R-3 Repository :: Browsing by Author "Kirienko, Natalia V."

Browsing by Author "Kirienko, Natalia V."

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A High-throughput, High-content, Liquid-based C. elegans Pathosystem
(JoVE, 2018) Anderson, Quinton L.; Revtovich, Alexey V.; Kirienko, Natalia V.
The number of new drugs identified by traditional, in vitro screens has waned, reducing the success of this approach in the search for new weapons to combat multiple drug resistance. This has led to the conclusion that researchers do not only need to find new drugs, but also need to develop new ways of finding them. Amongst the most promising candidate methods are whole-organism, in vivo assays that use high-throughput, phenotypic readouts and hosts that range from Caenorhabditis elegans to Danio rerio. These hosts have several powerful advantages, including dramatic reductions in false positive hits, as compounds that are toxic to the host and/or biounavailable are typically dropped in the initial screen, prior to costly follow up. Here we show how our assay has been used to interrogate host variation in the well-documented C. elegans—Pseudomonas aeruginosa liquid killing pathosystem. We also demonstrate several extensions of this well-worked out technique. For example, we are able to carry out high-throughput genetic screens using RNAi in 24- or 96-well plate formats to query host factors in this host-pathogen interaction. Using this assay, whole genome screens can be completed in only a few months, which can dramatically simplify the task of identifying drug targets, potentially without the need for laborious biochemical purification approaches. We also report here a variation of our method that substitutes the gram-positive bacterium Enterococcus faecalis for the gram-negative pathogen P. aeruginosa. Much as is the case for P. aeruginosa, killing by E. faecalis is time-dependent. Unlike previous C. elegans—E. faecalis assays, our assay for E. faecalis does not require preinfection, improving its safety profile and reducing the chances of contaminating liquid-handling equipment. The assay is highly robust, showing ~95% death rates 96 h post infection.
A mechanism for increased sensitivity of acute myeloid leukemia to mitotoxic drugs
(Springer Nature, 2019) Panina, Svetlana B.; Baran, Natalia; da Costa, Fabio H.Brasil; Konopleva, Marina; Kirienko, Natalia V.
Mitochondria play a central and multifunctional role in the progression of tumorigenesis. Although many recent studies have demonstrated correlations between mitochondrial function and genetic makeup or originating tissue, it remains unclear why some cancers are more susceptible to mitocans (anticancer drugs that target mitochondrial function to mediate part or all of their effect). Moreover, fundamental questions of efficacy and mechanism of action in various tumor types stubbornly remain. Here we demonstrate that cancer type is a significant predictor of tumor response to mitocan treatment, and that acute myeloid leukemias (AML) show an increased sensitivity to these drugs. We determined that AML cells display particular defects in mitochondrial metabolism that underlie their sensitivity to mitocan treatment. Furthermore, we demonstrated that combinatorial treatment with a mitocan (CCCP) and a glycolytic inhibitor (2-deoxyglucose) has substantial synergy in AML cells, including primary cells from patients with AML. Our results show that mitocans, either alone or in combination with a glycolytic inhibitor, display anti-leukemia effects in doses much lower than needed to induce toxicity against normal blood cells, indicating that mitochondria may be an effective and selective therapeutic target.
A Simple Nematode Infection Model for Studying Candida albicans Pathogenesis
(Wiley, 2020) Kim, Grace H.; Rosiana, Sierra; Kirienko, Natalia V.; Shapiro, Rebecca S.
Candida albicans is an opportunistic fungal pathogen and a model organism to study fungal pathogenesis. It exists as a harmless commensal organism and member of the healthy human microbiome, but can cause life‐threatening mucosal and systemic infections. A model host to study C. albicans infection and pathogenesis is the nematode Caenorhabditis elegans. C. elegans is frequently used as a model host to study microbial‐host interactions because it can be infected by many human pathogens and there are also close morphological resemblances between the intestinal cells of C. elegans and mammals, where C. albicans infections can occur. This article outlines a detailed methodology for exploiting C. elegans as a host to study C. albicans infection, including a C. elegans egg preparation protocol and an agar‐based C. elegans killing protocol to monitor fungal virulence. These protocols can additionally be used to study C. albicans genetic mutants in order to further our understanding of the genes involved in pathogenesis and virulence in C. albicans and the mechanisms of host‐microbe interactions.
