Center for Tropical and Emerging Global Diseases
University of Georgia
Dennis Kyle, director of the Center for Tropical and Emerging Global Diseases, was interviewed during the 4th Annual Amoeba Summit about his work with Naegleria fowleri. Listen to the interview at Outbreak News Today.
Antimicrobial treatment failure threatens our ability to control infections. In addition to antimicrobial resistance, treatment failures are increasingly understood to derive from cells that survive drug treatment without selection of genetically heritable mutations. Parasitic protozoa, such as Plasmodium species that cause malaria, Toxoplasma gondii and kinetoplastid protozoa, including Trypanosoma cruzi and Leishmaniaspp., cause millions of deaths globally. These organisms can evolve drug resistance and they also exhibit phenotypic diversity, including the formation of quiescent or dormant forms that contribute to the establishment of long-term infections that are refractory to drug treatment, which we refer to as ‘persister-like cells’. In this Review, we discuss protozoan persister-like cells that have been linked to persistent infections and discuss their impact on therapeutic outcomes following drug treatment.
Michael P. Barrett, Dennis E. Kyle, L. David Sibley, Joshua B. Radke & Rick L. Tarleton. Nat Rev Microbiol. 2019 Aug 23. doi: 10.1038/s41579-019-0238-x.
The ability to culture pathogenic organisms substantially enhances the quest for fundamental knowledge and the development of vaccines and drugs. Thus, the elaboration of a protocol for the in vitro cultivation of the erythrocytic stages of Plasmodium falciparum revolutionized research on this important parasite. However, for P. vivax, the most widely distributed and difficult to treat malaria parasite, a strict preference for reticulocytes thwarts efforts to maintain it in vitro. Cultivation of P. cynomolgi, a macaque-infecting species phylogenetically close to P. vivax, was briefly reported in the early 1980s, but not pursued further. Here, we define the conditions under which P. cynomolgi can be adapted to long term in vitro culture to yield parasites that share many of the morphological and phenotypic features of P. vivax. We further validate the potential of this culture system for high-throughput screening to prime and accelerate anti-P. vivax drug discovery efforts.
Chua ACY, Ong JJY, Malleret B, Suwanarusk R, Kosaisavee V, Zeeman AM, Cooper CA, Tan KSW, Zhang R, Tan BH, Abas SN, Yip A, Elliot A, Joyner CJ, Cho JS, Breyer K, Baran S, Lange A, Maher SP, Nosten F, Bodenreider C, Yeung BKS, Mazier D, Galinski MR, Dereuddre-Bosquet N, Le Grand R, Kocken CHM, Rénia L, Kyle DE, Diagana TT, Snounou G, Russell B, Bifani P. Nat Commun. 2019 Aug 12;10(1):3635. doi: 10.1038/s41467-019-11332-4.
Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
Beatriz Baragaña, Barbara Forte, Ryan Choi, Stephen Nakazawa Hewitt, Juan A. Bueren-Calabuig, João Pedro Pisco, Caroline Peet, David M. Dranow, David A. Robinson, Chimed Jansen, Neil R. Norcross, Sumiti Vinayak, Mark Anderson, Carrie F. Brooks, Caitlin A. Cooper, Sebastian Damerow, Michael Delves, Karen Dowers, James Duffy, Thomas E. Edwards, Irene Hallyburton, Benjamin G. Horst, Matthew A. Hulverson, Liam Ferguson, María Belén Jiménez-Díaz, Rajiv S. Jumani, Donald D. Lorimer, Melissa S. Love, Steven Maher, Holly Matthews, Case W. McNamara, Peter Miller, Sandra O’Neill, Kayode K. Ojo, Maria Osuna-Cabello, Erika Pinto, John Post, Jennifer Riley, Matthias Rottmann, Laura M. Sanz, Paul Scullion, Arvind Sharma, Sharon M. Shepherd, Yoko Shishikura, Frederick R. C. Simeons, Erin E. Stebbins, Laste Stojanovski, Ursula Straschil, Fabio K. Tamaki, Jevgenia Tamjar, Leah S. Torrie, Amélie Vantaux, Benoît Witkowski, Sergio Wittlin, Manickam Yogavel, Fabio Zuccotto, Iñigo Angulo-Barturen, Robert Sinden, Jake Baum, Francisco-Javier Gamo, Pascal Mäser, Dennis E. Kyle, Elizabeth A. Winzeler, Peter J. Myler, Paul G. Wyatt, David Floyd, David Matthews, Amit Sharma, Boris Striepen, Christopher D. Huston, David W. Gray, Alan H. Fairlamb, Andrei V. Pisliakov, Chris Walpole, Kevin D. Read, Wesley C. Van Voorhis, and Ian H. Gilbert. 2019. PNAS, https://doi.org/10.1073/pnas.1814685116
To discover leads for next-generation chemoprotective antimalarial drugs, we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1 micromolar). Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action.
