The microbiota’s influence on host (patho) physiology has gained interest in the context of Gulf War Illness (GWI), a chronic disorder featuring dysregulation of the gut-brain-immune axis. This study examined short- and long-term effects of GWI-related chemicals on gut health and fecal microbiota and the potential benefits of Lacto-N-fucopentaose-III (LNFPIII) treatment in a GWI model. Male C57BL/6J mice were administered pyridostigmine bromide (PB; 0.7 mg/kg) and permethrin (PM; 200 mg/kg) for 10 days with concurrent LNFPIII treatment (35 μg/mouse) in a short-term study (12 days total) and delayed LNFPIII treatment (2×/week) beginning 4 months after 10 days of PB/PM exposure in a long-term study (9 months total). Fecal 16S rRNA sequencing was performed on all samples post-LNFPIII treatment to assess microbiota effects of GWI chemicals and acute/delayed LNFPIII administration. Although PB/PM did not affect species composition on a global scale, it affected specific taxa in both short- and long-term settings. PB/PM elicited more prominent long-term effects, notably, on the abundances of bacteria belonging to Lachnospiraceae and Ruminococcaceae families and the genus Allobaculum. LNFPIII improved a marker of gut health (i.e., decreased lipocalin-2) independent of GWI and, importantly, increased butyrate producers (e.g., Butyricoccus, Ruminococcous) in PB/PM-treated mice, indicating a positive selection pressure for these bacteria. Multiple operational taxonomic units correlated with aberrant behavior and lipocalin-2 in PB/PM samples; LNFPIII was modulatory. Overall, significant and lasting GWI effects occurred on specific microbiota and LNFPIII treatment was beneficial.
Ryan S Mote, Jessica M Carpenter, Rachel L Dockman, Andrew J Steinberger, Garret Suen, Thomas Norberg, Donald A Harn, John J Wagner, Nikolay M Filipov. Int J Environ Res Public Health. 2020 Sep 27;17(19):E7081. doi: 10.3390/ijerph17197081.
Diseases caused by pathogenic free-living amoebae include primary amoebic meningoencephalitis (Naegleria fowleri), granulomatous amoebic encephalitis (Acanthamoeba spp.), Acanthamoeba keratitis, and Balamuthia amoebic encephalitis (Balamuthia mandrillaris). Each of these are difficult to treat and have high morbidity and mortality rates due to lack of effective therapeutics. Since repurposing drugs is an ideal strategy for orphan diseases, we conducted a high throughput phenotypic screen of 12,000 compounds from the Calibr ReFRAME library. We discovered a total of 58 potent inhibitors (IC50 <1 μM) against N. fowleri (n = 19), A. castellanii (n = 12), and B. mandrillaris (n = 27) plus an additional 90 micromolar inhibitors. Of these, 113 inhibitors have never been reported to have activity against Naegleria, Acanthamoeba or Balamuthia. Rapid onset of action is important for new anti-amoeba drugs and we identified 19 compounds that inhibit N. fowleri in vitro within 24 hours (halofuginone, NVP-HSP990, fumagillin, bardoxolone, belaronib, and BPH-942, solithromycin, nitracrine, quisinostat, pabinostat, pracinostat, dacinostat, fimepinostat, sanguinarium, radicicol, acriflavine, REP3132, BC-3205 and PF-4287881). These compounds inhibit N. fowleri in vitro faster than any of the drugs currently used for chemotherapy. The results of these studies demonstrate the utility of phenotypic screens for discovery of new drugs for pathogenic free-living amoebae, including Acanthamoeba for the first time. Given that many of the repurposed drugs have known mechanisms of action, these compounds can be used to validate new targets for structure-based drug design.
Christopher A Rice, Beatrice L Colon, Emily Chen, Mitchell V Hull, Dennis E Kyle. PLoS Negl Trop Dis. 2020 Sep 24;14(9):e0008353. doi: 10.1371/journal.pntd.0008353.
Unique lindenane sesquiterpenoid dimers from Chloranthecae spp. were recently identified with promising in vitro antiplasmodial activity and potentially novel mechanisms of action. To gain mechanistic insights to this new class of natural products, in vitro selection of Plasmodium falciparum resistance to the most active antiplasmodial compound, chlorajaponilide C, was explored. In all selected resistant clones, the half-maximal effective concentration (EC50) of chlorajaponilide C increased >250-fold, and whole genome sequencing revealed mutations in the recently discovered P. falciparum prodrug activation and resistance esterase (PfPARE). Chlorajaponilide C was highly potent (mean EC50 = 1.6 nM, n=34) against fresh Ugandan P. falciparum isolates. Analysis of the structure-resistance relationships revealed that in vitro potency of a subset of lindenane sesquiterpenoid dimers was not mediated by PfPARE mutations. Thus, chlorajaponilide C, but not some related compounds, required parasite esterase activity for in vitro potency, and those compounds serve as the foundation for development of potent and selective antimalarials.