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.
Box C/D small nucleolar ribonucleoproteins regulate mitochondrial surveillance and innate immunity
(Public Library of Science, 2022) Tjahjono, Elissa; Revtovich, Alexey V.; Kirienko, Natalia V.
Monitoring mitochondrial function is crucial for organismal survival. This task is performed by mitochondrial surveillance or quality control pathways, which are activated by signals originating from mitochondria and relayed to the nucleus (retrograde response) to start transcription of protective genes. In Caenorhabditis elegans, several systems are known to play this role, including the UPRmt, MAPKmt, and the ESRE pathways. These pathways are highly conserved and their loss compromises survival following mitochondrial stress. In this study, we found a novel interaction between the box C/D snoRNA core proteins (snoRNPs) and mitochondrial surveillance and innate immune pathways. We showed that box C/D, but not box H/ACA, snoRNPs are required for the full function of UPRmt and ESRE upon stress. The loss of box C/D snoRNPs reduced mitochondrial mass, mitochondrial membrane potential, and oxygen consumption rate, indicating overall degradation of mitochondrial function. Concomitantly, the loss of C/D snoRNPs increased immune response and reduced host intestinal colonization by infectious bacteria, improving host resistance to pathogenesis. Our data may indicate a model wherein box C/D snoRNP machinery regulates a “switch” of the cell’s activity between mitochondrial surveillance and innate immune activation. Understanding this mechanism is likely to be important for understanding multifactorial processes, including responses to infection and aging.
Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans
(Oxford University Press, 2021) Rosiana, Sierra; Zhang, Liyang; Kim, Grace H.; Revtovich, Alexey V.; Uthayakumar, Deeva; Sukumaran, Arjun; Geddes-McAlister, Jennifer; Kirienko, Natalia V.; Shapiro, Rebecca S.
Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.
Development and Characterization of High-Throughput Caenorhabditis elegans – Enterococcus faecium Infection Model
(Frontiers Media S.A., 2021) Revtovich, Alexey V.; Tjahjono, Elissa; Singh, Kavindra V.; Hanson, Blake M.; Murray, Barbara E.; Kirienko, Natalia V.
The genus Enterococcus includes two Gram-positive pathogens of particular clinical relevance: E. fae-calis and E. faecium. Infections with each of these pathogens are becoming more frequent, particular-ly in the case of hospital-acquired infections. Like most other bacterial species of clinical importance, antimicrobial resistance (and, specifically, multi-drug resistance) is an increasing threat, with both species considered to be of particular importance by the World Health Organization and the US Cen-ters for Disease Control. The threat of antimicrobial resistance is exacerbated by the staggering dif-ference in the speeds of development for the discovery and development of the antimicrobials versus resistance mechanisms . In the search for alternative strategies, modulation of host-pathogen interac-tions in general, and virulence inhibition in particular, has drawn substantial attention. Unfortunately, these approaches require a fairly comprehensive understanding of virulence determinants. This re-quirement is complicated by the fact that enterococcal infection models generally require vertebrates, making them slow, expensive, and ethically problematic, particularly when considering the thousands of animals that would be needed for the early stages of experimentation. To address this problem, we developed the first high-throughput C. elegans–E. faecium infection model involving host death. Im-portantly, this model recapitulates many key aspects of murine peritonitis models, including utilizing similar virulence determinants. Additionally, host death is independent of peroxide production, un-like other E. faecium–C. elegans virulence models, which allows the assessment of other virulence factors. Using this system, we analyzed a panel of lab strains with deletions of targeted virulence fac-tors. Although removal of certain virulence factors (e.g., Δfms15) was sufficient to affect virulence alone, multiple deletions were generally required to affect pathogenesis, suggesting that host-pathogen interactions are multifactorial. These data were corroborated by genomic analysis of select-ed isolates with high and low levels of virulence. We anticipate that this platform will be useful for identifying new treatments for E. faecium infection.
