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Category: publications

Regional Survey of Diamondback Moth (Lepidoptera: Plutellidae) Response to Maximum Dosages of Insecticides in Georgia and Florida

We conducted maximum dose bioassays of insecticide for the control of diamondback moth (DBM), Plutella xylostella (Linnaeus), in cole crops, from 2016 to 2019 at several commercial locations in Georgia and Florida. The nominal maximum dose was defined as the highest labeled rate of an insecticide at the beginning of the survey in the equivalent of 935 liters/ha dilution. The results indicated low insecticide efficacy for high labeled rates of the following insecticides by common name (Insecticide Resistance Action Committee group number in parentheses). Our 4-yr survey identified very low levels of DBM larval control (<47%) by lambda-cyhalothrin (3), methoxyfenozide (18), pyriproxyfen (7C), novaluron (15), bifenthrin (3), chlorantraniliprole (28), indoxacarb (22A), and methomyl (1A). The best products for DBM control (>74%) listed in decreasing average levels of efficacy were naled (1B), cyclaniliprole (28), tolfenpyrad (21A), emamectin benzoate (6), and cyantraniliprole (28). Intermediate levels of control (61-71%) were obtained with Bacillus thuringiensis subspecies aizawai (11A), Bacillus thuringiensis, subsp. kurstaki, strain ABTS-351 (11A), and spinetoram (5). This rapid bioassay provided the grower with a ranking of insecticide efficacy for the control the DBM population for that farm site. These data allowed growers to make an informed decision on control quickly and plan for resistance management rotations for DBM that season.

David Riley, Hugh Smith, John Bennett, Philip Torrance, Evan Huffman, Alton Sparks, Jr, Charles Gruver, Thomas Dunn, Donald Champagne. J Econ Entomol. 2020 Jun 19;toaa125. doi: 10.1093/jee/toaa125.

The Functional Characterization of TcMyoF Implicates a Family of Cytostome-Cytopharynx Targeted Myosins as Integral to the Endocytic Machinery of Trypanosoma cruzi

Of the pathogenic trypanosomatids, Trypanosoma cruzi alone retains an ancient feeding apparatus known as the cytostome-cytopharynx complex (SPC) that it uses as its primary mode of endocytosis in a manner akin to its free-living kinetoplastid relatives who capture and eat bacterial prey via this endocytic organelle. In a recent report, we began the process of dissecting how this organelle functions by identifying the first SPC-specific proteins in T. cruzi. Here, we continued these studies and report on the identification of the first enzymatic component of the SPC, a previously identified orphan myosin motor (MyoF) specifically targeted to the SPC. We overexpressed MyoF as a dominant-negative mutant, resulting in parasites that, although viable, were completely deficient in measurable endocytosis in vitro. To our surprise, however, a full deletion of MyoF demonstrated only a decrease in the overall rate of endocytosis, potentially indicative of redundant myosin motors at work. Thereupon, we identified three additional orphan myosin motors, two of which (MyoB and MyoE) were targeted to the preoral ridge region adjacent to the cytostome entrance and another (MyoC) which was targeted to the cytopharynx tubular structure similar to that of MyoF. Additionally, we show that the C-terminal tails of each myosin are sufficient for targeting a fluorescent reporter to SPC subregions. This work highlights a potential mechanism used by the SPC to drive the inward flow of material for digestion and unveils a new level of overlapping complexity in this system with four distinct myosin isoforms targeted to this feeding structure.

IMPORTANCE The parasite Trypanosoma cruzi is the etiological agent of Chagas disease and chronically infects upwards of 7 million people in the Americas. Current diagnostics and treatments remain grossly inadequate due, in part, to our general lack of understanding of this parasite’s basic biology. One aspect that has resisted detailed scrutiny is the mechanism employed by this parasite to extract nutrient resources from the radically different environments that it encounters as it transitions between its invertebrate and mammalian hosts. These parasites engulf food via a tubular invagination of its membrane, a strategy used by many protozoan species, but how this structure is formed or functions mechanistically remains a complete mystery. The significance of our research is in the identification of the mechanistic underpinnings of this feeding organelle that may bring to light new potential therapeutic targets to impede parasite feeding and thus halt the spread of this deadly human pathogen.

Nathan Michael Chasen, Menna Grace Etheridge, Ronald Drew Etheridge.

