Browsing by Author "Kang, Donghoon"

Now showing 1 - 11 of 11
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    An In Vitro Cell Culture Model for Pyoverdine-Mediated Virulence
    (MDPI, 2021) Kang, Donghoon; Kirienko, Natalia V.
    Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that utilizes a wide-range of virulence factors to cause acute, life-threatening infections in immunocompromised patients, especially those in intensive care units. It also causes debilitating chronic infections that shorten lives and worsen the quality of life for cystic fibrosis patients. One of the key virulence factors in P. aeruginosa is the siderophore pyoverdine, which provides the pathogen with iron during infection, regulates the production of secreted toxins, and disrupts host iron and mitochondrial homeostasis. These roles have been characterized in model organisms such as Caenorhabditis elegans and mice. However, an intermediary system, using cell culture to investigate the activity of this siderophore has been absent. In this report, we describe such a system, using murine macrophages treated with pyoverdine. We demonstrate that pyoverdine-rich filtrates from P. aeruginosa exhibit substantial cytotoxicity, and that the inhibition of pyoverdine production (genetic or chemical) is sufficient to mitigate virulence. Furthermore, consistent with previous observations made in C. elegans, pyoverdine translocates into cells and disrupts host mitochondrial homeostasis. Most importantly, we observe a strong correlation between pyoverdine production and virulence in P. aeruginosa clinical isolates, confirming pyoverdine’s value as a promising target for therapeutic intervention. This in vitro cell culture model will allow rapid validation of pyoverdine antivirulents in a simple but physiologically relevant manner.
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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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    High-Throughput Approaches for the Identification of Pseudomonas aeruginosa Antivirulents
    (American Society for Microbiology, 2021) Kang, Donghoon; Zhang, Liyang; Kirienko, Natalia V.
    Antimicrobial resistance is a serious medical threat, particularly given the decreasing rate of discovery of new treatments. Although attempts to find new treatments continue, it has become clear that merely discovering new antimicrobials, even if they are new classes, will be insufficient. It is essential that new strategies be aggressively pursued. Toward that end, the search for treatments that can mitigate bacterial virulence and tilt the balance of host-pathogen interactions in favor of the host has become increasingly popular. In this review, we will discuss recent progress in this field, with a special focus on synthetic small molecule antivirulents that have been identified from high-throughput screens and on treatments that are effective against the opportunistic human pathogen Pseudomonas aeruginosa.
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    High-Throughput Genetic Screen Reveals that Early Attachment and Biofilm Formation Are Necessary for Full Pyoverdine Production by Pseudomonas aeruginosa
    (Frontiers Media S.A., 2017) Kang, Donghoon; Kirienko, Natalia V.
    Pseudomonas aeruginosa is a re-emerging, multidrug-resistant, opportunistic pathogen that threatens the lives of immunocompromised patients, patients with cystic fibrosis, and those in critical care units. One of the most important virulence factors in this pathogen is the siderophore pyoverdine. Pyoverdine serves several critical roles during infection. 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. Inhibition of pyoverdine biosynthesis decreases P. aeruginosa pathogenicity in multiple host models. To better understand the regulation of pyoverdine production, we developed a high-throughput genetic screen that uses the innate fluorescence of pyoverdine to identify genes necessary for its biosynthesis. A substantial number of hits showing severe impairment of pyoverdine production were in genes responsible for early attachment and biofilm formation. In addition to genetic disruption of biofilm, both physical and chemical perturbations also attenuated pyoverdine production. This regulatory relationship between pyoverdine and biofilm is particularly significant in the context of P. aeruginosa multidrug resistance, where the formation of biofilm is a key mechanism preventing access to antimicrobials and the immune system. Furthermore, we demonstrate that the biofilm inhibitor 2-amino-5,6-dimethylbenzimidazole effectively attenuates pyoverdine production and rescues Caenorhabditis elegans from P. aeruginosa-mediated pathogenesis. Our findings suggest that targeting biofilm formation in P. aeruginosa infections may have multiple therapeutic benefits and that employing an unbiased, systems biology-based approach may be useful for understanding the regulation of specific virulence factors and identifying novel anti-virulence therapeutics or new applications for existing therapies for P. aeruginosa infections.