Examining Sporadic Cancer Mutations Uncovers a Set of Genes Involved in Mitochondrial Maintenance
(MDPI, 2023) Moreno, Armando; Taffet, Allison; Tjahjono, Elissa; Anderson, Quinton L.; Kirienko, Natalia V.
Mitochondria are key organelles for cellular health and metabolism and the activation of programmed cell death processes. Although pathways for regulating and re-establishing mitochondrial homeostasis have been identified over the past twenty years, the consequences of disrupting genes that regulate other cellular processes, such as division and proliferation, on affecting mitochondrial function remain unclear. In this study, we leveraged insights about increased sensitivity to mitochondrial damage in certain cancers, or genes that are frequently mutated in multiple cancer types, to compile a list of candidates for study. RNAi was used to disrupt orthologous genes in the model organism Caenorhabditis elegans, and a series of assays were used to evaluate these genes’ importance for mitochondrial health. Iterative screening of ~1000 genes yielded a set of 139 genes predicted to play roles in mitochondrial maintenance or function. Bioinformatic analyses indicated that these genes are statistically interrelated. Functional validation of a sample of genes from this set indicated that disruption of each gene caused at least one phenotype consistent with mitochondrial dysfunction, including increased fragmentation of the mitochondrial network, abnormal steady-state levels of NADH or ROS, or altered oxygen consumption. Interestingly, RNAi-mediated knockdown of these genes often also exacerbated α-synuclein aggregation in a C. elegans model of Parkinson’s disease. Additionally, human orthologs of the gene set showed enrichment for roles in human disorders. This gene set provides a foundation for identifying new mechanisms that support mitochondrial and cellular homeostasis.
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.
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.
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.
Interplay between mitochondria and diet mediates pathogen and stress resistance in Caenorhabditis elegans
(Public Library of Science, 2019) Revtovich, Alexey V.; Lee, Ryan; Kirienko, Natalia V.
Diet is a crucial determinant of organismal biology; interactions between the host, its diet, and its microbiota are critical to determining the health of an organism. A variety of genetic and biochemical means were used to assay stress sensitivity in C. elegans reared on two standard laboratory diets: E. coli OP50, the most commonly used food for C. elegans, or E. coli HT115, which is typically used for RNAi-mediated gene knockdown. We demonstrated that the relatively subtle shift to a diet of E. coli HT115 had a dramatic impact on C. elegans’s survival after exposure to pathogenic or abiotic stresses. Interestingly, this was independent of canonical host defense pathways. Instead the change arises from improvements in mitochondrial health, likely due to alleviation of a vitamin B12 deficiency exhibited by worms reared on an E. coli OP50 diet. Increasing B12 availability, by feeding on E. coli HT115, supplementing E. coli OP50 with exogenous vitamin B12, or overexpression of the B12 transporter, improved mitochondrial homeostasis and increased resistance. Loss of the methylmalonyl-CoA mutase gene mmcm-1/MUT, which requires vitamin B12 as a cofactor, abolished these improvements, establishing a genetic basis for the E. coli OP50-incurred sensitivity. Our study forges a mechanistic link between a dietary deficiency (nutrition/microbiota) and a physiological consequence (host sensitivity), using the host-microbiota-diet framework.
Long-Term Dominance of Carbapenem-Non-Susceptible Pseudomonas aeruginosa ST111 in Hematologic Malignancy Patients and Hematopoietic Cell Transplant Recipients
(Frontiers Media S.A., 2022) Zhang, Liyang; Tan, Filemon C.; Strasfeld, Lynne; Hakki, Morgan; Kirienko, Natalia V.
