Biology subjects

Zamanian, M.

Publications and source records attributed to Zamanian, M..

At least 19 recordsLinked to original sources

bioRxiv2026

High-throughput phenotypic profiling of insecticide responses in mosquito larvae

Extensive use of insecticides is increasing resistance risks that could severely reduce the control of mosquito vectors that transmit infectious agents of Neglected Tropical Diseases. Addressing resistance threats by discovering new insecticides can be challenging because of the limited throughput and translatability of existing in vitro screening pipelines. Acquiring multiple phenotypic endpoints of larvae could alleviate these restrictions by more thoroughly profiling drug effects on whole organisms, providing leads to compounds with novel mechanisms of action, and increasing screening throughput compared to adult-stage assays. Here, we establish a pair of assays for profiling motility and development traits in larval-stage mosquitoes at a higher scale than standard larvicidal screening techniques. We optimized assay parameters and developed novel image processing approaches that enable relatively high throughput screening of chemical compounds on single larvae within a screen with condition replicates. We tested the assay with an insect growth regulator (S-methoprene), a slow-acting pyrrole (chlorfenapyr), and two microbial larvicides (Spinosad and Lysinibacillus sphaericus). These measurements aligned well with known insecticide mechanisms, and dose response curves established assay baselines for comparison in future screens. Finally, we present ways in which the assay design can be modified across different imaging technologies, showing the flexibility of the screening approach.

pharmacology and toxicology↗

bioRxiv2026

CELeidoscope: quad-fluorescent Caenorhabditis elegans strain for tissue-specific spectral single-cell analyses

Cell and tissue-specific transcriptomic profiling of Caenorhabditis elegans is commonly achieved by fluorescence tagging or staining of targeted cell populations, often followed by fluorescence-activated cell sorting (FACS) and RNA sequencing. However, these approaches typically require separate strains for each labeled population, increasing labor and experimental variability while limiting direct comparison of multiple tissues within the same genetic background. To address this limitation and establish proof of concept, we engineered CELeidoscope, a multicolored C. elegans strain that enables spectral sorting of multiple major cell types within a single strain population. Strain construction was carried out using a high-throughput screening method that reduces the labor and plastic costs associated with transgene integration and outcrossing. Four primary tissues (body muscle, neurons, intestinal, and pharyngeal muscle cells) were tagged with spectrally distinct fluorescent proteins, allowing compatibility with viability and nucleic acid dyes. Using spectral flow cytometry, dissociated CELeidoscope cell suspensions could be sorted based on their spectral profiles, with cell recovery rates approximating the expected cell counts in whole organisms. Transcriptomic analysis of the sorted cell populations further validated the identity of the sorted populations, with recovered cells exhibiting gene expression signatures consistent with their intended cell and tissue identities. Together, these results establish CELeidoscope as a versatile tool for multiplexed cell-type isolation in C. elegans, providing a framework for tissue-specific analyses from a common strain background.

microbiology↗

bioRxiv2026

Bioactive Natural Products Produced by Streptomyces from the Microbiome of Cadaveric Fly Larvae

Streptomyces are prolific producers of bioactive compounds and increasingly recognized as members of insect microbiomes, yet the microbiome of cadaveric fly larvae remain an overlooked system for discovering metabolically versatile Streptomyces species. Here, we conduct targeted bacterial isolations from the microbiome of fly larvae collected from pig cadavers, generating 42 Streptomyces isolates of interest, and systematically evaluated their metabolic potential through genomic analysis, antimicrobial screening, biosynthetic gene cluster assessment, untargeted LC-MS/MS metabolomics, and compound purification. The Streptomyces isolates spanned nine species, including underrepresented lineages for which we added genomic representatives. Streptomyces from carrion fly larvae exhibited broad-spectrum antimicrobial activity and substantial BGC diversity, supported by metabolomic detection of antimycins, surugamides, and macrotetrolides. From a deep phylogenetic lineage, we purified JBIR-68 and Simamycin and demonstrated their potent anthelmintic activity against Brugia malayi microfilariae. GNPS molecular networking revealed three additional JBIR-68 analogs, establishing the first taxonomically resolved Streptomyces lineage capable of producing these rare metabolites. Our findings position cadaveric fly larvae as a rich ecological reservoir for discovering Streptomyces with the potential to produce chemically diverse natural products with biomedical applications.

