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Author: Donna Huber

Optimal 10-Aminoartemisinins With Potent Transmission-Blocking Capabilities for New Artemisinin Combination Therapies–Activities Against Blood Stage P. falciparum Including PfKI3 C580Y Mutants and Liver Stage P. berghei Parasites

We have demonstrated previously that amino-artemisinins including artemiside and artemisone in which an amino group replaces the oxygen-bearing substituents attached to C-10 of the current clinical artemisinin derivatives dihydroartemisinin (DHA), artemether and artesunate, display potent activities in vitro against the asexual blood stages of Plasmodium falciparum (Pf). In particular, the compounds are active against late blood stage Pf gametocytes, and are strongly synergistic in combination with the redox active drug methylene blue. In order to fortify the eventual selection of optimum amino-artemisinins for development into new triple combination therapies also active against artemisinin-resistant Pf mutants, we have prepared new amino-artemisinins based on the easily accessible and inexpensive DHA-piperazine. The latter was converted into alkyl- and aryl sulfonamides, ureas and amides. These derivatives were screened together with the comparator drugs DHA and the hitherto most active amino-artemisinins artemiside and artemisone against asexual and sexual blood stages of Pf and liver stage P. berghei (Pb) sporozoites. Several of the new amino-artemisinins bearing aryl-urea and -amide groups are potently active against both asexual, and late blood stage gametocytes (IC50 0.4-1.0 nM). Although the activities are superior to those of artemiside (IC50 1.5 nM) and artemisone (IC50 42.4 nM), the latter are more active against the liver stage Pb sporozoites (IC50 artemisone 28 nM). In addition, early results indicate these compounds tend not to display reduced susceptibility against parasites bearing the Pf Kelch 13 propeller domain C580Y mutation characteristic of artemisinin-resistant Pf. Thus, the advent of the amino-artemisinins including artemiside and artemisone will enable the development of new combination therapies that by virtue of the amino-artemisinin component itself will possess intrinsic transmission-blocking capabilities and may be effective against artemisinin resistant falciparum malaria.

Ho Ning Wong, Vivian Padín-Irizarry, Mariëtte E. van der Watt, Janette Reader, Wilna Liebenberg, Lubbe Wiesner, Peter Smith, Korina Eribez, Elizabeth A. Winzeler, Dennis E. Kyle, Lyn-Marie Birkholtz, Dina Coertzen, and Richard K. Haynes. Front Chem. 2020 Jan 10;7:901. doi: 10.3389/fchem.2019.00901. eCollection 2019.

Environmental Predictors of Schistosomiasis Persistent Hotspots following Mass Treatment with Praziquantel

Schistosomiasis control programs rely heavily on mass drug administration (MDA) campaigns with praziquantel for preventative chemotherapy. Areas where the prevalence and/or intensity of schistosomiasis infection remains high even after several rounds of treatment, termed “persistent hotspots” (PHSs), have been identified in trials of MDA effectiveness conducted by the Schistosomiasis Consortium for Operational Research and Evaluation (SCORE) in Kenya, Mozambique, Tanzania, and Côte d’Ivoire. In this analysis, we apply a previously developed set of criteria to classify the PHS status of 531 study villages from five SCORE trials. We then fit logistic regression models to data from SCORE and publically available georeferenced datasets to evaluate the influence of local environmental and population features, pre-intervention infection burden, and treatment scheduling on PHS status in each trial. The frequency of PHS in individual trials ranged from 35.3% to 71.6% in study villages. Significant relationships between PHS status and MDA frequency, distance to freshwater, rainfall, baseline schistosomiasis burden, elevation, land cover type, and village remoteness were each observed in at least one trial, although the strength and direction of these relationships was not always consistent among study sites. These findings suggest that PHSs are driven in part by environmental conditions that modify the risk and frequency of reinfection.

Joseph W. Walker, Nupur Kittur, Sue Binder, Jennifer D. Castleman, John M. Drake, Carl H. Campbell Jr., Charles H. King and Daniel G. Colley. Am J Trop Med Hyg. 2019 Dec 30. doi: 10.4269/ajtmh.19-0658.

Neurochemical and neuroinflammatory perturbations in two Gulf War Illness models: Modulation by the immunotherapeutic LNFPIII

