Browsing by Author "Bennett, George N"

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    Biofilm-Mediated Regulation of Siderophore Production in Pseudomonas aeruginosa
    (2019-06-04) Kang, Donghoon; Kirienko, Natalia V; Bennett, George N
    Pseudomonas aeruginosa is a gram-negative, multidrug-resistant, nosocomial pathogen that threatens the lives of immunocompromised patients. A key virulence factor in this pathogen is the siderophore pyoverdine. Due to its extremely high affinity for ferric iron, pyoverdine gives the pathogen a significant advantage over the host in their competition for iron. In addition, pyoverdine can regulate the production of multiple bacterial virulence factors and perturb host mitochondrial homeostasis. To elucidate the regulation of pyoverdine production, we developed a high-throughput genetic screen to identify genes necessary for its biosynthesis. Through this screen, we demonstrated that biofilm formation is necessary for pyoverdine production. Consistent with this result, upstream regulators of biofilm, notably intracellular c-di-GMP, regulated pyoverdine production in a biofilm-dependent manner. Furthermore, inhibiting biofilm formation using 2-amino-5,6-dimethylbenzimidazole was sufficient to attenuate pyoverdine production, suggesting that biofilm inhibitors may have multiple therapeutic benefits in addition to disrupting the pathogen’s main mechanism of antimicrobial resistance.
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    Insight into peroxisome biogenesis and metabolism from a microscopy-based screen for Arabidopsis thaliana mutants with deviations in peroxisomal morphology, distribution, or import
    (2016-04-20) Rinaldi, Mauro Adriel; Bartel, Bonnie; Bennett, George N; Raphael, Robert
    Key steps of vital metabolic pathways are housed in peroxisomes, essential organelles. Intensive research has advanced our understanding of peroxisomes, but there are still gaps in our models. For example, we do not fully understand the additional roles of β-oxidation and peroxisomal cofactor homeostasis. Therefore, I conducted a microscopy-based screen for aberrant distribution of peroxisomally-targeted fluorescence in Arabidopsis thaliana. This screen uncovered novel alleles defective in lipid body mobilization, fatty acid β-oxidation, the glyoxylate cycle, peroxisome fission, auxin metabolism, pexophagy and other peroxisomal processes. Weak mutants in lipid mobilization retained lipid bodies even without displaying other defects, suggesting that microscopy was sensitive to small deficiencies and that fatty acid β-oxidation happens at higher rates than those needed for normal growth. Moreover, analysis of these mutants revealed that fatty acid β-oxidation was needed for peroxisomal matrix protein import and wild-type peroxisome morphology. Epistasis analysis suggested that CoA and NAD+ import contributes to the peroxisomal pool and to β-oxidation. Mutants defective in PECTIN METHYLESTERASE 31 were also recovered, suggesting a role in lipid mobilization for this cytosolic protein. Peroxisomal processes rely on the action of peroxins (PEX proteins), factors needed to build and maintain peroxisomes. One of the most important peroxins is the ATPase PEX1, which together with the similar ATPase PEX6, recycles the peroxisomal matrix protein receptor PEX5 from the peroxisome membrane following cargo delivery. Mutations in human PEX1 can lead to lethal peroxisomal biogenesis disorders. However, very little is known about the role of PEX1 in plants. My screen yielded a PEX1 mutation that was lethal when homozygous, suggesting PEX1 is needed for peroxisomes and life. pex1 mutants accumulated reduced levels of PEX5 and PEX6, suggesting that PEX1 promotes PEX5 and PEX6 stability. PEX6 overexpression rescued a pex1 mutant. These data suggest that the heterohexamer model of PEX1-PEX6 function developed in other organisms is conserved in plants. Unlike PEX6 overexpression, PEX1 overexpression in Arabidopsis leads to growth defects, suggesting that PEX1 levels must remain within a narrow range. The screen performed for this thesis demonstrates the sustained power of forward genetic screens to uncover new factors in biological processes.
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    Regulation of the neurovascular patterning by growth factors and cytokines during anterior ocular development
    (2015-12-16) Ojeda Cardenas, Ana; Bennett, George N; Jacot, Jeffrey G; McNew, James A; Tao, Yizhi J
    The cornea is a transparent, avascular, and one of the most innervated tissue of the body. Corneal diseases including injuries, neovascularization, congenital eye defects and degenerations, represent a major public health burden. Although, studies have been focused on understanding the basis of transparency, innervation, and neovascularization of the adult cornea, little is known about the molecular mechanisms that lead to this specialized structure results in a highly innervated but avascular tissue during embryogenesis. The purpose of this work was to identify molecular regulators of the neurovascular patterning during cornea development. First, Sema3A, a well-known chemorepulser of axons, was identified as a key modulator in the establishment of cornea avascularity in both, avian and murine models. Moreover, I demonstrated that chemokines, initially described for their function in controlling immune cell migration, also play an important role in axon guidance and vasculogenesis during ocular development. Examination of the expression of the chemokine CXCL14 by in situ hybridization and immunohistochemistry revealed novel patterns of localization in the corneal stroma, iris, lens epithelium, retina and trigeminal ganglion. Comparison in the expression of CXCL14 and CXCL12 shows that they are expressed in complementary patterns in most tissues during ocular development, suggesting an interactive regulation of these chemokines. Visual examination of Retrovirus-mediated Knockdown of CXCL14 embryos revealed relatively smaller eyes compared to controls, and immunohistochemical analysis of ocular nerves indicated exacerbated projection of sensory nerves into the corneal stroma, corneal epithelium and iris, which subsequently elevated nerve density in these tissues. In vitro analyses revealed that CXCL14 has an inhibitory effect on CXCL12-induced axon growth of trigeminal ganglion sensory neurons. Furthermore, Knockdown of CXCL14 in Tg(tie1: H2B:eYFP) transgenic Japanese quail embryos resulted in ectopic migration of YFP fluorescently labeled angioblasts into the cornea and exogenous CXCL14 inhibits VEGF- and CXCL12-induced angioblast migration into the cornea. This is the first time that CXCL14 has been shown to have a critical function during embryogenesis that may be mediated through inhibition of CXCL12 signaling. Collectively, these results demonstrate that neurovascular patterning of the anterior eye during development depends on an intricate process and fine balance of growth factors and cytokines. These findings will contribute to a better understanding of the molecular mechanisms involved in pathological conditions such as cornea neovascularization, anterior segment ocular dysgeneses and wound healing, where angiogenesis and nerve regeneration are critically compromised.