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    In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores
    (American Society for Microbiology, 2024) Kang, Donghoon; Xu, Qi; Kirienko, Natalia V.
    The multidrug-resistant pathogen Pseudomonas aeruginosa is a common nosocomial respiratory pathogen that continues to threaten the lives of patients with mechanical ventilation in intensive care units and those with underlying comorbidities such as cystic fibrosis or chronic obstructive pulmonary disease. For over 20 years, studies have repeatedly demonstrated that the major siderophore pyoverdine is an important virulence factor for P. aeruginosa in invertebrate and mammalian hosts in vivo. Despite its physiological significance, an in vitro, mammalian cell culture model that can be used to characterize the impact and molecular mechanisms of pyoverdine-mediated virulence has only been developed very recently. In this study, we adapt a previously-established, murine macrophage-based model to use human bronchial epithelial (16HBE) cellsWe demonstrate that conditioned medium from P. aeruginosa induced rapid 16HBE cell death through the pyoverdine-dependent secretion of cytotoxic rhamnolipids. Genetic or chemical disruption of pyoverdine biosynthesis decreased rhamnolipid production and mitigated cell death. Consistent with these observations, chemical depletion of lipids or genetic disruption of rhamnolipid biosynthesis abrogated the toxicity of the conditioned medium. Furthermore, we also examine the effects of exposure to purified pyoverdine on 16HBE cells. While pyoverdine accumulated within cells, it was largely sequestered within early endosomes, resulting in minimal cytotoxicity. More membrane-permeable iron chelators, such as the siderophore pyochelin, decreased epithelial cell viability and upregulated several pro-inflammatory genes. However, pyoverdine potentiated these iron chelators in activating pro-inflammatory pathways. Altogether, these findings suggest that the siderophores pyoverdine and pyochelin play distinct roles in virulence during acute P. aeruginosa lung infection.
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    Novel Pyoverdine Inhibitors Mitigate Pseudomonas aeruginosa Pathogenesis
    (Frontiers, 2019) Kirienko, Daniel R.; Kang, Donghoon; Kirienko, Natalia V.
    Pseudomonas aeruginosa is a clinically important pathogen that causes a variety of infections, including urinary, respiratory, and other soft-tissue infections, particularly in hospitalized patients with immune defects, cystic fibrosis, or significant burns. Antimicrobial resistance is a substantial problem in P. aeruginosa treatment due to the inherent insensitivity of the pathogen to a wide variety of antimicrobial drugs and its rapid acquisition of additional resistance mechanisms. One strategy to circumvent this problem is the use of anti-virulent compounds to disrupt pathogenesis without directly compromising bacterial growth. One of the principle regulatory mechanisms for P. aeruginosa’s virulence is the iron-scavenging siderophore pyoverdine, as it governs in-host acquisition of iron, promotes expression of multiple virulence factors, and is directly toxic. Some combination of these activities renders pyoverdine indispensable for pathogenesis in mammalian models. Here we report identification of a panel of novel small molecules that disrupt pyoverdine function. These molecules directly act on pyoverdine, rather than affecting its biosynthesis. The compounds reduce the pathogenic effect of pyoverdine and improve the survival of Caenorhabditis elegans when challenged with P. aeruginosa by disrupting only this single virulence factor. Finally, these compounds can synergize with conventional antimicrobials, forming a more effective treatment. These compounds may help to identify, or be modified to become, viable drug leads in their own right. Finally, they also serve as useful tool compounds to probe pyoverdine activity.
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    PqsA Promotes Pyoverdine Production via Biofilm Formation
    (MDPI, 2018) Kang, Donghoon; Turner, Kelly E.; Kirienko, Natalia V.
