the past decade this class of phospholipid hydrolyzing enzymes has been shown to be considerably more complex than initially perceived and their impact on a wide range of basic cellular processes in eukaryotes, including oncogenesis, apoptosis, and inflammation has been increasingly appreciated. Likewise, there are many sundry and important functions for PLCs in microbial pathogenesis. We identified and characterized the first member of a novel class of homologous PLCs, the hemolytic phospholipase C (PIcH) of Pseudomonas aeruginosa. Members of this class of PLCs are produced by an array of opportunistic and frank pathogens, including potential bioterrorist agents. The genomes of some of these organisms encode as many as 4 homologs of this class of PLCs. Bacteria carrying genes encoding these PLCs (gene copy number shown in parentheses) include: P. aeruginosa (2), Mycobacterium tuberculosis (4), Francisella tularensis (1), Burkholderia pseudomallei and mallei (3 each) and Bordetella pertussis (1). We, as well as others, provided cogent evidence that members of this novel class of PLCs play significant and diverse roles in the infectious diseases caused by those agents. Although these PLCs share considerable amino acid homology, each member has distinct properties. There are some important differences in their substrate specificities, and many members have unique structural features that probably play a specific functional role in the pathogenesis of the organisms that produce them. This application will mainly focus on the paradigm of this novel class of PLCs (PIcH). In addition to its PLC activity, PIcH is the first prokaryotic or eukaryotic protein yet identified that has Sphingomyelin Synthase activity. The substrates (e.g. phosphatidylcholine & sphingomyelin) of PIcH or the products (e.g. diacylglycerol, ceramide or sphingomyelin) that it generates could have profound biological effects, particularly with respect to signaling processes in eukaryotic cells and the host responses to this infectious agent. We have also provided evidence that PIcH is highly cytotoxic for endothelial cells and probably enters these cells through interaction with integrin receptors. This research project will employ microbiological, genetic, biochemical, structural and cell biology methods to examine how PIcH and other members of this novel class of enzymes affect the virulence of the organisms that produce them. Furthermore, it is likely that information we derive from our efforts will also provide additional insights about the biochemistry and biology of PLCs in general.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL062608-11
Application #
6726336
Study Section
Special Emphasis Panel (ZRG1-BM-1 (01))
Program Officer
Peavy, Hannah H
Project Start
1993-06-01
Project End
2008-08-31
Budget Start
2003-09-26
Budget End
2004-08-31
Support Year
11
Fiscal Year
2003
Total Cost
$448,907
Indirect Cost
Name
University of Colorado Denver
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
041096314
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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