2015 |
Kozubowski, Lukasz |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
Mechanisms of Fluconazole-Induced Aneuploidy in Cryptococcus Neoformans
? DESCRIPTION (provided by applicant): Fungi are an increasingly important cause of death and morbidity in both immunocompetent and immunocompromised patients. Cryptococcal meningitis caused by Cryptococcus neoformans is the most common cause of fungal central nervous system infection in the world. One million cases of Cryptococcal infection occur globally, largely in the context of AIDS and constitute one-third of all AIDS-associated deaths. Despite these public health threats, effective treatments for cryptococcosis are inadequate. Recent reports indicate a high importance of genome plasticity in the pathogenicity of C. neoformans. For example, changes in chromosomal copy number are a major factor contributing to the resistance to the azole drug fluconazole in vitro and in vivo. The list of key resistance genes whose copy number increases in fluconazole-resistant C. neoformans isolates is well established. However, very little is known about the molecular mechanisms that govern changes in chromosomal copy number in this organism. The main objective of this proposal is to elucidate mechanisms responsible for generation of aneuploidy when C. neoformans is exposed to fluconazole. We will perform a detailed analysis of the effects of fluconazole on cell growth and nuclear division. In addition, we will elucidate basic architecture of the spindle assembly checkpoint (SAC) pathway in C. neoformans, and explore the possibility that the inhibition of this pathway is one of the causes of fluconazole-triggered aneuploidy leading to drug resistance. This work will contribute to our understanding of the mechanisms that are involved in chromosomal changes of a fungal pathogen during infection. This project will engage graduate and undergraduate students and allow for hands-on research experience. Students will be exposed to various laboratory techniques and will learn formulating and testing research hypotheses.
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0.957 |
2016 — 2020 |
Kozubowski, Lukasz |
P20Activity Code Description: To support planning for new programs, expansion or modification of existing resources, and feasibility studies to explore various approaches to the development of interdisciplinary programs that offer potential solutions to problems of special significance to the mission of the NIH. These exploratory studies may lead to specialized or comprehensive centers. |
Exploring the Mechanisms of Fluconazole-Induced Aneuploidy in Cryptococcus Neoformans
PROJECT SUMMARY Fungi are an increasingly important cause of death and morbidity in both immunocompetent and immunocompromised patients. Cryptococcal meningitis caused by Cryptococcus neoformans is the most common cause of fungal central nervous system infection in the world. One million cases of Cryptococcal infection occur globally, largely in the context of AIDS and constitute one-third of all AIDS-associated deaths. Despite these public health threats, effective treatments for cryptococcosis are inadequate. Recent reports indicate a high importance of genome plasticity in the pathogenicity of C. neoformans. Aneuploidy formed de- novo during meiotic divisions adds to the diversity of cryptococcal population potentially generating new virulent strains. During pulmonary infection, cryptococcal cells become polyploids, which protects them from the host immune response. Changes in chromosomal copy number are responsible for the resistance to the azole drug fluconazole in vitro and in vivo. Despite mounting evidence that aneuploidy is crucial in pathogenicity of C. neoformans very little is known about the molecular mechanisms that govern changes in chromosomal copy number in this organism. The main objective of this proposal is to elucidate mechanisms responsible for generation of aneuploidy in C. neoformans with a particular emphasis on the connection between fluconazole treatment and aneuploidy. We will perform a detailed analysis of the effects of fluconazole on cell growth and nuclear division. In addition, we will elucidate basic architecture of the spindle assembly checkpoint (SAC) pathway in C. neoformans, and explore the possibility that the inhibition of this pathway is one of the causes of fluconazole-triggered aneuploidy leading to drug resistance. This work will contribute to our understanding of the mechanisms that are involved in chromosomal changes of a fungal pathogen during infection.
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0.957 |
2022 — 2025 |
Brumaghim, Julia [⬀] Kozubowski, Lukasz |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Azole Antifungals Coordinate Metals and Create Reactive Oxygen Species That Damage Dna and Cause Chromosomal Instability
With the support of the Chemistry of Life Processes (CLP) Program in the Division of Chemistry, Julia Brumaghim and Lukasz Kozubowski of Clemson University are studying possible resistance mechanisms to commonly used azole antifungal compounds. In both agriculture and medicine, fungal development of resistance to azole compounds is a major worldwide problem, causing crop loss and agricultural-to-human-pathogen fungal resistance. Due to resistance development, azoles are increasingly failing as both agricultural and human antifungal treatments: the percentage of azole-resistant strains of one fungus increased from 5% to 20% in five years, and high patient mortalities are reported for azole-resistant fungal infections. This resistance may stem from DNA instability, yet little is known about the mechanisms that lead to changes to fungi DNA upon azole treatment. Understanding the development of resistance to antifungal compounds will impact the broad areas of biology, chemistry, agriculture, and medicine. This proposed work also will promote participation of a first-generation, economically disadvantaged graduate student, strengthen collaborative research efforts with Professor Ken Marcus (Clemson), and combat implicit bias in chemistry in a collaborative effort with Professor William Pennington (Clemson). In addition, Professor Brumaghim will introduce middle school and high school students to bioinorganic chemistry through teaching a DNA damage lab as part of a week-long residential summer chemistry camp. This research will train next-generation interdisciplinary researchers as it explores the chemistry behind a fundamental biological process with global implications. Results of this work have the potential to advance understanding of antifungal resistance mechanisms and to guide future antifungal development.<br/><br/>Azole compounds are the most widely used class of fungicides for crop protection as well as a first-line treatment for human fungal infections worldwide. Crop loss and agricultural-to-human-pathogen fungal resistance is a major global issue, since fungi are developing resistance to azole compounds. Azole resistance mechanisms include mutations in the ERG11 gene that encodes the antifungal target protein and upregulation of ERG11 or genes encoding azole efflux pumps. Azole resistance may stem from DNA instability and increases in chromosomal copy numbers (aneuploidy), yet the mechanisms that lead to these changes to fungi DNA upon azole treatment are unknown. Initial data from PI Brumaghim and co-PI Kozubowski indicate that despite sharing Erg11 as a common target, different azoles display a wide range of resistance development in the human pathogen Cryptococcus neoformans. In addition, the azole drug fluconazole binds to copper and iron, enhances reactive oxygen species (ROS) generation, and promotes metal-mediated DNA damage in vitro. Fluconazole also increases ROS and cellular damage in C. neoformans. The proposed work will test the hypothesis that ROS generation and DNA damage by azole antifungals interacting with copper and/or iron is a general mechanism for the DNA damage and genetic instability that causes azole resistance. This novel mechanism for azole antifungal resistance will be established by: 1) quantifying the ability of chemically diverse azole compounds to bind iron and copper, promote ROS generation, and damage DNA in vitro, and 2) determining the effects of the same azole compounds on cellular ROS, DNA integrity, and development of drug resistance in two model fungi Saccharomyces cerevisiae and C. neoformans under normal and elevated copper or iron conditions. This work aims to establish the role of metals and ROS in azole-mediated DNA damage and enable correlations of azole properties with their effects on DNA damage and resistance development. By investigating the hypothesis that azole-metal binding and DNA damage underlie azole antifungal resistance, the PI and co-PI is asking an important experimental question with potentially broad scientific implications for fungal resistance.<br/><br/>This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
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0.957 |