Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen associated with worsening disease outcomes in cystic fibrosis (CF) patients. P. aeruginosa uses quorum sensing (QS), a cell-cell signaling system, to control expression of a variety of genes including virulence factors. In P. aeruginosa, QS is mediated in part by acyl-homoserine lactone (AHL) signals that can diffuse in and out of cells. Once AHLs accumulate, they bind to a receptor regulator that activates gene transcription. P. aeruginosa has two complete AHL QS systems, LasI-LasR and RhlI-RhlR. The two systems are arranged in a hierarchy, with the las system controlling the rhl system. QS activation in P. aeruginosa is restrained by cellular proteins that dampen the QS response. These proteins, known as “anti-activators”, attenuate QS by preventing receptor activation. Three anti-activator proteins, QscR, QslA, and QteE, have been identified in P. aeruginosa. These anti-activator proteins have additive, overlapping roles in repressing expression of QS gene products in laboratory strains but their role in the QS dynamics of CF isolates is still unclear. This project used standard molecular cloning techniques to delete or overexpress anti-activator genes in a selection of clinical isolates from CF patients. A reporter plasmid with a fluorescent marker was used to track the activity of LasR and RhlR. These experiments were used to quantify differences in QS-controlled gene activation. To test the hypothesis that anti-activators decrease the amount of LasR in the cell, Western blots were used to assess the cellular levels of QS receptors. In strains with deleted anti-activator genes, LasR levels were higher and induction was earlier. Additional tests for phenotypes controlled by QS, such as protease and pyocyanin production, were also performed. Future research should focus on evaluating these effects in additional CF isolates.

The Toll-interacting protein (TOLLIP) is an adaptor protein involved in the signaling pathways of interleukin-1 (IL-1) and Toll-like receptors (TLRs) in innate immunity. Evidence in published literature suggests that TOLLIP acts as a negative regulator of IL-1 and TLR-mediated immune responses by inhibiting the activity of IL-1 receptor-associated kinase (IRAK1), a serine/threonine kinase in the IL-1 and TLR signaling pathway. Lipopolysaccharide (LPS) is a major component of the gram-negative bacteria cell wall that activates host immune response upon recognition by TLR4. We hypothesize that TOLLIP deficiency leads to impaired inhibition of the innate immune response, resulting in increased inflammation in LPS-induced lung injury. We treated wild type (WT) and TOLLIP knockout (KO) mice with LPS through intratracheal instillation and bronchial alveolar lavage fluid (BALF) was collected at 3 days post-injury. Lung inflammation was measured by BALF total white blood cell (WBC) count and cell differential, BALF total protein, and BALF cytokine levels. Contrary to our hypothesis, TOLLIP deficiency was associated with decreased inflammation in LPS-induced lung injury as demonstrated by lower polymorphonuclear (PMN) cell count and significantly lower levels of cytokines in KO mice. In future studies, we will examine the mechanisms by which TOLLIP positively regulates inflammation in the LPS model of lung injury.

Each year, nearly 6 million deaths worldwide are caused by lower respiratory tract infections, diarrhoeal diseases, and tuberculosis. These infectious diseases are leading causes of death worldwide. Currently, the pharmacological treatment of infection is encumbered by the presence of inaccessible intracellular pathogen reservoirs, and the need for prolonged treatment regimes. Drug delivery systems can be engineered to overcome these biological barriers for effective treatment by facilitating intracellular delivery and tailored release. Extended release of drugs alleviates the need for exhaustive treatment regimes and increases patient compliance. Furthermore, this can decrease treatment duration, reduce the cost of treatment, and improve access for disadvantaged populations. Our research utilizes modular polymeric prodrugs composed of molecular targeting agents, cleavable linkers, and antimicrobial drugs. This platform permits facile alteration of functional modalities, enabling custom tailored treatments for each disease setting. By utilizing tunable linkers, we can control the precise delivery mechanism and therefore direct the localized release of drugs. To characterize the controlled release of antimicrobial drugs from our polymeric prodrugs, we are designing high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS) assays. The developed assay will evaluate the release mechanism with stability and release studies. Furthermore, the robust methodology will enable the determination of the pharmacokinetics of the polymeric prodrug delivery system. The assay and results from this study will ultimately support the development of improved therapies.