Dennis Kyle leads the UGA Center for Tropical and Emerging Diseases, and his endowment enables him to run a 16-person lab of student researchers, postdocs, and research scientists fighting a host of parasitic diseases around the world. (Photo by Andrew Davis Tucker/UGA)
GRA Endowment helps researchers save lives through drug discovery
The Amoeba Summit in Orlando last year is where the importance of her work on drug discovery for deadly amoebae really hit home for Cassiopeia Russell.
It was there she learned the story of an 11-year-old boy who had gone on a family vacation to Costa Rica and was having a great time going down a waterslide at a hot spring there. Days later, he started complaining of a terrible headache. Then he started vomiting. Within a week, he was dead. The cause was a microscopic organism, Naegleria fowleri, living in the warm waters of the spring.
Cassiopeia Russell is a doctoral student working in Dennis Kyle’s lab on treatments to combat Naegleria fowleri, a rare but deadly brain-eating microscopic organism that can be 99% fatal if contracted. “This parasite infects mainly young children,” she says. “Hundreds have died.”
Russell, a doctoral student, had been working in the lab of Dennis Kyle, director of the UGA Center for Tropical and Emerging Global Diseases, for about a year when she was able to attend the conference thanks to a Georgia Research Alliance (GRA) endowment and learn how the research she does in the lab is making a real impact on people’s lives. The GRA was founded with the goal of expanding Georgia universities’ ability to conduct high-level research with the potential of bringing new and innovative products to market. Kyle is the GRA Eminent Scholar in Antiparasitic Drug Discovery, and his endowment enables him to run a 16-person staff of student researchers, postdocs, and research scientists.
“I asked myself at the beginning if I really wanted to work on a parasite this rare,” Russell says. “But if you look at the statistics, this parasite infects mainly young children. Hundreds have died.” She thinks about that every day in the lab.
More commonly referred to as the brain-eating amoeba, Naegleria fowleri is a surprisingly common amoeba found in warm water lakes, ponds, and rivers. When water is forced up the nose—like when diving into a body of water or repeatedly riding a waterslide—the parasite travels to the brain, where it attacks the organ’s cells. Though infections are rare, the brain-eating amoeba kills almost everyone it infects.
One reason Naegleria fowleri is so deadly is because symptoms of the infection resemble those of viral meningitis, a much more common and more treatable disease. “That misdiagnosis and waiting to see if the patient gets better after beginning treatment is wasting valuable time,” says Russell. She’s committed to finding faster, more effective ways to diagnose the condition so patients can get the right medications in time to stop the disease’s progression.
The Georgia Research Alliance really helped me set up this whole operation when I got here. Without the GRA, there’s no way that I could have had this team going for three years.” — Dennis Kyle, GRA Eminent Scholar in Antiparasitic Drug Discovery
As a member of Kyle’s lab, Russell also tests drug compounds to see which ones can kill an amoeba without destroying the human cells it infects. Current drugs used to treat the infection aren’t very effective and are highly toxic.
Despite being almost 99% fatal, not many federal dollars go toward research on brain-eating and other kinds of amoebae. That’s where the Georgia Research Alliance comes in.
“The Georgia Research Alliance really helped me set up this whole operation when I got here,” says Kyle. “Without the GRA, there’s no way that I could have had this team going for three years. This is something that we have concerns about in Georgia. Every summer, we hear of Naegleria fowleri cases on the news. But we don’t have many people worldwide working on it and very few doing the drug discovery needed to come up with a new drug that could save lives. And that’s really our goal.”
The other main area of research in the Kyle lab is malaria and how the parasite becomes resistant to the drugs commonly used to treat it. Additionally, a less commonly studied strain of malaria can go dormant inside its host, effectively hiding in the liver until flaring up weeks, months, or even years later. Kyle and his team were able to develop a model that simulates that dormant phase to test a variety of drugs to find a way to kill the parasite.
But in order to use that model, researchers in the lab must collect parasites from the field. The endowment helped the lab send assistant research scientist Steven Maher to Asia 25 times over the past four years to work with partners in Cambodia and Thailand.
“International travel has definitely changed my life,” Maher says. “Right now, we’re supporting people in Asia and their families, and I think having that human connection is really important. I think all too often we do research and we forget about ‘how is what I’m doing helping real people?’ I think a lot of researchers would benefit from that type of experience.”
