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Spatial transcriptomics reveals antiparasitic targets associated with essential behaviors in the human parasite Brugia malayi

['Paul M. Airs', 'Department Of Pathobiological Sciences', 'University Of Wisconsin-Madison', 'Madison', 'Wisconsin', 'United States Of America', 'Kathy Vaccaro', 'Kendra J. Gallo', 'Nathalie Dinguirard', 'Zachary W. Heimark']

Date: 2022-05

Lymphatic filariasis (LF) is a chronic debilitating neglected tropical disease (NTD) caused by mosquito-transmitted nematodes that afflicts over 60 million people. Control of LF relies on routine mass drug administration with antiparasitics that clear circulating larval parasites but are ineffective against adults. The development of effective adulticides is hampered by a poor understanding of the processes and tissues driving parasite survival in the host. The adult filariae head region contains essential tissues that control parasite feeding, sensory, secretory, and reproductive behaviors, which express promising molecular substrates for the development of antifilarial drugs, vaccines, and diagnostics. We have adapted spatial transcriptomic approaches to map gene expression patterns across these prioritized but historically intractable head tissues. Spatial and tissue-resolved data reveal distinct biases in the origins of known drug targets and secreted antigens. These data were used to identify potential new drug and vaccine targets, including putative hidden antigens expressed in the alimentary canal, and to spatially associate receptor subunits belonging to druggable families. Spatial transcriptomic approaches provide a powerful resource to aid gene function inference and seed antiparasitic discovery pipelines across helminths of relevance to human and animal health.

Lymphatic filariasis (LF) is mosquito-borne parasitic disease that infects tens of millions of people, causing significant morbidity and disability. Disease elimination is complicated by a lack of vaccines and suboptimal drugs that are ineffective against adult stage filarial nematode parasites. Many essential adult parasite behaviors are controlled by tissues that are located within the anterior-most tip of the nematode body plan. This head region includes structures that control parasite feeding, sensory, secretory, and reproductive behaviors. We paired spatial transcriptomics and microscopy approaches to map gene expression patterns across these prioritized parasite tissues. These data were used to map biases in the origins of known drug targets and antigens, identify potential new drug and vaccine targets, including putative hidden antigens expressed in the intestinal tract, and to spatially associate receptor subunits belonging to druggable protein families. Ultimately these approaches provide a powerful means to identify and map potential new antiparasitic targets in medically significant but understudied parasites.

Funding: This work was supported by National Institutes of Health NIAID grant R01 AI151171 to M.Z (NIH.gov). K.J.G. was supported by a UW SciMed GRS Fellowship (scimedgrs.wisc.edu) and NIH Parasitology and Vector Biology Training grant T32 AI007414 (NIH.gov). N.J.W. was supported by NIH Ruth Kirschstein NRSA fellowship F32 AI152347 (NIH.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Airs et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Bulk transcriptomics in filarial parasites has thus far been used to explore changes in gene expression associated with development [ 20 – 23 ] and environmental or drug perturbations [ 24 – 27 ]. While proteomics has shed light on large and accessible tissues in B. malayi [ 28 ], small head-associated structures are massively underrepresented in whole-parasite omics and have yet to be characterized. Here, we adapt spatial transcriptomic and microscopy approaches to profile the head region of B. malayi and resolve gene expression patterns in critical tissues at the host-parasite interface. RNA tomography [ 29 , 30 ] and tissue-specific transcriptomes are leveraged to map the distributions of current drug targets and known antigens, as well as to prioritize putative antiparasitic and vaccine targets. The first application of these complementary methods in a human parasitic nematode provides a template for the localization of gene transcripts and targets of therapeutic and diagnostic value in similarly intractable parasitic nematodes of human and veterinary significance.

In adult filarial parasites, vital tissues and interfaces for host-parasite communication are concentrated within the anteriormost region of the body plan. The first millimeter of the B. malayi female head region (~3% of the length) contains cells and tissues that control parasite feeding, sensory, secretory, and reproductive behaviors [ 19 ]. Transcriptomic profiling of this region can aid the prioritization of new antifilarial targets, localize the targets of existing drugs, and provide clues to the origins of immunomodulatory molecules released into the host environment. This effort is currently impeded by a lack of scalable transgenesis and in situ localization techniques in this two-host parasite system.