biochemistry↗

bioRxiv2026

Deep phenotypic profiling uncovers cryptic effects of antifilarial drugs

The anthelmintics ivermectin, albendazole, and diethylcarbamazine are the backbone of mass drug administration (MDA) campaigns targeting human filariasis, yet their direct effects on parasites are still not fully defined or understood. The clinical effects of these drugs are stage dependent, resulting in effective clearance of circulating microfilariae but only limited activity against adult worms, a pattern that complicates disease surveillance and elimination efforts. Although molecular targets have been identified or proposed for some antifilarial drugs, their precise modes of action remain opaque, and conventional in vitro assays of motility or viability have generally failed to reflect pharmacologically relevant effects. There is growing evidence that cryptic phenotypes involving altered host-parasite interactions, including changes in parasite secretions, may help reconcile these discrepancies. Focusing on the causative species of lymphatic filariasis, we used high content imaging and quantitative mass spectrometry to enable deeper phenotypic profiling of drug responses in microfilariae and adult worms exposed to antifilarial compounds. In microfilariae, altered environmental conditions (temperature and salinity) lead to modest ivermectin effects on motility at therapeutic concentrations. In adult parasites, we show that drug responses vary with worm age and that different anthelmintics induce distinct changes in the secretory proteome. This improved phenotypic resolution advances our understanding of drug action in intra-host stages and highlights how antifilarial drugs can alter secretory cargo relevant to the detection of adult parasites that persist after drug treatment.

microbiology↗

bioRxiv2026

Nematicidal indole oxazoles and chemoattractants from soil bacteria

Ecological interactions between bacteria and nematodes in many environments provide a basis for the prediction that diverse bacteria produce anti-nematode compounds. The discovery of microbial secondary metabolites with broad-spectrum nematostatic or nematicidal properties can be hastened by drug screening approaches that include several nematode species and phenotypes. We cultured a collection of 22 soil-derived bacterial isolates that carry in their genomes putative pathways for production of unknown secondary metabolites. Isolates were cultured in various media to enhance natural product diversity and yield, and we evaluated culture filtrates for activity against two evolutionarily distinct nematode species: Clade V free-living nematode Caenorhabditis elegans and Clade III mammalian parasitic nematodes in the genus Brugia. Partitioned extracts from Pseudomonas sp. strain TE4607 stunted C. elegans development and caused motility defects in both blood-circulating larval and adult stages of Brugia. The primary active compound was identified as labradorin 1, an indole with known antibacterial and anticancer properties that had not been previously described as affecting nematodes. Notably, filtrates of Pseudomonas sp. TE4607 cultures attracted free-living nematodes in sensory assays, adding to evidence that certain Pseudomonas species modulate the behavior of free-living nematodes. These findings underscore the need to further explore the link between nematode sensory responses and whole-organism effects of microbial metabolites, with potential applications in anthelmintic discovery. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/700618v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@1909e29org.highwire.dtl.DTLVardef@17f9badorg.highwire.dtl.DTLVardef@c59d79org.highwire.dtl.DTLVardef@1e5abbf_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Survey of benzimidazole resistance in ascarid parasites of poultry

Ascarid parasites, such as Ascaridia galli and Heterakis gallinarum, are nearly ubiquitous in poultry and can cause serious production losses. H. gallinarum is of particular concern because of its role as a vector for the protozoan Histomonas meleagridis, the cause of blackhead disease. Currently, only the benzimidazole anthelmintic, fenbendazole (FBZ), is approved for use in poultry, and recently, FBZ resistance has been discovered and validated in populations of the turkey ascarid Ascaridia dissimilis and in ascarid of gallinaceous birds H. gallinarum. Here, we further explore the prevalence of resistance in poultry ascarids by testing FBZ efficacy against thirteen isolates of A. galli and eight isolates of H. gallinarum. Isolates were used to infect day-old naive chickens. Four weeks after infection, animals to be treated received the label-recommended dosage of FBZ (SafeGuard Aquasol) for five days, per the manufacturers directions. One week after the fifth day of treatment, animals were euthanized and parasite burdens were counted to determine treatment efficacy between the untreated and treated groups. Resistance was identified and validated in a single isolate of A. galli, marking the first confirmed case in the species. All isolates of H. gallinarum were found to be resistant. The emergence of resistance in A. galli and the high prevalence of resistance in H. gallinarum highlight the growing concern of resistance in parasites of poultry. Without approved alternative treatments, the detrimental effects of infections cannot be mitigated in resistant populations, significantly impacting profit margins. Diagnostics that enable broader surveys are necessary to understand the full scope of the problem. However, we show that resistance is present across production species and should act as an impetus for the discovery of new treatments and the adoption of new management strategies.

microbiology↗

bioRxiv2025

An optimized rabies vaccine vehicle for orotopical administration to wild vampire bats.

