Browsing by Subject "Quorum sensing"
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Item Characterization and microfabrication of environmentally sensitive materials for studying bacterial group behaviors(2012-08) Connell, Jodi Lynn; Shear, Jason B.; Ellington, Andrew D.This dissertation describes the development and application of an approach for creating multiphoton crosslinked protein microchambers to characterize bacterial group behaviors in small populations (~10¹ - 10⁵ cells). Porous protein cavities of desired size and geometry are made with sub-micrometer three-dimensional (3D) resolution using a dynamic mask-based multiphoton lithography (MPL) technique previously developed in the Shear Group. One aspect of this dissertation focuses on basic characterizations of properties of these materials key to their utility in studying entrapped bacteria. Studies are presented on the mass transport across microcavity walls (important for growth and signaling), and the temperature- and light-induced volume response (used to open/close microchamber apertures for cell entry/exit). Fabrication parameters are optimized to trap and manipulate small populations under in vitro conditions that are relevant to in vivo environments. The ability to culture bacteria at physiologic growth rates within protein microstructures has provided a unique platform to study the group behaviors of quorum sensing (QS) and antibiotic resistance in biologically relevant population sizes, a platform I have exploited to study group behaviors in the opportunistic pathogen, Pseudomonas aeruginosa. This work presents the first experimental evidence supporting the efficiency sensing QS model by showing that QS-dependent gene expression is affected by both the population size and density, as well the external flow rate in the surrounding environment. The onset of antibiotic resistance is observed in as few as ~150 P. aeruginosa cells, and is shown to increase with cell density. Lastly, the development of a gelatin-based MPL approach that is demonstrated in situ to create confined populations of non-motile cells, free-floating 3D cultures, nested colonies, and spatially patterned polymicrobial communities of P. aeruginosa and Staphylococcus aureus.Item Influence of autoinducer 2 (ai-2) and ai-2-like inhibitors generated from ground beef on escherichia coli o157:h7 protein expression(2009-05-15) Soni, Kamleshkumar A.Autoinducer 2 (AI-2) molecules produced by bacterial cells are thought to be involved in controlling a variety of bacterial cellular processes by coordinated gene and protein expression. Previous work in our laboratory has shown that ground beef contains compounds that can interfere with AI-2-mediated bioluminescence expression in Vibrio. harveyi. The underlying hypothesis of this work was that AI-2 molecules affect the protein expression in Escherichia coli O157:H7 and AI-2 inhibitory molecules negate the influence of AI-2 molecules. The main objectives of this study were to identify, characterize, and isolate the factors responsible for inhibition of AI-2 molecules from ground beef extracts, elucidate the role of LuxS/AI-2 cell signaling system in E. coli O157:H7 protein expression, and determine if inhibitory factors present in ground beef extract can negate the influence of AI-2 molecules on the protein expression. Using a solvent extraction procedure and gas chromatography analysis, AI-2 inhibitory factors present in ground beef extracts were identified as both medium and long chain fatty acids. When identified fatty acids were tested at different concentrations for AI-2 inhibition, AI-2 inhibition ranging from 25% to 90% was observed. Both ground beef extracts and mixture of selected fatty acids also resulted in 2- to 4-fold reduced AI-2 influenced biofilm formation by E. coli K12 cells. Identification of LuxS/AI-2-mediated protein expression in E. coli O157:H7 was conducted using two dimensional gel electrophoresis. Protein expression analysis showed that the LuxS/AI-2 system modulates the expression of proteins involved in different cellular processes such as carbohydrate and amino acid metabolism, stress response, and formation of flagella and motility. When AI-2 inhibitory factors were added along with AI-2 molecules, the expression patterns of three AI-2-influenced proteins (GlmS, SpeE, and NikA) were changed suggesting that AI-2 inhibitors can negate the influence of AI-2 molecules on protein expression of selected proteins. Collectively, these results highlight that proteins associated with different cellular processes in E. coli O157:H7 can be modulated depending on whether cells are in contact with AI-2 molecules in the presence or absence of AI-2 inhibitory factors.Item Synthesis and biological research of the pseudomonas aeruginosa signaling compounds PQS and N-3-oxo-dodecanoyl-L-homoserine lactone(2011-05) Olmos, Aaron J.; Li, Guigen; Rumbaugh, Kendra P.; Fuertes, Michael J.In order to obtain a better understanding of the complex host-pathogen interactions involved in chronic infections, the Pseudomonas aeruginosa signaling molecules 3-oxo-C12-homoserine lactone and Pseudomonas quinolone signal were synthesized so that the mammalian cellular responses to their presence via an infection could be studied. It was hypothesized that differing combinations of the signaling molecules would result in the up regulation of host cytokines that are involved in the disease process. Previous studies have not taken into consideration the combined effect of the major signal molecules that would be present in a Pseudomonas aeruginosa infection. Cultured human A549 lung epithelial cells were taken as a model for the study of an invitro cystic fibrosis type infection, where the change in genetic transcription could be easily determined. The presence of cytokines produced during Pseudomonas aeruginosa infections was established based upon reverse transcriptase PCR followed by the amplification of cytokine cDNA and analysis by gel electrophoresis.