Control of LF relies on routine mass drug administration with anthelmintics, which effectively clear microfilariae but are ineffective against adult stages and are contraindicated in areas co-endemic for other filarial parasites [ 1 , 2 , 7 ]. Anthelmintic resistance is widespread in veterinary medicine and also represents a threat to filariasis control efforts in both animals and humans [ 8 – 12 ]. To address these challenges and accelerate LF elimination there is a need to generate new antifilarial therapies, particularly drugs effective against adult stage parasites. Current anthelmintics target or dysregulate parasite cell integrity, neuromuscular control, reproductive potential, and the secretion of parasite molecules necessary for the establishment and maintenance of parasitism [ 13 – 18 ]. The development of macrofilaricidal (adult-killing) drugs can be hastened by an improved knowledge of tissues that underpin survival in adult parasites.

Lymphatic filariasis (LF) is a chronic and debilitating neglected tropical disease (NTD) recognized as a leading global cause of long-term disability. Over 60 million people are currently infected with LF and ~900 million people are at risk of infection across 72 endemic countries [ 1 – 3 ]. LF is caused by the filarial nematodes Brugia malayi, Brugia timori, and Wuchereria bancrofti, which reside as adults in the lymphatics producing microfilariae that migrate to the blood and undergo cyclodevelopmental transmission in competent blood-feeding mosquito vectors [ 4 ]. Adult stage parasites cause blockage and inflammation of lymphatic vessels that can result in disfiguring and stigmatizing manifestations, including lymphedema (most notably elephantiasis) and hydrocele that afflicts an estimated 36 million individuals [ 1 , 2 , 5 , 6 ].

Results

The adult filarial head region expresses prominent antigens and known drug targets Adult stage filariae cause incurable chronic illnesses. To develop new therapies that aid parasite elimination, we must learn more about tissues and structures underlying adult behaviors. Adult female B. malayi are ~34.6 mm (31.8–39.8 mm) in length when reared in Mongolian jirds, but the vast majority of their body plan is composed of mid-body structures including the body wall muscle, the reproductive tract, and intestine [19]. The anterior-most 3% (~1 mm) of the parasite head region contains vital structures including the buccal cavity, amphid neurons, nerve ring, vulva, pharynx, esophageal-intestinal junction, and the excretory-secretory (ES) apparatus. These tissues control essential parasite behaviors and include host-parasite interfaces where drug and antigen interactions likely occur. To identify head-enriched gene transcripts, individual adult female B. malayi head regions were dissected from the body at the vulva (~0.6 mm from anterior) using ultra-fine probes (Fig 1A). The vulva was chosen as a visible marker to ensure head tissues were captured and isolated from the reproductive tract, which would be contaminated with microfilaria. Low-input RNA-seq was carried out using paired head and body tissues isolated from three individual parasites. Biological replicates displayed high concordance (Fig 1B), with 70–80% reads from head and body region samples uniquely mapping to the B. malayi genome. Analysis of differentially-expressed genes (DEGs) identified 2,406 head-enriched genes (log 2 (FC) > 1 and p-value < 0.01) with at least 30 total raw reads from the six samples (S1 Table). Transcripts associated with secreted proteins [31,32] are distributed evenly across both head and body region tissues, suggesting mixed origins for what are classically referred to as “ES products” (Fig 1C). In striking contrast, the majority (86%) of prominent filarial antigens with known immunomodulatory capacity, including those that have been pursued as vaccine candidates [33], are head-enriched (Fig 1C). Immunization with recombinant proteins encoded by many genes on this list, including Bm97 and Bma-far-1, confers significant protection against filarial nematode infection in animal challenge studies [34,35]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. The B. malayi head transcriptome reveals enrichment of anthelmintic targets, prominent antigens, and vaccine candidates. (A) Illustration of head region tissues and point of dissection adjacent to the vulva (black probes). (B) Principal component analysis (PCA) plot of head and corresponding body transcriptome replicates. Each point represents tissue from a different parasite. (C) Volcano plot highlighting head enrichment of prominent antigens and immunomodulatory genes (25 of 29 genes; purple) [16,33,94–101], compared to a more diffuse pattern of expression for proteins identified in secretome studies (141 of 244 genes; teal) [31,32]. (D) Heatmap of gene expression patterns shows head-enrichment of candidate vaccine targets, as well as both head- and body-enrichment for the targets of antifilarial drugs (benzimidazoles: β-tubulins, macrocyclic lactones: GluCls, and emodepside: BK channel) (* p-value < 0.5, ** p-value < 0.01, *** p-value < 0.001). https://doi.org/10.1371/journal.ppat.1010399.g001 Antifilarial targets from existing classes of drugs show different distributions (Fig 1D). The putative glutamate-gated chloride channel (GluCl) targets of ivermectin, Bma-avr-14 and Bma-glc-2 [36], show higher relative expression in body tissues consistent with Bma-avr-14 localization to the reproductive tract and developing embryos [37]. Bma-glc-3 and Bma-glc-4 channel subunits are more enriched in the head and may also play a role in macrocyclic lactone responses. Bma-slo-1, a putative target of emodepside, an emerging candidate adulticide for treatment of river blindness [38], is more highly expressed in the body. Conversely, the likely β-tubulin target of albendazole (Bma-btub-1), based on homology to Caenorhabditis elegans ben-1, is head-enriched.