Rabies vaccination of vampire bats (Desmodus rotundus) has been proposed as a superior control method to culling but has yet to be implemented. Success of rabies vaccination depends on a topical vehicle that spreads through a bat colony via allogrooming while additionally preserving vaccine immunogenicity. This work describes the in vitro and in vivo optimization of a new orotopical gel for rabies vaccine delivery to vampire bats. Autonomous transferability of our carboxymethyl cellulose (CMC) gel vaccine delivery formulation was tested in a microchipped vampire bat colony in rural Jalisco, Mexico. Intra-colony gel uptake was traced using the fluorescent biomarker rhodamine B. Importantly, application of topical treatment of [~]20% of the bat colony resulted in estimated uptake by over 85% of the colony. The in vitro stability of our raccoon poxviral-vectored rabies vaccine candidate within CMC was measured at time points up to 3 months at 40 {degrees}C, 23 {degrees}C, and 4 {degrees}C. Extended storage at 4 {degrees}C and short exposure at higher temperatures of potential vampire bat environments preserved vaccine titers within CMC. Furthermore, physical properties of our CMC formulation were compared to the previously used glycerin jelly at 40 {degrees}C, 20 {degrees}C, and 0 {degrees}C using rheological tests. These tests indicate that CMC exhibits optimal properties for topical application to bats even at extreme temperatures possible during field vaccination. This study advances our rabies vaccination strategy for vampire bats by providing a topical vehicle suitable for field application that may additionally be employed for other significant bat diseases.

ecology↗

bioRxiv2025

A graphical user interface for wrmXpress 2.0 streamlines helminth phenotypic screening

Image-based phenotypic screening is a fundamental technique used to better understand the basic biology of helminths and advance discovery of new anthelmintics. Miniaturization of screening platforms and automated microscopy have led to a surge in imaging data and necessitated software to organize and analyze these data. Traditionally, these analyses are performed remotely on high-performance computers, often requiring an understanding of a command-line interface (CLI) and the ability to write scripts to control the software or job scheduler. Requiring access to specialized computing equipment and advanced computational skills raises the barrier to entry for these sorts of studies. The development of efficient, performant computer and graphical processing units for personal computers and cheaper imaging solutions has made the requirement of remote servers superfluous for many small to medium-scale screens, but most analytical software still require interaction with a CLI. To democratize the analysis of image-based phenotypic screens, we have developed a graphical user interface (GUI) for wrmXpress, a tool that integrates many popular computational pipelines for analyzing imaging data of parasitic and free-living worms. The GUI operates on any personal computer using the operating systems native web browser, allowing users to configure and run analyses using a point-and-click approach. Containerization of the application eliminates the need to install specialized programming libraries and dependencies, further increasing the ease of use. GUI development required a substantial reorganization of the wrmXpress backend codebase, which allowed for the addition a new pipeline for high-resolution tracking of worm behavior, and we demonstrate its functionality by showing that praziquantel modulates the behavior of Schistosoma mansoni miracidia. These advances make cutting-edge analyses of image-based phenotyping of worms more equitable and accessible.

bioinformatics↗

bioRxiv2024

Transcriptional phenotype of the anti-parasitic benzodiazepine meclonazepam on the blood fluke Schistosoma mansoni