Discovery of Anti-Amoebic Inhibitors from Screening the MMV Pandemic Response Box on Balamuthia mandrillaris, Naegleria fowleri, and Acanthamoeba castellanii

Pathogenic free-living amoebae, Balamuthia mandrillarisNaegleria fowleri, and several Acanthamoeba species are the etiological agents of severe brain diseases, with case mortality rates > 90%. A number of constraints including misdiagnosis and partially effective treatments lead to these high fatality rates. The unmet medical need is for rapidly acting, highly potent new drugs to reduce these alarming mortality rates. Herein, we report the discovery of new drugs as potential anti-amoebic agents. We used the CellTiter-Glo 2.0 high-throughput screening methods to screen the Medicines for Malaria Ventures (MMV) Pandemic Response Box in a search for new active chemical scaffolds. Initially, we screened the library as a single-point assay at 10 and 1 µM. From these data, we reconfirmed hits by conducting quantitative dose–response assays and identified 12 hits against B. mandrillaris, 29 against N. fowleri, and 14 against A. castellanii ranging from nanomolar to low micromolar potency. We further describe 11 novel molecules with activity against B. mandrillaris, 22 against N. fowleri, and 9 against A. castellanii. These structures serve as a starting point for medicinal chemistry studies and demonstrate the utility of phenotypic screening for drug discovery to treat diseases caused by free-living amoebae.

Christopher A. Rice, Emma V. Troth, A. Cassiopeia Russell, Dennis E. Kyle. Pathogens. 2020 Jun 16;9(6):E476. doi: 10.3390/pathogens9060476.

Evaluation of Morbidity in Schistosoma Mansoni-Positive Primary and Secondary School Children After Four Years of Mass Drug Administration of Praziquantel in Western Kenya

Background: World Health Organization guidelines recommend preventive chemotherapy with praziquantel to control morbidity due to schistosomiasis. The primary aim of this cross-sectional study was to determine if 4 years of annual mass drug administration (MDA) in primary and secondary schools lowered potential markers of morbidity in infected children 1 year after the final MDA compared to infected children prior to initial MDA intervention.

Methods: Between 2012 and 2016 all students in two primary and three secondary schools within three kilometers of Lake Victoria in western Kenya received annual mass praziquantel administration. To evaluate potential changes in morbidity we measured height, weight, mid-upper arm circumference, hemoglobin levels, abdominal ultrasound, and quality of life in children in these schools. This study compared two cross-sectional samples of Schistosoma mansoni egg-positive children: one at baseline and one at year five, 1 year after the fourth annual MDA. Data were analyzed for all ages (6-18 years old) and stratified by primary (6-12 years old) and secondary (12-18 years old) school groups.

Results: The prevalence of multiple potential morbidity markers did not differ significantly between the egg-positive participants at baseline and those at 5 years by Mann Whitney nonparametric analysis and Fisher’s exact test for continuous and categorical data, respectively. There was a small but significantly higher score in school-related quality of life assessment by year five compared to baseline by Mann Whitney analysis (P = 0.048) in 13-18 year olds where malaria-negative. However, anemia was not positively impacted by four annual rounds of MDA, but registered a significant negative outcome.

Conclusions: We did not detect differences in morbidity markers measured in a population of those infected or re-infected after multiple MDA. This could have been due to their relative insensitivity or a failure of MDA to prevent morbidity among those who remain infected. High malaria transmission in this area and/or a lack of suitable methods to measure the more subtle functional morbidities caused by schistosomiasis could be a factor. Further research is needed to identify and develop well-defined, easily quantifiable S. mansoni morbidity markers for this age group.

Bernard O. Abudho, Bernard Guyah, Bartholomew N. Ondigo, Eric M. Ndombi, Edmund Ireri, Jennifer M. Carter, Diana K. Riner, Nupur Kittur, Diana M. S. Karanja & Daniel G. Colley. Evaluation of morbidity in Schistosoma mansoni-positive primary and secondary school children after four years of mass drug administration of praziquantel in western Kenya. Infect Dis Poverty 9, 67 (2020). https://doi.org/10.1186/s40249-020-00690-7

Potent Tetrahydroquinolone Eliminates Apicomplexan Parasites

Apicomplexan infections cause substantial morbidity and mortality, worldwide. New, improved therapies are needed. Herein, we create a next generation anti-apicomplexan lead compound, JAG21, a tetrahydroquinolone, with increased sp3-character to improve parasite selectivity. Relative to other cytochrome b inhibitors, JAG21 has improved solubility and ADMET properties, without need for pro-drug. JAG21 significantly reduces Toxoplasma gondii tachyzoites and encysted bradyzoites in vitro, and in primary and established chronic murine infections. Moreover, JAG21 treatment leads to 100% survival. Further, JAG21 is efficacious against drug-resistant Plasmodium falciparum in vitro. Causal prophylaxis and radical cure are achieved after P. berghei sporozoite infection with oral administration of a single dose (2.5 mg/kg) or 3 days treatment at reduced dose (0.625 mg/kg/day), eliminating parasitemia, and leading to 100% survival. Enzymatic, binding, and co-crystallography/pharmacophore studies demonstrate selectivity for apicomplexan relative to mammalian enzymes. JAG21 has significant promise as a pre-clinical candidate for prevention, treatment, and cure of toxoplasmosis and malaria.