In order for cells to generate copies of themselves they must undergo a highly complex process called mitosis. During mitosis, many enzymes called protein kinases work together to ensure both daughter cells inherit the correct number of chromosomes when the cell divides. Polo-like kinase 1 (Plk1) is a protein kinase that regulates several events during mitosis including centrosome maturation, spindle assembly, sister chromatid cohesion, and cytokinesis. Recently, the A-kinase anchoring protein Gravin (AKAP12) has been implicated in regulating Plk1 function at mitotic centrosomes. Specifically, loss of Gravin has been linked to defective protein signaling at centrosomes, chromosome misalignment, and increased incidence of micronuclei (small nuclei, an aberration often seen in cancer). However, while previous studies used shRNA-mediated knockdown to reduce Gravin levels in cells, it remains unclear how complete loss of this scaffold in human cells influences mitotic signaling events. To test this, our lab generated Gravin knockout U2OS (osteosarcoma) cells using CRISPR/Cas9 genome editing. First, I employed a combination of immunohistochemical staining and quantitative imaging tools to assess how Gravin loss affected chromosome alignment, micronuclei formation, and gamma tubulin accumulation at centrosomes. I found that loss of Gravin in U2OS and HeLa cells caused misaligned chromosomes and micronuclei. Additional experiments I conducted revealed that Gravin-depleted U2OS, HeLa, and MEF cells presented aberrant gamma tubulin accumulation at mitotic spindle poles. Next, a local drug-targeting approach was used to specifically inhibit Plk1 activity at mitotic spindle poles in U2OS cells. I determined that localized inhibition of Plk1 produced similar mitotic defects as observed in cells lacking Gravin. Collectively, these findings suggest that Gravin is required for coordinating proper Plk1 signaling at centrosomes during mitosis while the loss of this scaffold protein leads to mitotic defects. Future work will uncover downstream substrates of Gravin-anchored Plk1 that becomes dysregulated in cells lacking Gravin.

The mitochondria are important organelles that regulate various processes such as oxidative phosphorylation, fatty acid oxidation and calcium homeostasis. They communicate with the rest of the cell to coordinate these functions. Mitochondrial communication is mediated by calcium signaling in which calcium ions pass through a calcium uniporter in the mitochondrial inner membrane. This signaling is altered when the cell undergoes stress and the mitochondria initiates the unfolded protein response (UPR^mito). We are interested in understanding the regulation of the mitochondrial calcium uniporter during UPR^mito. We previously have shown that calcium uptake becomes inhibited when UPR^mito is induced in HeLa cells. It is not known how the unfolded proteins initiate the UPR^mito gene expression pathway. However, we hypothesize that the calcium uniporter is involved in this process. To further characterize the behavior of the uniporter, we induced UPR^mito in another cell line, A549 cells, using different pharmacological agents. After the treatment, I analyzed and performed calcium uptake assays to determine the activity of the mitochondrial calcium uniporter. The results revealed that, similar to HeLa cells, UPR^mito inhibits the uniporter in A549 cells. We hypothesize that uniporter inhibition affects gene expression during UPRmito, especially in disease states where chronic UPR^mito is observed, such as Alzheimer’s and Parkinson’s diseases. To test this hypothesis, we want to develop a more physiologically relevant system for UPR^mito induction, the focus of my project is to induce chronic UPR^mito using low dose, long term drug treatment to be able to mimic diseases conditions better and to determine the effects of chronic UPR^mito on mitochondrial calcium uptake. This study has the potential to implicate the uniporter as a clinical drug target for treatment of diseases with chronic UPR^mito involvement.

Pseudomonas aeruginosa, an opportunistic pathogen that commonly infects cystic fibrosis patients, uses quorum sensing (QS), a form of cell-cell communication, to regulate the production of some virulence factors and public resources. P. aeruginosa QS consists of N-acyl homoserine lactone (AHL) signal molecules that activate two separate regulatory molecules, LasR and RhlR, which in turn activates the transcription of other target genes in their respective regulons. Three anti-activator proteins, QscR, QteE, and QslA, restrict the expression of these two QS regulons. However, it is not clear how P. aeruginosa anti-activators contribute to synchronizing the activities of individual cells in the population. Discovering precisely how this mechanism functions could help establish novel methods of treating infections based on using anti-activators. To investigate this problem, we over-expressed each anti-activator in P. aeruginosa, and monitored the activity of two important QS genes: lasI, which is LasR-regulated, and rhlA, a RhlR regulated gene. Using reporter gene fusion to measure the activity of the lasI and rhlA promoters, we noticed that expression of both genes is reduced significantly in strains with over-expressed anti-activators. In addition, we measured a delay in expression from both promoters when compared to wild type P. aeruginosa. These results indicate that the three anti-activators both raise the QS threshold and slow the rate that the threshold is reached. Future directions of this project include over-expressing anti-activators in LasR and LasI deletion mutants, as well as using flow cytometry to analyze QS activity in individual cells.