There are limited control measures for the disease schistosomiasis, despite the fact that infection with parasitic blood flukes affects hundreds of millions of people worldwide. The current treatment, praziquantel, has been in use since the 1980s and there is a concern that drug resistance may emerge with continued monotherapy. Given the need for additional antischistosomal drugs, we have re-visited an old lead, meclonazepam. In comparison to praziquantel, there has been relatively little work on its antiparasitic mechanism. Recent findings indicate that praziquantel and meclonazepam act through distinct receptors, making benzodiazepines a promising chemical series for further exploration. Previous work has profiled the transcriptional changes evoked by praziquantel treatment. Here, we examine in detail schistosome phenotypes evoked by in vitro and in vivo meclonazepam treatment. These data confirm that meclonazepam causes extensive tegument damage and directly kills parasites, as measured by pro-apoptotic caspase activation. In vivo meclonazepam exposure results in differential expression of many genes that are divergent in parasitic flatworms, as well as several gene products implicated in blood feeding and regulation of hemostasis in other parasites. Many of these transcripts are also differentially expressed with praziquantel exposure, which may reflect a common schistosome response to the two drugs. However, despite these similarities in drug response, praziquantel-resistant parasites retain susceptibility to meclonazepams schistocidal effects. These data provide new insight into the mechanism of antischistosomal benzodiazepines, resolving similarities and differences with the current frontline therapy, praziquantel.

pharmacology and toxicology↗

bioRxiv2024

Naturally occurring variation in a cytochrome P450 modifies thiabendazole responses independent of beta-tubulin

Widespread anthelmintic resistance has complicated the management of parasitic nematodes. Resistance to the benzimidazole (BZ) drug class is nearly ubiquitous in many species and is associated with mutations in beta-tubulin genes. However, mutations in beta-tubulin alone do not fully explain all BZ resistance. We performed a genome-wide association study using a genetically diverse panel of Caenorhabditis elegans strains to identify loci that contribute to resistance to the BZ drug thiabendazole (TBZ). We identified a quantitative trait locus (QTL) on chromosome V independent of all beta-tubulin genes and overlapping with two promising candidate genes, the cytochrome P450 gene cyp-35d1 and the nuclear hormone receptor nhr-176, identified by another mapping technique. Both genes were previously demonstrated to play a role in TBZ metabolism. NHR-176 binds TBZ and induces the expression of CYP-35D1, which metabolizes TBZ. We generated single gene deletions of nhr-176 and cyp-35d1 and found that both genes play a role in TBZ response. A predicted high-impact lysine-to-glutamate substitution at position 267 (K267E) in CYP-35D1 was identified in a sensitive parental strain, and reciprocal allele replacement strains in both genetic backgrounds were used to show that the lysine allele conferred increased TBZ resistance. Using competitive fitness assays, we found that neither allele is deleterious, but the lysine allele is selected in the presence of TBZ. Additionally, we found that the lysine allele significantly increased the rate of TBZ metabolism compared to the glutamate allele. Moreover, yeast expression assays showed that the lysine version of CYP-35D1 had twice the enzymatic activity of the glutamate allele. To connect our results to parasitic nematodes, we analyzed four Haemonchus contortus cytochrome P450 orthologs but did not find variation at the 267 position in fenbendazole-resistant populations. Overall, we confirmed that variation in this cytochrome P450 gene is the first locus independent of beta-tubulin to play a role in BZ resistance. Author SummaryBenzimidazoles (BZs) are the most common drug class used to control parasitic nematodes, but because of overuse, resistance is widespread. The known genetic causes of BZ resistance are associated with mutations in beta-tubulin and are the most well understood of any anthelmintic class. However, BZ response varies significantly and differential levels of resistance likely require mutations in genes independent of beta-tubulin. We used the free-living model nematode Caenorhabditis elegans to identify and characterize a novel cytochrome P450 gene, cyp-35d1, associated with natural resistance to the BZ drug thiabendazole (TBZ). We demonstrated that a lysine at position 267 confers TBZ resistance and is selected over multiple generations after TBZ treatment. This allele significantly increased the rate of TBZ metabolism in both C. elegans and yeast. In conclusion, we have characterized the role of variation in a cytochrome P450 that contributes to TBZ resistance, independent of mutations in beta-tubulin.

genetics↗

bioRxiv2024

Development of non-sedating antischistosomal benzodiazepines

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970s. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABAARs) are not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index, and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.

pharmacology and toxicology↗

bioRxiv2023

Wolbachia-mediated resistance to Zika virus infection in Aedes aegypti is dominated by diverse transcriptional regulation and weak evolutionary pressures