The Georgia Research Alliance, private donors, and the UGA Athletic Association are committed to providing researchers like Russell and Maher with opportunities to advance their work on deadly infectious diseases threatening nations around the world.
Assistant research scientist Steven Maher (far right) with the team he works with in Cambodia. GRA funding has allowed him to travel to Asia 25 times over the past four years battling malaria.
Melissa Sleda, a Ph.D. trainee is Silvia Moreno’s laboratory, is in her third year at UGA. She is originally from Sandusky, Michigan and attended Lawrence Technological University where she majored in Molecular and Cell Biology with a minor in Chemistry. At UGA, she has held positions as the Secretary for the Cell Bio Grad Student Association (2019-2020), and as Treasurer (2019-2020) and current President (2020-2021) of the CTEGD grad student association.
Melissa Sleda has been awarded a T32 Trainee Fellowship for the 2020-2021 academic year.
Why did you choose UGA?
I chose UGA because of the Integrated Life Sciences Umbrella program. As an incoming graduate student, I was not set on studying a particular organism, and I was excited for the opportunity to rotate in labs across different departments.
What is your research project?
My project seeks to characterize enzymes of the isoprenoid biosynthetic pathway in Toxoplasma gondii and to investigate these enzymes as potential chemotherapeutic targets. The current chemotherapy for Toxoplasmosis is ineffective because it does not eliminate the chronic stage of infection. My project seeks to test drugs that target enzymes of the isoprenoid pathway in both the acute and chronic forms of infection in order to find a more effective chemotherapy.
What are your future professional plans?
My future career goal is to stay in academia and become a professor at a smaller institution with a higher emphasis on teaching and leading smaller research projects. I want to help students at smaller universities gain research experience through classroom labs and one-on-one research projects.
What do you hope to do for your Capstone Experience?
For my capstone experience, I hope to be able to do research in another country to gain a wider perspective of how research is done in other countries. I hope that I am able to do research in a lab that I can learn new techniques that will translate into my research project.
What is your favorite thing about Athens?
My favorite thing about Athens is the warm weather and the great sense of community.
What advice do you have for students interested in this field?
Do things out of your comfort zone because it will help you develop as a scientist.
Support trainees like Melissa by giving today to the Center for Tropical & Emerging Global Diseases.
The recent identification of isolates of D. immitis with confirmed resistance to the macrocyclic lactone preventatives presents an opportunity for comparative genomic studies using these isolates, and examining the genetic diversity within and between them. We studied the genomes of Wolbachia endosymbionts of five isolates of D. immitis maintained at the University of Georgia. Missouri and Georgia-2 are maintained as drug susceptible isolates, and JYD-27, Yazoo-2013 and Metairie-2014 are resistant to the macrocyclic lactone preventatives. We used whole genome amplification followed by Illumina-based sequencing from 8 to 12 individual microfilariae from each of the five isolates, obtaining a depth of coverage of approximately 40–75 fold for each. The Illumina sequences were used to create new genome assemblies for all the Wolbachia isolates studied. Comparisons of the Wolbachia sequences revealed more than 3000 sequence variations in each isolate. We identified 67 loci specific in resistant isolates but not in susceptible isolates, including 18 genes affected. Phylogenetic analysis suggested that the endosymbionts of the drug-susceptible isolates are more closely related to each other than to those from any of the resistant parasites. This level of variation in the Wolbachia endosymbionts of D. immitis isolates suggests a potential for selection for resistance against drugs targeting them.
Pei-Tsz Shin, Rodrigo de Paula Baptista, Connor M. O’Neill, Connor Wallis, Barbara J.Reaves, Adrian J. Wolstenholme. Veterinary Parasitology, Volume 286, October 2020, 109225. https://doi.org/10.1016/j.vetpar.2020.109225
Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewisx antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewisx antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.
