| Popis: |
The gram-negative bacteria, Pseudomonas aeruginosa, is an opportunistic pathogen. P. aeruginosa forms biofilms by altering gene expression, through cellular signaling, to protect the cells from environmental stressors. Our goal is to investigate the role of biomolecules and genes contributing to the formation of P. aeruginosa biofilms. Insight into the pathways associated with biofilm production can illuminate targetable methods to fight future P. aeruginosa infections.Genes encode proteins that control cellular function in biological systems. Each gene contributes to specific pathways during cellular development. To understand the role of each gene, targeted gene knockout methodologies have been developed for bacterial and eukaryotic systems. We have designed a protocol for the creation of specific gene knockouts in P. aeruginosa with the use of plasmids incorporated with the lambda-red recombinase system. We have proven the effectiveness of these methodologies with the generation of a PA14 knockout mutant.Matrix assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) has emerged as a useful for the identification of molecules associated with microorganisms.The diversity of molecules in bacterial cultures leads to difficult analysis in whole cell samples. We have tested multiple common MALDI matrices to determine ionization preference for major molecules including rhamnolipids, phospholipids, and quinolones in P. aeruginosa. We have putatively identified nominal ions by collision induced dissociation (CID) fragmentation in all matrices tested.Biofilms are complex aggregations of cells encased in an extracellular matrix. This matrix assists in cellular signaling by small molecules in the event of environmental signals to produce a response. Different cell subpopulations are localized throughout the biofilm to respond to these environmental signals. We have identified 3 different cell subpopulations in a P. aeruginosa biofilm. We have determined that these subpopulations display different morphologies and phenotypes. Through RNA-seq analysis we have also determined these subpopulations have varying expression levels of genes associated with the intracellular second messenger 3’,5’-cyclic diguanosine monophosphate (cyclic-di-GMP). High levels of intracellular cyclic-di-GMP have been associated with biofilms and there is evidence that subpopulations have varying levels. Extracellular concentrations of cyclic-di-GMP are less researched. We have developed an extraction method and liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitation of cyclic-di-GMP from both cell and supernatant samples. Similar extraction methods have also been applied for the extraction and analysis of nicotinic acid adenine nucleotide diphosphate (NAADP) from Jurkat cell lysates. We have developed LC-MS methods that can be used for direct quantification of the potent Ca2+ releasing second messenger NAADP. These analyses and methods will help elucidate how these second messengers contribute to cellular functionality. |
