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Browsing by Author "Coffey, Barbara M."
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Item Development of a New in vitro System for Cystic Fibrosis Research(Office of the Vice Chancellor for Research, 2013-04-05) Coffey, Barbara M.; Anderson, Gregory G.Individuals with cystic fibrosis (CF) have a life expectancy of 40 years and require daily treatments to mitigate the effects of the disease. CF impacts organs throughout the body, especially the lungs, where thick mucus builds up, impairs breathing, and provides an environment for bacterial growth. Chronic lung infection is the leading cause of mortality in CF. The majority of CF lung infections are caused by Pseudomonas aeruginosa, a common bacterium which typically does not cause disease in healthy individuals. In the CF lung, however, P. aeruginosa burrows into the thick mucus layer, evades the immune system, and resists antibiotic therapy by encasing itself in a protective matrix called a biofilm. Laboratory methods for studying biofilm are not true replicas of the CF lung environment, leaving a knowledge gap between how bacteria grow in a test tube (in vitro) and how they grow in the lungs of a person with CF. The focus of this work is to develop an improved laboratory model which combines artificial sputum (as a surrogate for mucus in the CF lung) and cultured CF airway epithelial cells. To assess the potential of this model, we have performed experiments to compare P. aeruginosa in artificial sputum versus standard laboratory media. Results demonstrate that P. aeruginosa in artificial sputum exhibits differences in growth, biofilm formation, toxin production, cytotoxicity, and protein expression, compared to results in standard media. These data suggest that our model system can contribute new information to the understanding of CF airway infection. The aim of future studies is to use this system to identify sputum components and bacterial proteins which have not been recognized previously by standard methods. It is our ultimate goal to contribute knowledge leading to improved longevity and quality of life for people with CF.Item DOES LOW MAGNESIUM IN CYSTIC FIBROSIS CONTRIBUTE TO BACTERIAL PATHOGENICITY?(2012-04-13) Coffey, Barbara M.; Anderson, Gregory G.Cystic fibrosis (CF) is a genetic disease for which there is currently no cure. Individuals with CF are plagued by myriad symptoms, including chronic pneumonia, which diminishes quality of life and reduces life expectancy to 40 years. The most common bacterium in CF patients’ lungs is Pseudomonas aeruginosa, a highly adaptable organism capable of surviving robust antibi-otic treatment. At the heart of developing improved treatments for CF pa-tients is the need to better understand P. aeruginosa pathogenicity. To this end, we have been studying the role of magnesium, which is often found at below normal levels in CF patients. Magnesium is an essential element in numerous cellular functions in both bacteria and humans. In previous re-search, we developed a P. aeruginosa strain with a deletion of the magnesi-um transport protein MgtE, as well as 16 plasmids carrying different muta-tions of the mgtE gene. Experiments with these constructs demonstrated a relationship between magnesium transport and bacterial toxin production. In the research presented here, we hypothesize that lower levels of magnesium may trigger a bacterial response, causing a change in P. aeruginosa patho-genicity. Changes may include differential growth, toxin release, and for-mation of biofilms, which are surface-adhered, antibiotic tolerant bacterial communities in a protective polysaccharide matrix. Using various magnesi-um levels, we have measured P. aeruginosa growth rates, motility, biofilm formation, and cytotoxicity toward cultured cells derived from the CF bron-chial epithelium. Preliminary results suggest that lower magnesium contrib-utes to changes in the bacterium that favor persistence in the CF lung. On-going studies include the effect of long-term growth of P. aeruginosa in low magnesium and how this impacts a number of virulence factors. We antici-pate that our research will elucidate the relationship between magnesium and P. aeruginosa pathogenicity and potentially lead to improved treatments for CF patients.Item Dual Functions of the Protein MgtE in Pseudomonas aeruginosa(2012-07-03) Coffey, Barbara M.; Anderson, Gregory G.; Marrs, James A.; Randall, Stephen K.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen which readily establishes itself in the lungs of people with cystic fibrosis (CF). Most CF patients have life-long P. aeruginosa infections. By modulating its own virulence and forming biofilms, P. aeruginosa is able to evade both host immune responses and antibiotic treatments. Previous studies have shown that the magnesium transporter MgtE plays a role in virulence modulation by inhibiting transcription of the type III secretion system, a mechanism by which bacteria inject toxins directly into the eukaryotic host cell. MgtE had already been identified as a magnesium transporter, and thus its role in regulating cytotoxicity was indicative of dual functions for this protein. This research focused on a structure-function analysis of MgtE, with the hypothesis that the magnesium transport and cytotoxicity functions could be exerted independently. Cytotoxicity assays were conducted using a co-culture model system of cystic fibrosis bronchial epithelial cells and a ∆mgtE strain of P. aeruginosa transformed with plasmids carrying wild type or mutated mgtE. Magnesium transport was assessed using the same mgtE plasmids in a Salmonella strain deficient in all magnesium transporters. Through analysis of a number of mgtE mutants, we found two constructs – a mutation in a putative magnesium binding site, and an N-terminal truncation – which demonstrated a separation of functions. We further demonstrated the uncoupling of functions by showing that different mgtE mutants vary widely in their ability to regulate cytotoxicity, whether or not they are able to transport magnesium. Overall, these results support the hypothesis of MgtE as a dual function protein and may lead to a better understanding of the mechanisms underlying P. aeruginosa virulence. By understanding virulence mechanisms, we may be able to develop treatments to reduce infections and pave the way to better health for people with cystic fibrosis.Item Successful Integration of Distributed Drug Discovery (D3) Components: Computational, Synthetic, and Biological Evaluation of Phenylalanine Derivatives as Potential Biofilm Inhibitors(Office of the Vice Chancellor for Research, 2013-04-05) Abraham, Milata M.; LaCombe, Jonathan M.; Carnahan, Jon M.; O'Donnell, Martin J. O.; Scott, William L.; Denton, Ryan E.; Samaritoni, J. Geno; Harper, Richard; Anderson, Gregory G.; Marrs, Kathleen A.; Coffey, Barbara M.Distributed Drug Discovery (D3) is a multidisciplinary approach to identifying molecules that exhibit activity in the treatment of neglected diseases such as malaria, leishmaniasis, and tuberculosis as well as recalcitrant cystic fibrosis (CF) airway infections. D3 seeks to accomplish this task by combining computational chemistry, synthetic chemistry, and biological screening all within an educational framework. Recent reports suggest that D-amino acids are effective in the disassembly and inhibition of bacterial biofilms, which are important for a number of bacterial infections, including those in the CF lung. Utilizing chemical drawing software, we constructed (enumerated) target phenylalanine derivatives from commercially available benzyl halides by substitution at the α position of an amino acid scaffold. A subset of these enumerated molecules was computationally selected for synthesis based on chemical properties. These compounds were synthesized using simple, solid-phase techniques in an undergraduate organic chemistry laboratory class. The resulting racemic unnatural amino acid derivatives were then screened for activity in a biofilm assay. The results show biofilm inhibition with synthesized phenylalanine derivatives. Analysis of the results reveals a trend between lipophilicity and the degree of biofilm inhibition. These new molecules may lead to an avenue for therapy for those CF individuals suffering with bacterial lung infection. As a part of the undergraduate curriculum, this work provides the first example of D3-linked undergraduate student computational analysis, synthesis, and biological evaluation.