Gulf War Illness (GWI) manifests a multitude of symptoms, including neurological and immunological, and approximately a third of the 1990–1991 Gulf War (GW) veterans suffer from it. This study sought to characterize the acute neurochemical (monoamine) and neuroinflammatory profiles of two established GWI animal models and examine the potential modulatory effects of the novel immunotherapeutic Lacto-N-fucopentaose III (LNFPIII). In Model 1, male C57BL/6 J mice were treated for 10 days with pyridostigmine bromide (PB) and permethrin (PM). In Model 2, a separate cohort of mice were treated for 14 days with PB and N,N-Diethyl-methylbenzamide (DEET), plus corticosterone (CORT) via drinking water on days 8–14 and diisopropylfluorophosphate (DFP) on day 15. LNFPIII was administered concurrently with GWI chemicals treatments. Brain and spleen monoamines and hippocampal inflammatory marker expression were examined by, respectively, HPLC-ECD and qPCR, 6 h post treatment cessation. Serotonergic (5-HT) and dopaminergic (DA) dyshomeostasis caused by GWI chemicals was apparent in multiple brain regions, primarily in the nucleus accumbens (5-HT) and hippocampus (5-HT, DA) for both models. Splenic levels of 5-HT (both models) and norepinephrine (Model 2) were also disrupted by GWI chemicals. LNFPIII treatment prevented many of the GWI chemicals induced monoamine alterations. Hippocampal inflammatory cytokines were increased in both models, but the magnitude and spread of inflammation was greater in Model 2; LNFPIII was anti-inflammatory, more so in the apparently milder Model 1. Overall, in both models, GWI chemicals led to monoamine disbalance and neuroinflammation. LNFPIII co-treatment prevented many of these disruptions in both models, which is indicative of its promise as a potential GWI therapeutic.

J.M. Carpenter, H.E. Gordon, H.D. Ludwig, J.J. Wagner, D.A. Harn, T. Norberg, N.M. Filipov. Neurotoxicology. 2019 Dec 19;77:40-50. doi: 10.1016/j.neuro.2019.12.012

Conservation of Ancient Genetic Pathways for Intracellular Persistence Among Animal Pathogenic Bordetellae

Animal and human pathogens of the genus Bordetella are not commonly considered to be intracellular pathogens, although members of the closely related classical bordetellae are known to enter and persist within macrophages in vitro and have anecdotally been reported to be intracellular in clinical samples. B. bronchiseptica, the species closest to the ancestral lineage of the classical bordetellae, infects a wide range of mammals but is known to have an alternate life cycle, persisting, replicating and disseminating with amoeba. These observations give rise to the hypothesis that the ability for intracellular survival has an ancestral origin and is common among animal-pathogenic and environmental Bordetella species. Here we analyzed the survival of B. bronchiseptica and defined its transcriptional response to internalization by murine macrophage-like cell line RAW 264.7. Although the majority of the bacteria were killed and digested by the macrophages, a consistent fraction survived and persisted inside the phagocytes. Internalization prompted the activation of a prominent stress response characterized by upregulation of genes involved in DNA repair, oxidative stress response, pH homeostasis, chaperone functions, and activation of specific metabolic pathways. Cross species genome comparisons revealed that most of these upregulated genes are highly conserved among both the classical and non-classical Bordetella species. The diverse Bordetella species also shared the ability to survive inside RAW 264.7 cells, with the single exception being the bird pathogen B. avium, which has lost several of those genes. Knock-out mutations in genes expressed intracellularly resulted in decreased persistence inside the phagocytic cells, emphasizing the importance of these genes in this environment. These data show that the ability to persist inside macrophage-like RAW 264.7 cells is shared among nearly all Bordetella species, suggesting that resisting phagocytes may be an ancient mechanism that precedes speciation in the genus and may have facilitated the adaptation of Bordetella species from environmental bacteria to mammalian respiratory pathogens.

Israel Rivera, Bodo Linz, Kalyan K. Dewan, Longhuan Ma, Christopher A. Rice, Dennis E. Kyle and Eric T. Harvill. Front Microbiol. 2019 Dec 11;10:2839. doi: 10.3389/fmicb.2019.02839. eCollection 2019.

Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.

The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing “tried and true” methods of our lab who pioneered the first use of GECI’s in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

Stephen A. Vella, Abigail Calixto, Beejan Asady, Zhu-Hong Li, Silvia N. J. Moreno. Methods Mol Biol. 2020;2071:187-207. doi: 10.1007/978-1-4939-9857-9_11.

The genomes of two parasitic wasps that parasitize the diamondback moth

Background

Parasitic insects are well-known biological control agents for arthropod pests worldwide. They are capable of regulating their host’s physiology, development and behaviour. However, many of the molecular mechanisms involved in host-parasitoid interaction remain unknown.

Results

We sequenced the genomes of two parasitic wasps (Cotesia vestalis, and Diadromus collaris) that parasitize the diamondback moth Plutella xylostella using Illumina and Pacbio sequencing platforms. Genome assembly using SOAPdenovo produced a 178 Mb draft genome for C. vestalis and a 399 Mb draft genome for D. collaris. A total set that contained 11,278 and 15,328 protein-coding genes for C. vestalis and D. collaris, respectively, were predicted using evidence (homology-based and transcriptome-based) and de novo prediction methodology. Phylogenetic analysis showed that the braconid C. vestalis and the ichneumonid D. collaris diverged approximately 124 million years ago. These two wasps exhibit gene gains and losses that in some cases reflect their shared life history as parasitic wasps and in other cases are unique to particular species. Gene families with functions in development, nutrient acquisition from hosts, and metabolism have expanded in each wasp species, while genes required for biosynthesis of some amino acids and steroids have been lost, since these nutrients can be directly obtained from the host. Both wasp species encode a relative higher number of neprilysins (NEPs) thus far reported in arthropod genomes while several genes encoding immune-related proteins and detoxification enzymes were lost in both wasp genomes.