An epidemiological study uncovered that fluoroquinolone (FQ) neutropenic prophylaxis in hematopoietic cell transplant and hematologic malignancy (HCT/HM) patients was associated with breakthrough Pseudomonas aeruginosa bloodstream infections (BSIs) with isolates non-susceptible to both FQs and meropenem. The molecular epidemiology of the FQ/meropenem-non-susceptible P. aeruginosa isolates causing FQ-breakthrough BSIs in the HCT/HM patients remains unclear. Through whole genome sequencing on 57 P. aeruginosa isolates from 54 patients diagnosed with HM or receiving an HCT, we found that ST111 strains predominated, accounting for 22 (38.6%) of the isolates. 17 of 33 (51.5%) FQ-breakthrough BSIs were caused by ST111 strains, of which 15 (88.2%) were meropenem non-susceptible. ST111 strains, but not other oprD-deficient, meropenem-non-susceptible clinical strains, were found to have a colonization advantage over P. aeruginosa strain PA14 in C. elegans and to outcompete PA14 in in vitro co-culture assays. Together, we found that breakthrough P. aeruginosa BSIs during FQ prophylaxis in HCT/HM patients are dominated by clonally-related FQ/meropenem non-susceptible strains, predominantly ST111 type, and that the dominance of ST111 strains may be explained by a relative fitness advantage over other clinical strains. Additional work is necessary to better understand the factors driving the dominance and persistence of these ST111 strains.
Mitochondria-affecting small molecules ameliorate proteostasis defects associated with neurodegenerative diseases
(Springer Nature, 2021) Tjahjono, Elissa; Pei, Jingqi; Revtovich, Alexey V.; Liu, Terri-Jeanne E.; Swadi, Alisha; Hancu, Maria C.; Tolar, Joe G.; Kirienko, Natalia V.
Macroautophagic recycling of dysfunctional mitochondria, known as mitophagy, is essential for mitochondrial homeostasis and cell viability. Accumulation of defective mitochondria and impaired mitophagy have been widely implicated in many neurodegenerative diseases, and loss-of-function mutations of PINK1 and Parkin, two key regulators of mitophagy, are amongst the most common causes of heritable parkinsonism. This has led to the hypothesis that pharmacological stimulation of mitophagy may be a feasible approach to combat neurodegeneration. Toward this end, we screened ~ 45,000 small molecules using a high-throughput, whole-organism, phenotypic screen that monitored accumulation of PINK-1 protein, a key event in mitophagic activation, in a Caenorhabditis elegans strain carrying a Ppink-1::PINK-1::GFP reporter. We obtained eight hits that increased mitochondrial fragmentation and autophagosome formation. Several of the compounds also reduced ATP production, oxygen consumption, mitochondrial mass, and/or mitochondrial membrane potential. Importantly, we found that treatment with two compounds, which we named PS83 and PS106 (more commonly known as sertraline) reduced neurodegenerative disease phenotypes, including delaying paralysis in a C. elegans β-amyloid aggregation model in a PINK-1-dependent manner. This report presents a promising step toward the identification of compounds that will stimulate mitochondrial turnover.
Novel Immune Modulators Enhance Caenorhabditis elegans Resistance to Multiple Pathogens
(American Society for Microbiology, 2021) Hummell, Nicholas A.; Revtovich, Alexey V.; Kirienko, Natalia V.
