Browsing by Author "Kirienko, Natalia V"

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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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    ESRE Network Activation and Regulation Provide Insight into Mitochondrial Surveillance
    (2020-04-22) Tjahjono, Elissa; Bartel, Bonnie; Kirienko, Natalia V
    All living organisms exist in a precarious state of homeostasis that requires constant maintenance. A wide variety of stresses, including hypoxia, heat, and infection by pathogens perpetually threaten to imbalance this state. Organisms use a battery of defenses to mitigate damage and restore normal function. In a pyoverdine-dependent Caenorhabditis elegans-Pseudomonas aeruginosa assay, C. elegans’ defense response utilizes the phylogenetically conserved ESRE (Ethanol and Stress Response Element) network, which has previously been shown to mitigate damage from a variety of abiotic stresses. It is intriguing that this network is involved in innate immunity; it indicates that host innate immune responses overlap with responses to abiotic stresses. I further investigated the ESRE defense network following exposure to P. aeruginosa and showed that mitochondrial damage leads to ESRE activation both in C. elegans and in mammals. Mitochondrial dysfunction contributes to a wide variety of pathologies, including neurodegenerative diseases, cancer, metabolic diseases, and aging. Mitochondrial status is actively surveilled by several different systems to ensure the preservation of cellular viability. I characterized the ESRE pathway, a novel mitochondrial surveillance system, and showed that it is robustly activated in response to increased superoxide radical. This differentiates ESRE from other existing mitochondrial surveillance pathways, such as the mitochondrial unfolded protein response (UPRmt) that monitors disruption of mitochondrial protein import and the mitochondrial MAPK (MAPKmt) pathway that responds to mitochondrial bioenergetic disruption. I also observed multiple interactions between the ESRE, UPRmt, and MAPKmt pathways. Understanding mitochondrial surveillance and the interplay that occurs among these pathways is crucial for the advancement and understanding of cell biology. Considering that the ESRE motif and its role in response to mitochondrial damage is conserved in humans, modulation of mitochondrial pathways could confer resistance to stress and improve health span, leading to a direct benefit to human health.
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    Siderophore-Mediated Virulence and its Inhibition in Pseudomonas aeruginosa
    (2023-11-03) Kang, Donghoon; Kirienko, Natalia V
    Pseudomonas aeruginosa is a Gram-negative, nosocomial pathogen that causes life-threatening, acute infections in immunocompromised patients in intensive care units and debilitating chronic infections in those with cystic fibrosis. The rising prevalence of drug-resistant P. aeruginosa demands the exploration of new therapeutic avenues such as targeting pathogen virulence factors through small molecule inhibitors (‘antivirulents’). One key virulence factor for P. aeruginosa is the siderophore pyoverdine, which not only provides the bacterium with iron during infection, but also regulates the production of several secreted toxins, including the translational inhibitor exotoxin A and protease PrpL. We have further developed various in vivo and in vitro pathosystems using Caenorhabditis elegans, murine alveolar macrophages, and human bronchial epithelial cells to model P. aeruginosa pathogenesis and discovered several novel mechanisms of pyoverdine-dependent virulence. Against the nematode host, pyoverdine directly exerted virulence by translocating into host tissue and disrupting iron and mitochondrial homeostasis. Against mammalian cells, pyoverdine promoted the production of highly cytotoxic rhamnolipids. Due to a combination of these functions, pyoverdine production was necessary for P. aeruginosa virulence during acute murine lung infection. We also surveyed several panels of P. aeruginosa clinical isolates and demonstrated that pyoverdine production strongly correlated with pathogen virulence. Importantly, these models allowed us to characterize several novel small molecules that target either pyoverdine biosynthesis or function. In all aforementioned pathosystems, including murine lung infection, 5-FC curtailed pyoverdine production and significantly mitigated pathogenesis without overtly affecting bacterial titer, consistent with an antivirulent mechanism of action. Importantly, 5-FC was an effective therapeutic against not only laboratory-adapted strains of P. aeruginosa, but also highly virulent clinical isolates. 5-FC also synergized with the antipseudomonal agent gallium nitrate to inhibit bacterial growth without substantially increasing its own selective pressure for resistance. Moreover, from a small molecule diversity library screen, we identified compounds that directly interacted with the siderophore, attenuated the production of pyoverdine-regulated virulence factors (exotoxin A and protease PrpL), and rescued C. elegans during pathogen exposure. Altogether, findings in this dissertation demonstrate the promise of antivirulence therapeutics, specifically those that target pyoverdine, in treating multidrug-resistant P. aeruginosa infections.