Conclusions

We present the annotated genome sequence of two parasitic wasps C. vestalis and D. collaris, which parasitize a common host, the diamondback moth, P. xylostella. These data will provide a fundamental source for studying the mechanism of host control and will be used in parasitoid comparative genomics to study the origin and diversification of the parasitic lifestyle.

Min Shi, Zhizhi Wang, Xiqian Ye, Hongqing Xie, Fei Li, Xiaoxiao Hu, Zehua Wang, Chuanlin Yin, Yuenan Zhou, Qijuan Gu, Jiani Zou, Leqing Zhan, Yuan Yao, Jian Yang, Shujun Wei, Rongmin Hu, Dianhao Guo, Jiangyan Zhu, Yanping Wang, Jianhua Huang, Francesco Pennacchio, Michael R. Strand & Xuexin Chen. The genomes of two parasitic wasps that parasitize the diamondback moth. BMC Genomics 20893 (2019) doi:10.1186/s12864-019-6266-0.

Chagas Disease Drug Discovery: Multiparametric Lead Optimization against Trypanosoma cruzi in Acylaminobenzothiazole Series

Acylaminobenzothiazole hits were identified as potential inhibitors of Trypanosoma cruzi replication, a parasite responsible for Chagas disease. We selected compound 1 for lead optimization, aiming to improve in parallel its anti-T. cruzi activity (IC50 = 0.63 μM) and its human metabolic stability (human clearance = 9.57 mL/min/g). A total of 39 analogues of 1 were synthesized and tested in vitro. We established a multiparametric structure-activity relationship, allowing optimization of antiparasite activity, physicochemical parameters, and ADME properties. We identified compound 50 as an advanced lead with an improved anti-T. cruzi activity in vitro (IC50 = 0.079 μM) and an enhanced metabolic stability (human clearance = 0.41 mL/min/g) and opportunity for the oral route of administration. After tolerability assessment, 50 demonstrated a promising in vivo efficacy.

Charlotte Fleau, Angel Padilla, Juan Miguel-Siles, Maria T. Quesada-Campos, Isabel Saiz-Nicolas, Ignacio Cotillo, Juan Cantizani Perez, Rick L. Tarleton, Maria Marco, Gilles Courtemanche. J Med Chem. 2019. doi: 10.1021/acs.jmedchem.9b01429.

Field Relevant Variation in Ambient Temperature Modifies Density-Dependent Establishment of Plasmodium falciparum Gametocytes in Mosquitoes

The relationship between Plasmodium falciparum gametocyte density and infections in mosquitoes is central to understanding the rates of transmission with important implications for control. Here, we determined whether field relevant variation in environmental temperature could also modulate this relationship. Anopheles stephensi were challenged with three densities of P. falciparum gametocytes spanning a ~10-fold gradient, and housed under diurnal/daily temperature range (“DTR”) of 9°C (+5°C and -4°C) around means of 20, 24, and 28°C. Vector competence was quantified as the proportion of mosquitoes infected with oocysts in the midguts (oocyst rates) or infectious with sporozoites in the salivary glands (sporozoite rates) at peak periods of infection for each temperature to account for the differences in development rates. In addition, oocyst intensities were also recorded from infected midguts and the overall study replicated across three separate parasite cultures and mosquito cohorts. While vector competence was similar at 20 DTR 9°C and 24 DTR 9°C, oocyst and sporozoite rates were also comparable, with evidence, surprisingly, for higher vector competence in mosquitoes challenged with intermediate gametocyte densities. For the same gametocyte densities however, severe reductions in the sporozoite rates was accompanied by a significant decline in overall vector competence at 28 DTR 9°C, with gametocyte density per se showing a positive and linear effect at this temperature. Unlike vector competence, oocyst intensities decreased with increasing temperatures with a predominantly positive and linear association with gametocyte density, especially at 28 DTR 9°C. Oocyst intensities across individual infected midguts suggested temperature-specific differences in mosquito susceptibility/resistance: at 20 DTR 9°C and 24 DTR 9°C, dispersion (aggregation) increased in a density-dependent manner but not at 28 DTR 9°C where the distributions were consistently random. Limitations notwithstanding, our results suggest that variation in temperature could modify seasonal dynamics of infectious reservoirs with implications for the design and deployment of transmission-blocking vaccines/drugs.

Ashutosh K. Pathak, Justine C. Shiau, Matthew B. Thomas and Courtney C. Murdock, Front Microbiol. 2019 Nov 15;10:2651. doi: 10.3389/fmicb.2019.02651. eCollection 2019.