Martin J. McPhillie, Ying Zhou, Mark R. Hickman, James A. Gordon, Christopher R. Weber, Qigui Li, Patty J. Lee, Kangsa Amporndanai, Rachel M. Johnson, Heather Darby, Stuart Woods, Zhu-hong Li, Richard S. Priestley, Kurt D. Ristroph, Scott B. Biering, Kamal El Bissati, Seungmin Hwang, Farida Esaa Hakim, Sarah M. Dovgin, Joseph D. Lykins, Lucy Roberts, Kerrie Hargrave, Hua Cong, Anthony P. Sinai, Stephen P. Muench, Jitender P. Dubey, Robert K. Prud’homme, Hernan A. Lorenzi, Giancarlo A. Biagini, Silvia N. Moreno, Craig W. Roberts, Svetlana V. Antonyuk, Colin W. G. Fishwick, and Rima McLeod. Front. Cell. Infect. Microbiol., 09 June 2020 | https://doi.org/10.3389/fcimb.2020.00203

Plastid Biogenesis in Malaria Parasites Requires the Interactions and Catalytic Activity of the Clp Proteolytic System

The human malaria parasite, Plasmodium falciparum, contains an essential plastid called the apicoplast. Most apicoplast proteins are encoded by the nuclear genome and it is unclear how the plastid proteome is regulated. Here, we study an apicoplast-localized caseinolytic-protease (Clp) system and how it regulates organelle proteostasis. Using null and conditional mutants, we demonstrate that the P. falciparum Clp protease (PfClpP) has robust enzymatic activity that is essential for apicoplast biogenesis. We developed a CRISPR/Cas9-based system to express catalytically dead PfClpP, which showed that PfClpP oligomerizes as a zymogen and is matured via transautocatalysis. The expression of both wild-type and mutant Clp chaperone (PfClpC) variants revealed a functional chaperone-protease interaction. Conditional mutants of the substrate-adaptor (PfClpS) demonstrated its essential function in plastid biogenesis. A combination of multiple affinity purification screens identified the Clp complex composition as well as putative Clp substrates. This comprehensive study reveals the molecular composition and interactions influencing the proteolytic function of the apicoplast Clp system and demonstrates its central role in the biogenesis of the plastid in malaria parasites.

Anat Florentin, Dylon R. Stephens, Carrie F. Brooks, Rodrigo P. Baptista, and Vasant Muralidharan. Proc Natl Acad Sci USA. 2020 Jun 1;201919501. doi: 10.1073/pnas.1919501117.

Limonoids From Cipadessa baccifera

Eighteen new limonoids, including eight methyl angolensates (18) and 10 cipadesins (918), were isolated from the leaves of Cipadessa baccifera. Their structures were characterized by means of spectroscopic data analyses, single-crystal X-ray diffraction, and quantum chemistry computational methods. The C-6 configurations in those compounds possessing a C-6 hydroxy group were all assigned as S regardless of the magnitude of J5,6, and the C-2′ configuration in those bearing a 2-methylbutyryl residue was defined by single-crystal X-ray diffraction and NMR data. Compounds 156711, and 12 showed moderate antimalarial activities with IC50 values ranging from 12 to 28 μM.

Jin-Hai Yu, Hua Zhang, Bin Zhou, Flavia M. Zimbres, Seema Dalal, Qun-Fang Liu, Maria B. Cassera, and Jian-Min Yue. J Nat Prod. 2020 May 29. doi: 10.1021/acs.jnatprod.9b00666.