Microscopic investigation of the adult head region and putative excretory-secretory apparatus Organizational knowledge of prominent head structures can scaffold spatial transcriptomic data. The distances of the buccal cavity (~5 μm), vulva (~657–667μm) and esophageal-intestinal junction (~861–1010 μm) from the anterior have been measured in adult female B. malayi [19], but locating the excretory-secretory (ES) system in adult stage B. malayi and other filaria has been notoriously difficult [39–43]. In microfilariae, the ES apparatus is a hallmark and essential structure consisting of a pore and vesicle leading to a single excretory cell via a cytoplasmic bridge [44]. Ivermectin is thought to disrupt microfilarial ES protein and exosome release through binding to ion channels in the vicinity of the ES vesicle [14,16,17]; however, these structures become inconspicuous through development [39,40,42,45,46]. To help pinpoint the ES in adult female B. malayi, the relative organization of head structures across Clade III [47,48] nematodes was collected from available literature (Fig 2A). Among Clade III parasites, the ES pore and/or cell are located posterior to the nerve ring in 23/24 species, anterior to reproductive openings (24/24 species), and anterior to the esophageal-intestinal junction (23/24 species) in at least one life stage surveyed. The conservation of structural organization across developmental and evolutionary time indicates the presence of ES structures between the nerve ring and vulva in adult female B. malayi. PPT PowerPoint slide