A promising candidate for arbovirus control and prevention relies on replacing arbovirus-susceptible Aedes aegypti populations with mosquitoes that have been colonized by the intracellular bacterium Wolbachia and thus have a reduced capacity to transmit arboviruses. This reduced capacity to transmit arboviruses is mediated through a phenomenon referred to as pathogen blocking. Pathogen blocking has primarily been proposed as a tool to control dengue virus (DENV) transmission, however it works against a range of viruses, including Zika virus (ZIKV). Despite years of research, the molecular mechanisms underlying pathogen blocking still need to be better understood. Here, we used RNA-seq to characterize mosquito gene transcription dynamics in Ae. aegypti infected with the wMel strain of Wolbachia that are being released by the World Mosquito Program in Medellin, Colombia. Comparative analyses using ZIKV-infected, uninfected tissues, and mosquitoes without Wolbachia revealed that the influence of wMel on mosquito gene transcription is multifactorial. Importantly, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to pathogen blocking. Therefore, to understand the influence of Wolbachia on within-host ZIKV evolution, we characterized the genetic diversity of molecularly barcoded ZIKV virus populations replicating in Wolbachia-infected mosquitoes and found that within-host ZIKV evolution was subject to weak purifying selection and, unexpectedly, loose anatomical bottlenecks in the presence and absence of Wolbachia. Together, these findings suggest that there is no clear transcriptional profile associated with Wolbachia-mediated ZIKV restriction, and that there is no evidence for ZIKV escape from this restriction in our system. Author SummaryWhen Wolbachia bacteria infect Aedes aegypti mosquitoes, they dramatically reduce the mosquitoes susceptibility to infection with a range of arthropod-borne viruses, including Zika virus (ZIKV). Although this pathogen-blocking effect has been widely recognized, its mechanisms remain unclear. Furthermore, because Wolbachia limits, but does not completely prevent, replication of ZIKV and other viruses in coinfected mosquitoes, there is a possibility that these viruses could evolve resistance to Wolbachia-mediated blocking. Here, we use host transcriptomics and viral genome sequencing to examine the mechanisms of ZIKV pathogen blocking by Wolbachia and viral evolutionary dynamics in Ae. aegypti mosquitoes. We find complex transcriptome patterns that do not suggest a single clear mechanism for pathogen blocking. We also find no evidence that Wolbachia exerts detectable selective pressures on ZIKV in coinfected mosquitoes. Together our data suggest that it may be difficult for ZIKV to evolve Wolbachia resistance, perhaps due to the complexity of the pathogen blockade mechanism.

molecular biology↗

bioRxiv2023

Mapping resistance-associated anthelmintic interactions in the model nematode Caenorhabditis elegans

Parasitic nematodes infect billions of people and are mainly controlled by anthelmintic mass drug administration (MDA). While there are growing efforts to better understand mechanisms of anthelmintic resistance in human and animal populations, it is unclear how resistance mechanisms that alter susceptibility to one drug affect the interactions and efficacy of drugs used in combination. Mutations that alter drug permeability across primary nematode barriers have been identified as potential resistance mechanisms using the model nematode Caenorhabditis elegans. We leveraged high-throughput assays in this model system to measure altered anthelmintic susceptibility in response to genetic perturbations of potential cuticular, amphidial, and alimentary routes of drug entry. Mutations in genes associated with these tissue barriers differentially altered susceptibility to the major anthelmintic classes (macrocyclic lactones, benzimidazoles, and nicotinic acetylcholine receptor agonists) as measured by animal development. We investigated two-way anthelmintic interactions across C. elegans genetic backgrounds that confer resistance or hypersensitivity to one or more drugs. We observe that genetic perturbations that alter susceptibility to a single drug can shift the drug interaction landscape and lead to the appearance of novel synergistic and antagonistic interactions. This work establishes a framework for investigating combinatorial therapies in model nematodes that can potentially be translated to amenable parasite species.

microbiology↗

bioRxiv2023

A high-throughput nematode sensory assay reveals an inhibitory effect of ivermectin on parasite gustation

Sensory pathways first elucidated in Caenorhabditis elegans are conserved across free-living and parasitic nematodes, even though each species responds to a diverse array of compounds. Most nematode sensory assays are performed by tallying observations of worm behavior on two-dimensional planes using agarose plates. These assays have been successful in the study of volatile sensation but are poorly suited for investigation of water-soluble gustation or parasitic nematodes without a free-living stage. In contrast, gustatory assays tend to be tedious, often limited to the manipulation of a single individual at a time. We have designed a nematode sensory assay using a microfluidics device that allows for the study of gustation in a 96-well, three-dimensional environment. This device is suited for free-living worms and parasitic worms that spend their lives in an aqueous environment, and we have used it to show that ivermectin inhibits the gustatory ability of vector-borne parasitic nematodes.

