Dennis Kyle & CTEGD featured in Georgia Trend’s Article
Director Dennis Kyle was interviewed about CTEGD and his research as part of Georgia Trend’s feature article “Moving Innovation Beyond the Walls”. Read the article now.
Shotgun Kinetic Target-Guided Synthesis Approach Enables the Discovery of Small-Molecule Inhibitors against Pathogenic Free-Living Amoeba Glucokinases
Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat the rise of infection from these pathogens. Naegleria fowleri glucokinase (NfGlck), a key metabolic enzyme involved in generating glucose-6-phosphate, was previously identified as a potential target due to its limited sequence similarity with human Glck (HsGlck). Herein, we used our previously demonstrated multifragment kinetic target-guided synthesis (KTGS) screening strategy to identify inhibitors against pFLA glucokinases. Unlike the majority of previous KTGS reports, our current study implements a “shotgun” approach, where fragments were not biased by predetermined binding potentials. The study resulted in the identification of 12 inhibitors against 3 pFLA glucokinase enzymes─NfGlck, Balamuthia mandrillaris Glck (BmGlck), and Acanthamoeba castellanii Glck (AcGlck). This work demonstrates the utility of KTGS to identify small-molecule binders for biological targets where resolved X-ray crystal structures are not readily accessible.
Mintesinot Kassu, Prakash T Parvatkar, Jillian Milanes, Neil P Monaghan, Chungsik Kim, Matthew Dowgiallo, Yingzhao Zhao, Ami H Asakawa, Lili Huang, Alicia Wagner, Brandon Miller, Karissa Carter, Kayleigh F Barrett, Logan M Tillery, Lynn K Barrett, Isabelle Q Phan, Sandhya Subramanian, Peter J Myler, Wesley C Van Voorhis, James W Leahy, Christopher A Rice, Dennis E Kyle, James Morris, Roman Manetsch. ACS Infect Dis. 2023 Oct 11. doi: 10.1021/acsinfecdis.3c00284.
Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri
Introduction: As global temperatures rise to unprecedented historic levels, so too do the latitudes of habitable niches for the pathogenic free-living amoeba, Naegleria fowleri. This opportunistic parasite causes a rare, but >97% fatal, neurological infection called primary amoebic meningoencephalitis. Despite its lethality, this parasite remains one of the most neglected and understudied parasitic protozoans.
Methods: To better understand amoeboid intercellular communication, we elucidate the structure, proteome, and potential secretion mechanisms of amoeba-derived extracellular vesicles (EVs), which are membrane-bound communication apparatuses that relay messages and can be used as biomarkers for diagnostics in various diseases.
Results and discussion: Herein we propose that N. fowleri secretes EVs in clusters from the plasma membrane, from multivesicular bodies, and via beading of thin filaments extruding from the membrane. Uptake assays demonstrate that EVs are taken up by other amoebae and mammalian cells, and we observed a real-time increase in metabolic activity for mammalian cells exposed to EVs from amoebae. Proteomic analysis revealed >2,000 proteins within the N. fowleri-secreted EVs, providing targets for the development of diagnostics or therapeutics. Our work expands the knowledge of intercellular interactions among these amoebae and subsequently deepens the understanding of the mechanistic basis of PAM.
A Cassiopeia Russell, Peter Bush, Gabriela Grigorean, Dennis E Kyle. Front Microbiol. 2023 Sep 27:14:1264348. doi: 10.3389/fmicb.2023.1264348.
In the News: Dennis Kyle is quoted in “brain-eating” amoeba news story
A Nexstar Media wire story on Naegleria fowleri (aka “brain-eating” amoeba) featuring CTEGD director Dennis Kyle has been picked up by news outlets.
Brain-eating amoeba: How do most infections occur? (NewsNation)
Brain-eating amoeba: How do infections occur, and where are they most common? (The Hill)
Increasing the knowledge base on brain-eating amoeba
Cassie Russell, a graduate student in the Department of Infectious Diseases, was an undergraduate when she first heard of Naegleria fowleri, also known as the brain-eating amoeba. While whole lectures in her parasitology course had been dedicated to other parasites, N. fowleri was barely a mention.
“I remember maybe 15 minutes was spent on it,” said Russell. “I was shocked that was all that was known about this deadly organism.”
N. fowleri causes the acute neurological disease primary amoebic meningoencephalitis (PAM). There have been hundreds of reported cases of PAM, but only seven survivors worldwide, according to the Centers for Disease Control and Prevention.
“I had the opportunity to speak with families in Florida who had lost someone to Naegleria fowleri infection,” she said. “The fear they had in not knowing what was wrong with their loved one and then learning that there was very little that could be done—their stories were just heartbreaking.”After arriving at UGA, Russell was pleased to find out that N. fowleri was one of the parasites being studied in Dennis Kyle’s laboratory at the Center for Tropical and Emerging Global Diseases.