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    Pyoverdine Inhibitors and Gallium Nitrate Synergistically Affect Pseudomonas aeruginosa
    (American Society for Microbiology, 2021) Kang, Donghoon; Revtovich, Alexey V.; Deyanov, Alexander E.; Kirienko, Natalia V.
    Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that frequently causes ventilator-associated pneumonia in intensive care units and chronic lung infections in cystic fibrosis patients. The rising prevalence of drug-resistant bacteria demands the exploration of new therapeutic avenues for treating P. aeruginosa infections. Perhaps the most thoroughly explored alternative is to use novel treatments to target pathogen virulence factors, like biofilm or toxin production. Gallium(III) nitrate is one such agent. It has been recognized for its ability to inhibit pathogen growth and biofilm formation in P. aeruginosa by disrupting bacterial iron homeostasis. However, irreversible sequestration by pyoverdine substantially limits its effectiveness. In this report, we show that disrupting pyoverdine production (genetically or chemically) potentiates the efficacy of gallium nitrate. Interestingly, we report that the pyoverdine inhibitor 5-fluorocytosine primarily functions as an antivirulent, even when it indirectly affects bacterial growth in the presence of gallium, and that low selective pressure for resistance occurs. We also demonstrate that the antibiotic tetracycline inhibits pyoverdine at concentrations below those required to prevent bacterial growth, and this activity allows it to synergize with gallium to inhibit bacterial growth and rescue Caenorhabditis elegans during P. aeruginosa pathogenesis.
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    Pyoverdine, a siderophore from Pseudomonas aeruginosa, translocates into C. elegans, removes iron, and activates a distinct host response
    (Taylor & Francis, 2018) Kang, Donghoon; Kirienko, Daniel R.; Webster, Phillip; Fisher, Alfred L.; Kirienko, Natalia V.
    Pseudomonas aeruginosa, a re-emerging, opportunistic human pathogen, encodes a variety of virulence determinants. Pyoverdine, a siderophore produced by this bacterium, is essential for pathogenesis in mammalian infections. This observation is generally attributed to its roles in acquiring iron and/or regulating other virulence factors. Here we report that pyoverdine translocates into the host, where it binds and extracts iron. Pyoverdine-mediated iron extraction damages host mitochondria, disrupting their function and triggering mitochondrial turnover via autophagy. The host detects this damage via a conserved mitochondrial surveillance pathway mediated by the ESRE network. Our findings illuminate the pathogenic mechanisms of pyoverdine and highlight the importance of this bacterial product in host-pathogen interactions.
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    Pyoverdine-Dependent Virulence ofﾠPseudomonas aeruginosaﾠIsolates From Cystic Fibrosis Patients
    (Frontiers, 2019) Kang, Donghoon; Revtovich, Alexey V.; Chen, Qingquan; Shah, Kush N.; Cannon, Carolyn L.; Kirienko, Natalia V.
    The development of therapies that modulate or prevent pathogen virulence may be a key strategy for circumventing antimicrobial resistance. Toward that end, we examined the production of pyoverdine, a key virulence determinant, in ∼70 Pseudomonas aeruginosa isolates from pediatric cystic fibrosis patients. Pyoverdine production was heterogeneous and showed a clear correlation with pathogenicity in Caenorhabditis elegans and an acute murine pneumonia model. Examination showed pyoverdine accumulation in host tissues, including extrapharyngeal tissues of C. elegans and lung tissues of mice, where accumulation correlated with host death. Many of the isolates tested were resistant to multiple antimicrobials, so we assayed the ability of pyoverdine inhibitors to mitigate virulence and rescue pyoverdine-mediated host pathology. Representatives from three different classes of pyoverdine inhibitors (gallium, fluoropyrimidines, and LK11) significantly improved survival. Our findings highlight the utility of targeting virulence factors in general, and pyoverdine in particular, as a promising method to control bacterial pathogenesis as the utility of antimicrobials continues to diminish.
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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.