Using Population Genetics to Examine Relationships of Dirofilaria Immitis Based on Both Macrocyclic Lactone-Resistance Status and Geography

Prevention of infection with canine heartworm (Dirofilaria immitis) is based on the compliant administration of macrocyclic lactone (ML) drugs. Resistance to ML drugs is well documented in D. immitis; however, there remains a paucity of information on the spatial distribution and prevalence of resistant isolates. This project aims to improve understanding of ML-resistance by using a population genetic approach. We developed a large panel of microsatellite loci and identified 12 novel highly polymorphic markers. These 12, and five previously published markers were used to screen pools of microfilariae from 16 confirmed drug-susceptible, 25 confirmed drug-resistant, and from 10 suspected drug-resistant field isolates. In isolates where microfilarial suppression testing indicated resistance, Spatial Principal Component Analysis (sPCoA), Neighbor Joining Trees and Bayesian clustering all revealed high genetic similarity between pre- and post-treatment samples. Somewhat surprisingly, the Neighbor Joining tree and sPCoA generated using pairwise Nei’s distances did not reveal clustering for resistant isolates, nor did it reveal state-level geographic clustering from samples collected in Georgia, Louisiana or Mississippi. In contrast, Discriminant Analysis of Principle Components was able to discriminate between susceptible, suspected-resistant and resistant samples. However, no resistance-associated markers were detected, and this clustering was driven by the combined effects of multiple alleles across multiple loci. Additionally, we measured unexpectedly large genetic distances between different passages of laboratory strains that originated from the same source infection. This finding strongly suggests that the genetic makeup of laboratory isolates can change substantially with each passage, likely due to genetic bottlenecking. Taken together, these data suggest greater than expected genetic variability in the resistant isolates, and in D. immitis overall. Our results also suggest that microsatellite genotyping lacks the sensitivity to detect a specific genetic signature for resistance. Future investigations using genomic analyses will be required to elucidate the genetic relationships of ML-resistant isolates.

Julie Sanchez, Guha Dharmarajan, Melissa M. George, Cassan Pulaski, Adrian J. Wolstenholme, John S. Gilleard, Ray M. Kaplan. 2020 Veterinary Parasitology; 283:109125. doi: 10.1016/j.vetpar.2020.109125.

Combination of Serological, Antigen Detection, and DNA Data for Plasmodium Falciparum Provides Robust Geospatial Estimates for Malaria Transmission in Haiti

Microscopy is the gold standard for malaria epidemiology, but laboratory and point-of-care (POC) tests detecting parasite antigen, DNA, and human antibodies against malaria have expanded this capacity. The island nation of Haiti is endemic for Plasmodium falciparum (Pf) malaria, though at a low national prevalence and heterogenous geospatial distribution. In 2015 and 2016, serosurveys were performed of children (ages 6-7 years) sampled in schools in Saut d’Eau commune (n = 1,230) and Grand Anse department (n = 1,664) of Haiti. Children received malaria antigen rapid diagnostic test and provided a filter paper blood sample for further laboratory analysis of the Pf histidine-rich protein 2 (HRP2) antigen, Pf DNA, and anti-Pf IgG antibodies. Prevalence of Pf infection ranged from 0.0-16.7% in 53 Saut d’Eau schools, and 0.0-23.8% in 56 Grand Anse schools. Anti-Pf antibody carriage exceeded 80% of students in some schools from both study sites. Geospatial prediction ellipses were created to indicate clustering of positive tests within the survey areas and overlay of all prediction ellipses for the different types of data revealed regions with high likelihood of active and ongoing Pf malaria transmission. The geospatial utilization of different types of Pf data can provide high confidence for spatial epidemiology of the parasite.

Adan Oviedo, Alaine Knipes, Caitlin Worrell, LeAnne M Fox, Luccene Desir, Carl Fayette, Alain Javel, Franck Monestime, Kimberly Mace, Michelle A Chang, Venkatachalam Udhayakumar, Jean F Lemoine, Kimberly Won, Patrick J Lammie, Eric Rogier. Scientific Reports volume 10, Article number: 8443 (2020). https://doi.org/10.1038/s41598-020-65419-w

Nationwide Remapping of Schistosoma mansoni Infection in Rwanda Using Circulating Cathodic Antigen Rapid Test: Taking Steps Toward Elimination

Eugene Ruberanziza, Udo Wittmann, Aimable Mbituyumuremyi, Alphonse Mutabazi, Carl H. Campbell Jr, Daniel G. Colley, Fiona M. Fleming, Giuseppina Ortu, Govert J. van Dam, Irenee Umulisa, Jamie Tallant, Michee Kabera, Muhammed Semakula, Paul L. A. M. Corstjens, Tharcisse Munyaneza, Warren Lancaster, Jean Bosco Mbonigaba and Michelle N. Clements. Am J Trop Med Hyg. 2020 May 18. doi: 10.4269/ajtmh.19-0866.