molecular biology↗

bioRxiv2022

Phenotypic profiling of macrocyclic lactones on parasitic Schistosoma flatworms

Macrocyclic lactones are front-line therapies for parasitic roundworm infections, but there are no comprehensive studies on the activity of this drug class against parasitic flatworms. Ivermectin is well known to be inactive against flatworms. However, the structure-activity relationship of macrocyclic lactones may vary across phyla, and it is entirely possible other members of this drug class do in fact show antiparasitic activity on flatworms. For example, there are several reports hinting at the anti-schistosomal activity of doramectin and moxidectin. To explore this class further, we developed an automated imaging assay combined with measurement of lactate levels from worm media. This assay was applied to the screening of 21 macrocyclic lactones (avermectins, milbemycins and others such as spinosyns) against adult schistosomes. These in vitro assays identified several macrocyclic lactones (emamectin, milbemycin oxime, and the moxidectin metabolite 23-ketonemadectin) that caused contractile paralysis and lack of lactate production. Several of these were also active against miracidia, a juvenile life cycle stage of the parasite. Hits prioritized from these in vitro assays were administered to mice harboring patent schistosome infections. However, no reduction in worm burden was observed. Nevertheless, these data show the utility of a multiplexed in vitro screening platform to quantitatively assess drug action and prioritize hits in a chemical series for in vivo studies. While the prototypical macrocyclic lactone ivermectin displays minimal activity against adult Schistosoma mansoni, this family of compounds does contain schistocidal compounds which may serve as a starting point for development of new anti-flatworm chemotherapies.

pharmacology and toxicology↗

bioRxiv2022

Pharmacological profiling of a Brugia malayi muscarinic acetylcholine receptor as a putative antiparasitic target

The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor (Bma-GAR-3) that is highly expressed across intra-mammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Lastly, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.

molecular biology↗

bioRxiv2022

Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution.

Nematode excretory-secretory (ES) products are essential for the establishment and maintenance of infections in mammals and are valued as therapeutic and diagnostic targets. While parasite effector proteins contribute to host immune evasion and anthelmintics have been shown to modulate secretory behaviors, little is known about the cellular origins of ES products or the tissue distributions of drug targets. We leveraged single-cell approaches in the human parasite Brugia malayi to generate an annotated cell expression atlas of microfilariae. We show that prominent antigens are transcriptionally derived from both secretory and non-secretory cell and tissue types, and anthelmintic targets display distinct expression patterns across neuronal, muscular, and other cell types. While the major classes of anthelmintics do not affect the viability of isolated cells at pharmacological concentrations, we observe cell-specific transcriptional shifts in response to ivermectin. Finally, we introduce a microfilariae cell culture model to enable future functional studies of parasitic nematode cells. We expect these methods to be readily adaptable to other parasitic nematode species and stages.

microbiology↗

bioRxiv2022

Multivariate chemogenomic screening prioritizes new macrofilaricidal leads

Development of direct acting macrofilaricides for the treatment of human filariases is hampered by limitations in screening throughput imposed by the parasite life cycle. Efforts to circumvent arduous screening of adult filariae include drug repurposing and high-throughput screens that target commensal bacteria. In vitro adult screens typically assess single phenotypes without prior enrichment for chemicals with antifilarial potential. We developed a multivariate screen that identified dozens of compounds with submicromolar macrofilaricidal activity, achieving a hit rate of >50% by leveraging abundantly accessible microfilariae. Adult assays were multiplexed to thoroughly characterize compound activity across relevant parasite fitness traits, including neuromuscular control, fecundity, metabolism, and viability. 17 compounds from a diverse chemogenomic library elicited strong effects on at least one adult trait, with differential potency against microfilariae and adults. Stage-specific drug effects may be crucial to limiting adverse events in endemic regions, and our screen identified five compounds with high potency against adults but low potency or slow-acting microfilaricidal effects, at least one of which acts through a novel mechanism. We show that the use of microfilariae in a primary screen outperforms model nematode developmental assays and virtual screening of protein structures inferred with deep-learning. These data provide new leads for drug development, and the high-content and multiplex assays set a new foundation for antifilarial discovery.

microbiology↗