In animals, the response to chronic hypoxia is mediated by prolyl-hydroxylases (PHDs) that regulate the levels of hypoxia inducible transcription factor a (HIFα). PHD homologues exist in other types of eukaryotes and prokaryotes where they act on non-HIF substrates. To gain insight into the factors underlying different PHD substrates and properties, we carried out biochemical and biophysical studies on PHD homologues from the slime mold, Dictyostelium discoideum, and the protozoan parasite, Toxoplasma gondii, both lacking HIF. The respective prolyl-hydroxylases (DdPhyA and TgPhyA) catalyze prolyl-hydroxylation of S-Phase Kinase Associated Protein 1 (Skp1), a reaction enabling adaptation to different dioxygen availability. Assays with full length Skp1 substrates reveal substantial differences in the kinetic properties of DdPhyA and TgPhyA, both with respect to each other and compared with human PHD2; consistent with cellular studies TgPhyA is more active at low dioxygen concentrations than DdPhyA. TgSkp1 is a DdPhyA substrate and DdSkp1 is a TgPhyA substrate. No cross-reactivity was detected between DdPhyA/TgPhyA substrates and human PHD2. The human Skp1 E147P variant is a DdPhyA and TgPhyA substrate, suggesting some retention of ancestral interactions. Crystallographic analysis of DdPhyA enables comparisons with homologues from humans, Trichoplax adhaerens, and prokaryotes, TgPhyA informing on differences in mobile elements involved in substrate binding and catalysis. In DdPhyA, two mobile loops that enclose substrates in the PHDs are conserved, but the C-terminal helix of the PHDs is strikingly absent. The combined results support the proposal that PHD homologues have evolved kinetic and structural features suited to their specific sensing roles.
Tongri Liu, Martine I Abboud, Rasheduzzaman Chowdhury, Anthony Tumber, Adam P Hardy, Kerstin Lippl, Christopher T Lohans, Elisabete Pires, James Wickens, Michael A McDonough, Christopher M West, Christopher J Schofield. J Biol Chem. 2020 Sep 15;jbc.RA120.013998. doi: 10.1074/jbc.RA120.013998.
Diego Huet studies the organelles involved in the metabolic adaptations of the apicomplexan parasite Toxoplasma gondii. In this mSphere of Influence article, he reflects on how the paper “Absolute quantification of matrix metabolites reveals the dynamics of mitochondrial metabolism” by Chen et al. (W. W. Chen, E. Freinkman, T. Wang, K. Birsoy, and D. M. Sabatini, Cell 166:1324–1337.e11, 2016, https://doi.org/10.1016/j.cell.2016.07.040) shaped his research by providing an approach for rapidly and specifically isolating mitochondria to probe the metabolism of these organelles.
The antimalarial candidate MMV008138 (1a) is of particular interest because its target enzyme (IspD) is absent in human. To achieve higher potency, and to probe for steric demand, a series of analogs of 1a were prepared that featured methyl-substitution of the B- and C-rings, as well as ring-chain transformations. X-ray crystallography, NMR spectroscopy and calculation were used to study the effects of these modifications on the conformation of the C-ring and orientation of the D-ring. Unfortunately, all the B- and C-ring analogs explored lost in vitro antimalarial activity. The possible role of steric effects and conformational changes on target engagement are discussed.
Sha Ding, Maryam Ghavami, Joshua H.Butler, Emilio F. Merino, Carla Slebodnick, Maria B. Cassera, Paul R. Carlier. Bioorg Med Chem Lett. 2020 Sep 5;127520. doi: 10.1016/j.bmcl.2020.127520
The microbiota’s influence on host (patho) physiology has gained interest in the context of Gulf War Illness (GWI), a chronic disorder featuring dysregulation of the gut-brain-immune axis. This study examined short- and long-term effects of GWI-related chemicals on gut health and fecal microbiota and the potential benefits of Lacto-N-fucopentaose-III (LNFPIII) treatment in a GWI model. Male C57BL/6J mice were administered pyridostigmine bromide (PB; 0.7 mg/kg) and permethrin (PM; 200 mg/kg) for 10 days with concurrent LNFPIII treatment (35 μg/mouse) in a short-term study (12 days total) and delayed LNFPIII treatment (2×/week) beginning 4 months after 10 days of PB/PM exposure in a long-term study (9 months total). Fecal 16S rRNA sequencing was performed on all samples post-LNFPIII treatment to assess microbiota effects of GWI chemicals and acute/delayed LNFPIII administration. Although PB/PM did not affect species composition on a global scale, it affected specific taxa in both short- and long-term settings. PB/PM elicited more prominent long-term effects, notably, on the abundances of bacteria belonging to Lachnospiraceae and Ruminococcaceae families and the genus Allobaculum. LNFPIII improved a marker of gut health (i.e., decreased lipocalin-2) independent of GWI and, importantly, increased butyrate producers (e.g., Butyricoccus, Ruminococcous) in PB/PM-treated mice, indicating a positive selection pressure for these bacteria. Multiple operational taxonomic units correlated with aberrant behavior and lipocalin-2 in PB/PM samples; LNFPIII was modulatory. Overall, significant and lasting GWI effects occurred on specific microbiota and LNFPIII treatment was beneficial.