Individuals, most commonly young children, become infected when they inhale warm freshwater contaminated with N. fowleri. This typically occurs during the late summer months when people are participating in recreational activities in rivers and lakes, but it can also occur when people use unsterilized tap water in nasal irrigation devices. It is more likely to occur in the southern United States, but infection is very rare. Between 2011 and 2020 only 33 cases were reported in the United States, according the CDC.
N. fowleri is one of the most neglected of the neglected tropical diseases. However, knowledge about the parasite has been growing since the 1960s as scientists build on new data and apply new technology. Russell is doing her part and was the lead on a study recently published in Microbiology Spectrum where, for the first time, drug susceptibility was tested across 11 clinical isolates.
“Current drug treatment is a cocktail of six different drugs,” said Russell. “However, only a few isolates have been tested in the lab for susceptibility. We don’t know if some drugs work better for different strains.”
A big question facing researchers is why these drugs show effectiveness in the lab when so few real-world cases have been successfully treated. Russell suspected that other factors were at play in treatment failure, such as genetic differences among geographically distinct amoeba populations.
The 11 isolates used in the study came from patients who contracted N. fowleri in different geographic regions. Russell found that these isolates had significant differences in susceptibility to seven of the eight drugs currently used to treat the infection.
The need for effective and fast-acting treatments is especially great. PAM is almost always fatal, with death occurring about a week after the initial onset of symptoms.
Doctors are racing against the clock as there is often a delay in diagnosis: The symptoms mimic meningitis, and N. fowleri is a rare infection. The drugs used can also be pretty toxic, so identifying the safest and most effective drug treatment could significantly improve outcomes.
Russell’s findings are another stepping stone to propel N. fowleri research toward increased understanding of this parasite and ultimately better treatments. For example, she realized that there is not a gold standard for genotyping.
“Researchers could be talking about genetically different isolates but not realize it,” said Russell.
In addition to creating a genotyping standard, she has identified combinational drug studies to test for synergism as a next step. For now, though, Russell is focusing on another need in the fight against N. fowleri—diagnostics.
“Awareness, improved diagnostic techniques and faster-acting drugs are needed to improve outcomes,” she said.
This story first appeared at https://research.uga.edu/news/increasing-the-knowledge-base-on-brain-eating-amoeba/
EdU Incorporation To Assess Cell Proliferation and Drug Susceptibility in Naegleria fowleri
Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri, and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study, we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2′-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor-linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy, and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (7-deaza-2′-deoxy-7-ethynyladenosine), an adenine analog, also was incorporated by N. fowleri but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.
Emma V Troth, Dennis E Kyle. Antimicrob Agents Chemother. 2021 Jun 17;65(7):e0001721. doi: 10.1128/AAC.00017-21.
Dennis Kyle Featured Guest on People, Parasites & Plagues Podcast
Dr. Dennis Kyle, director of CTEGD and professor in the departments of cellular biology and infectious diseases, is the featured guest on Episode 5 of the People, Parasites & Plagues Podcast. He talks about a deadly disease caused by Naegleria fowleri, also known as the brain-eating amoeba.
The podcast is also available at Amazon, iTunes, Google, Spotify, Stitcher, Audible, and TuneIn
People, Parasites & Plagues is a podcast aimed at delivering information about the fascinating pathogens among us from the impressive professionals who study them.
Join hosts Dr. David Peterson and Dr. Liliana Salvador, two infectious disease researchers from the University of Georgia, as they explore the past, present, and future of science.
Tune in every other week for a new and enlightening episode as they unpack the details surrounding some of Earth’s most perplexing diseases. Look for the People, Parasites & Plagues Podcast on your favorite Podcast service!
EdU incorporation to assess cell proliferation and drug susceptibility in Naegleria fowleri
Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2′-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro EdA (5’ethynyl-2′-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.
Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba
Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.
Logan Tillery, Kayleigh Barrett, Jenna Goldstein, Jared W Lassner, Bram Osterhout, Nathan L Tran, Lily Xu, Ryan M Young, Justin Craig, Ian Chun, David M Dranow, Jan Abendroth, Silvia L Delker, Douglas R Davies, Stephen J Mayclin, Brandy Calhoun, Madison J Bolejack, Bart Staker, Sandhya Subramanian, Isabelle Phan, Donald D Lorimer, Peter J Myler, Thomas E Edwards, Dennis E Kyle, Christopher A Rice, James C Morris, James W Leahy, Roman Manetsch, Lynn K Barrett, Craig L Smith, Wesley C Van Voorhis (2021) Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba. PLoS ONE 16(3): e0241738. https://doi.org/10.1371/journal.pone.0241738