Browsing by Author "Revtovich, Alexey V."
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Item 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.Item 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.Item 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.Item 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.Item 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.Item 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.Item 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.Item 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.Item 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.