Yevgeniya Antonova-Koch, Stephan Meister, Matthew Abraham, Madeline R. Luth, Sabine Ottilie, Amanda K. Lukens, Tomoyo Sakata-Kato, Manu Vanaerschot, Edward Owen, Juan Carlos Jado, Steven P. Maher, Jaeson Calla, David Plouffe, Yang Zhong, Kaisheng Chen, Victor Chaumeau, Amy J. Conway, Case W. McNamara, Maureen Ibanez, Kerstin Gagaring, Fernando Neria Serrano, Korina Eribez, Cullin McLean Taggard, Andrea L. Cheung, Christie Lincoln, Biniam Ambachew, Melanie Rouillier, Dionicio Siegel, François Nosten, Dennis E. Kyle, Francisco-Javier Gamo, Yingyao Zhou, Manuel Llinás, David A. Fidock, Dyann F. Wirth, Jeremy Burrows, Brice Campo, Elizabeth A. Winzeler. 2018. Science; 362(6419):eaat9446. http://science.sciencemag.org/content/362/6419/eaat9446
Life cycles of spirorchiids that infect the vascular system of turtles are poorly understood. Few life cycles of these blood flukes have been elucidated and all intermediate hosts reported are gastropods (Mollusca), regardless of whether the definitive host is a freshwater or a marine turtle. During a recent survey of blood fluke larvae in polychaetes on the coast of South Carolina, USA, spirorchiid-like cercariae were found to infect the polychaetes Amphitrite ornata (Terebellidae) and Enoplobranchus sanguineus (Polycirridae). Cercariae were large, furcate, with a ventral acetabulum, but no eyespots were observed. Partial sequences of D1–D2 domains of the large ribosomal subunit, the internal transcribed spacer 2, and the mitochondrial cytochrome oxidase 1 genes allowed the identification of sporocysts and cercariae as belonging to two unidentified Neospirorchis species reported from the green turtle, Chelonia mydas, in Florida: Neospirorchis sp. (Neogen 13) in A. ornata and Neospirorchis sp. (Neogen 14) in E. sanguineus. Phylogenetic analysis suggests that infection of annelids by blood flukes evolved separately in aporocotylids and spirorchiids. Our results support the contention that the Spirorchiidae is not a valid family and suggest that Neospirorchis is a monophyletic clade within the paraphyletic Spirorchiidae. Since specificity of spirorchiids for their intermediate hosts is broader than it was thus far assumed, surveys of annelids in turtle habitats are necessary to further our understanding of the life history of these pathogenic parasites.
Isaure de Buron, Beatrice L. Colon, Sasha V. Siegel, Jenna Oberstaller, Andrea Rivero, Dennis E. Kyle. 2018. International Journal for Parasitology; 48(14):1097-1106. https://doi.org/10.1016/j.ijpara.2018.08.002
Naegleria fowleri is the causative agent of primary amoebic meningoencephalitis (PAM), which is fatal in >97% of cases. In this study, we aimed to identify new, rapidly acting drugs to increase survival rates. We conducted phenotypic screens of libraries of Food and Drug Administration–approved compounds and the Medicines for Malaria Venture Pathogen Box and validated 14 hits (defined as a 50% inhibitory concentration of <1 μM). The hits were then prioritized by assessing the rate of action and efficacy in combination with current drugs used to treat PAM. Posaconazole was found to inhibit amoeba growth within the first 12 hours of exposure, which was faster than any currently used drug. In addition, posaconazole cured 33% of N. fowleri–infected mice at a dose of 20 mg/kg and, in combination with azithromycin, increased survival by an additional 20%. Fluconazole, which is currently used for PAM therapy, was ineffective in vitro and vivo. Our results suggest posaconazole could replace fluconazole in the treatment of PAM.
Beatrice L Colon, Christopher A Rice, R Kiplin Guy, Dennis E Kyle. 2018. The Journal of Infectious Diseases. https://doi.org/10.1093/infdis/jiy622
Background:Plasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available.
Results: Here, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite.
Conclusions: The α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.
Carola Schafer, Nicholas Dambrauskas, Ryan W. Steel, Sara Carbonetti, Vorada Chuenchob, Erika L. Flannery, Vladimir Vigdorovich, Brian G. Oliver, Wanlapa Roobsoong, Steven P. Maher, Dennis Kyle, Jetsumon Sattabongkot, Stefan H. I. Kappe, Sebastian A. Mikolajczak and D. Noah Sather. 2018. Malaria Journal; 17:370. https://doi.org/10.1186/s12936-018-2519-7
Beatrice Colon, an Illinois native, is a Ph.D. trainee in Dennis Kyle’s laboratory. She holds a Bachelor of Science degree from the University of Illinois at Urbana-Champaign and a Master of Science degree from the University of South Florida (USF). She began her Ph.D. at USF as well.
Beatrice moved to the University of Georgia in January 2017 with the Kyle Lab.
“I decided to transfer universities because of the excellent infectious disease department,” said Beatrice.
“My favorite thing about the CTEGD is the openness for collaborations; the center is also very focused on training a new generation of scientists. “
Beatrice is currently working on a drug discovery project for the brain-eating amoeba, Naegleria fowleri. The disease was the major factor that drew her to the project. Primary amoebic meningoencephalitis is nearly always fatal and affects young healthy children. Moreover, there is not an effective drug treatment for people that do get infected with the amoeba.
In her short time at UGA, Beatrice has won first place for a poster presentation at the graduate student and postdoc symposium. She was also selected for the Biology of Parasitism course at Woods Hole, MA this past summer.
“This course was definitely a career-changing experience – I was able to work with a variety of infectious diseases and learn techniques that were not available for the parasite I work on.”
Beatrice is interested in staying in drug discovery for infectious diseases and currently looking at positions in both academia and industry.
Support trainees like Beatrice Colon. Give TODAY!