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TIFF original image Download: Fig 2. Coordinating the elusive excretory-secretory system in the adult B. malayi head region. (A) Comparative anatomy of Clade III nematode head structures from published descriptions (detailed in S1 File). Positions of the nerve ring = NR, excretory-secretory pore and/or vesicle = EP, excretory cell = EC, genital primordium of larvae = G (larvae), vulva = V, and esophageal-intestinal junction = I shown as rank order for larvae and average micron distances from anterior in adult stages (A = anterior). (B) Light sheet maximum intensity projection of ES pore pulsing activity (arrowhead) in DRAQ5 stained live adult male B. malayi. Scale bars = 100 μm and 10 μm for insets. (C) Single section from adult female SBF-SEM showing multinucleated (arrowheads) epidermis within the lateral cord and membranous structures (pseudocolor purple) that are embedded within and surround the lateral cord (structures highlighted for one of two lateral cords). Scale bar = 10 μm. (D) TEM of lateral cord in adult male showing likely seam cell homolog (pseudocolor orange), membranous processes enriched in microtubules (pseudocolor purple), membranous processes lacking obvious microtubules (pseudocolor blue), and Wolbachia endosymbionts (asterisks). (d’) Closeup of seam cell, identifiable by the position on the median ridge of the lateral cord and the presence of adherens junctions (arrows) connecting to surrounding epidermis. Scale bar = 2 μm. (d”) Closeup of membranous process enriched in microtubules and surrounded by epidermis. Scale bar = 400 nm. https://doi.org/10.1371/journal.ppat.1010399.g002 To identify the ES pore in adults we optimized live 4D light sheet microscopy as well as multiple electron microscopy methods. Critical point drying scanning electron microscopy (SEM) of adult Brugia and the closely-related but much larger filarial parasite Dirofilaria immitis allowed clear visualization of the vulva, but not the ES pore (S1 Fig). This may be due to the small size and angle of the pore opening [49]. Light sheet imaging was adapted for live adult males partially paralyzed with 1 mM levamisole to restrict gross muscle movement, and adults were monitored for up to 1 hr at 10 s intervals. Males were chosen to avoid confusion with the confounding activity of the vulva, which is proximal in females. Nuclei stained head regions revealed instances of pulsing during which stain condensed into a large cell with a pore-like tubular structure that was then cleared from the worm ~430 μm from the anterior (Fig 2B and S1 Video). This location is consistent with the ES pore location (397–537 μm) in the fur seal parasite Acanthocheilonema odendhali [50], the only filarial nematode where the adult stage ES pore has been morphometrically characterized. To our knowledge, these pulses represent the first evidence of dynamic ES pore opening events in a mammalian parasitic nematode. To obtain a finer description of head structures and potential ES channels, we utilized high-pressure freeze fixation with serial block face-SEM (SBF-SEM) to obtain approximately ~1000 serial sections (~70 nm/section) from the anterior of an adult female (Fig 2C, see also data deposited on https://doi.org/10.6084/m9.figshare.16441689.v1). The ventral nerve cord and pharynx were present throughout and we observed 30 pharyngeal, 21 body wall muscle (~5 per muscle quadrant), 5 ventral nerve cord, and 83 lateral cord (~40 per cord) nuclei. Similar to C. elegans, the lateral cords appear to be partially composed of epidermal syncytia with multiple closely apposed nuclei (Fig 2C) that were evident along the anterior-posterior axis in SBF-SEM sections 1–37, 52–92, 189–250, 431–449, 516–543, 660–683, 737–800. We did not observe any nuclei within the dorsal cord itself. The C. elegans excretory canal [51] is visible in EM sections immediately ventral of the lateral cords, while in Onchocerca volvulus a glomerulus-like excretory-structure [52] is suggested to be embedded within the lateral cords. Neither canal-type was observed in our SBF-SEM data. Their absence is possibly due to individual variation in the position of ES structures or the excretory system may be greatly reduced in size, as proposed previously [40,52]. Within lateral cords we observed membrane bound processes along the lateral and basal edges. These processes were described previously as axons or infolded membranes [43,53,54]. In some regions processes are also embedded within the lateral cord, while others appear to bisect the lateral cord. Similar processes embedded in the lateral cord are not seen in C. elegans [55]. To better define the lateral cord in this region we turned to transmission electron microscopy (TEM) (Fig 2D). As previously suggested, membrane processes embedded within the lateral cord appear to be neuronal, as evidenced by numerous microtubules. However the processes located on the basal boundary of the lateral cord lacked microtubules. The absence of consistent microtubules argues against a solely commissure identity. Another possibility is that these structures towards the interior of the worm comprise a modified excretory system. Their position adjacent to the pseudocoelom would be consistent with an excretory system; however, additional serial data are needed to identify the nature of these structures. TEM also demonstrated the presence of likely homologs to the C. elegans seam cells along the median ridge surrounded by putative epidermal syncytia (Fig 2D). These are readily identifiable by their position and the presence of adherens junctions connecting the seam to the syncytial epidermis. In C. elegans and other nematodes, the seam cells have stem cell-like properties and act to contribute nuclei to the growing epidermal syncytia [56,57]. As previously shown, the lateral cords were also enriched in Wolbachia endosymbionts [54].

[END]

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