Traditionally, treatments for bacterial infection have focused on killing the microbe or preventing its growth. As antimicrobial resistance becomes more ubiquitous, the feasibility of this approach is beginning to wane and attention has begun to shift toward disrupting the host-pathogen interaction by improving the host defense. Using a high-throughput, fragment-based screen to identify compounds that alleviate Pseudomonas aeruginosa-mediated killing of Caenorhabditis elegans, we identified over 20 compounds that stimulated host defense gene expression. Five of these molecules were selected for further characterization. Four of five compounds showed little toxicity against mammalian cells or worms, consistent with their identification in a phenotypic, high-content screen. Each of the compounds activated several host defense pathways, but the pathways were generally dispensable for compound-mediated rescue in liquid killing, suggesting redundancy or that the activation of unknown pathway(s) may be driving compound effects. A genetic mechanism was identified for LK56, which required the Mediator subunit MDT-15/MED15 and NHR-49/HNF4 for its function. Interestingly, LK32, LK34, LK38, and LK56 also rescued C. elegans from P. aeruginosa in an agar-based assay, which uses different virulence factors and defense mechanisms. Rescue in an agar-based assay for LK38 entirely depended upon the PMK-1/p38 MAPK pathway. Three compounds—LK32, LK34, and LK56—also conferred resistance to Enterococcus faecalis, and the two lattermost, LK34 and LK56, also reduced pathogenesis from Staphylococcus aureus. This study supports a growing role for MDT-15 and NHR-49 in immune response and identifies five molecules that have significant potential for use as tools in the investigation of innate immunity. IMPORTANCE Trends moving in opposite directions (increasing antimicrobial resistance and declining novel antimicrobial development) have precipitated a looming crisis: the nearly complete inability to safely and effectively treat bacterial infections. To avert this, new approaches are needed. One idea is to stimulate host defense pathways to improve the clearance of bacterial infection. Here, we describe five small molecules that promote resistance to infectious bacteria by activating C. elegans’ innate immune pathways. Several are effective against both Gram-positive and Gram-negative pathogens. One of the compounds was mapped to the action of MDT-15/MED15 and NHR-49/HNF4, a pair of transcriptional regulators more generally associated with fatty acid metabolism, potentially highlighting a new link between these biological functions. These studies pave the way for future characterization of the anti-infective activity of the molecules in higher organisms and highlight the compounds’ potential utility for further investigation of immune modulation as a novel therapeutic approach.
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.
Pathogen-induced dormancy in liquid limits gastrointestinal colonization of Caenorhabditis elegans
(Taylor & Francis, 2023) Zhang, Liyang; Gade, Vyshnavi; Kirienko, Natalia V.
Colonization is generally considered a prerequisite for infection, but this event is context-dependent, as evidenced by the differing ability of the human pathogen Pseudomonas aeruginosa to efficiently colonize Caenorhabditis elegans on agar but not in liquid . In this study, we examined the impact of the environment, pathogen, host, and their interactions on host colonization. We found that the transition to a liquid environment reduces food uptake by about two-fold. Also expression of specific adhesins was significantly altered in liquid-based assays for P. aeruginosa, suggesting that it may be one factor driving diminished colonization. Unexpectedly, host immune pathways did not appear to play a significant role in decreased colonization in liquid. Although knocking down key immune pathways (e.g. daf-16 or zip-2), either alone or in combination, significantly reduced survival, the changes in colonization were very small. In spite of the limited bacterial accumulation in the liquid setting, pathogenic colonization was still required for the virulence of Enterococcus faecalis. In addition, we found that a pathogen-induced dormancy was displayed by C. elegans in liquid medium after pathogen exposure, resulting in cessation of pharyngeal pumping and a decrease in bacterial intake. We conclude that poor colonization in liquid is likely due to a combination of environmental factors and host-pathogen interactions. These results provide new insights into mechanisms for colonization in different models, enabling pathogenesis models to be fine-tuned to more accurately represent the conditions seen in human infections so that new tools for curbing bacterial and fungal infections can be developed.
PqsA Promotes Pyoverdine Production via Biofilm Formation
(MDPI, 2018) Kang, Donghoon; Turner, Kelly E.; Kirienko, Natalia V.
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.
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.

Browsing by Author "Kirienko, Natalia V."

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