Resolving the origins of secretory products and anthelmintic responses in a human parasitic nematode at single-cell resolution
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eLife assessment
In this important study, the authors generate and analyse single-cell datasets for the human parasitic nematode Brugia malayi. The new resource has the potential to uncover new details of the biology of secretory systems in this filarial nematode but the main claims are only partially supported and strengthening them would require additional experimental support and new analyses. With the methodological part strengthened, the new resource would be of broad interest to parasitologists and nematode biologists and would have the potential to accelerate research in the search of new anthelmintics and vaccines.
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Abstract
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.
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Author Response
Reviewer #1 (Public Review):
The authors generated a detailed single-cell RNAseq dataset for the microfilariae stage of the human nematode parasite Brugia malayi. This is an impressive and important achievement, given that it is difficult to obtain sufficient material from human parasites and the microfilariae are protected by a chitin sheath. The authors collected microfilariae from jirds and carefully worked out a protocol of digestion, dissociation and filtering, to obtain single-cell material for sequencing.
The single-cell resource was complemented with a dataset derived from FACS-sorted large secretory cells, allowing the identification of several specific proteins expressed in this unique microfilarial cell-type important for immune evasion.
The authors also generated new data for secretory cells of …
Author Response
Reviewer #1 (Public Review):
The authors generated a detailed single-cell RNAseq dataset for the microfilariae stage of the human nematode parasite Brugia malayi. This is an impressive and important achievement, given that it is difficult to obtain sufficient material from human parasites and the microfilariae are protected by a chitin sheath. The authors collected microfilariae from jirds and carefully worked out a protocol of digestion, dissociation and filtering, to obtain single-cell material for sequencing.
The single-cell resource was complemented with a dataset derived from FACS-sorted large secretory cells, allowing the identification of several specific proteins expressed in this unique microfilarial cell-type important for immune evasion.
The authors also generated new data for secretory cells of Caenorhabditis elegans and concluded that there is limited similarity between the composition of Brugia and C. elegans secretory cell types.
In a further set of experiments, the authors analysed gene expression changes in dissociated Brugia cells to the commonly used anthelminthic drug ivermectin. This revealed specific gene expression changes across various cell types, providing new insights into how the drug effects the parasite.
Finally, the authors developed a method to keep dissociated Brugia cells alive in culture for two days. This method will aid cellular studies of this parasite.
The authors may want to explore the new resource in more detail to reach more specific biological conclusions. For example, the authors mention that the large secretory cells are critical to parasite survival and immune evasion. With a more complete list of genes expressed in these cells the authors could try to reach more specific conclusions or predictions. Are there newly identified secreted factors that could contribute to immune evasion? It would be important to read in more detail about such proteins (including an analysis of the sequences and phylogenies), especially if the authors could identify new candidates as potential vaccine or diagnostic targets. Likewise, can the data be used to understand in more detail the mechanism of immune evasion or ivermectin action?
Thank you for this comment. We have since added a source data file with the list of secretory cell DEGs along with gene ontology (GO) analysis. We have added a main figure to the revised manuscript that takes a deeper look at transcripts enriched in the secretory cell compared to other annotated cell types. Lastly, we included a deeper look at the paralogous expansion of C2H2 transcription factors that localize near exclusively to the secretory cell. This family of transcription factors is diverse and the significant presence in the secretory cell may play a role in adapting to varying host environmental conditions or in the expression of proteins contributing to immune evasion. Our single-cell data show specific transcriptional shifts in cells expressing putative IVM targets and recapitulate changes identified in whole-parasite drug exposure experiments and highlight the importance of cell connectivity to the in vitro phenotype. These supplemental analyses of the secretory cell will seed future lines of investigation about secretion and aid in further dissecting anthelmintic mode of action.
The authors searched for known secreted proteins, including antigens, vaccine targets, and diagnostic markers and mapped the expression of these to the single-cell atlas. It is not clear from the paper how comprehensive previous studies to identify secretory proteins were. With the new resource in hand, the authors could look at all secreted proteins (with a signal peptide) expressed in the ES and other cells. The paper would benefit from a more comprehensive overview of the classes of secretory proteins and their expression.
Thank you for this suggestion. We have completed a computational prediction of signal peptides in differentially expressed secretory cell transcripts (Figure 4) and show that although there is an enrichment of signal peptide-containing sequences enriched in the secretory cell compared to other cell types, less than half of the proteins identified contained signal peptide sequences.
This was unsurprising as most secreted proteins identified in the literature (diagnostic and vaccine targets) do not have a signal peptides. The routes of exit for these prominent circulating targets remain murky. We also carried out transmembrane prediction on protein-coding genes that are differentially expressed in the secretory cell (and other cell types) and note that some of these are established components of exosome-like vesicles, emerging as important players in host modulation. This additional analysis has been added to a new figure (Figure 4) and the accompanying results section.
The authors show that an abundance of C2H2 transcription factors is localizing almost exclusively to the secretory cells. It would be useful to see a classification of these proteins and phylogenetic analysis relating them to C2H2 from C. elegans and other animals.
The C. elegans genome contains 106 annotated C2H2 zinc finger transcription factors. Based on a reverse phylogenetic approach, we identified a total of 241 orthologous C2H2 zinc finger transcription factors in B. malayi, many of which exhibit strong and/or exclusive expression in the secretory cell. This analysis has been added to an additional figure (Figure 4) describing the secretory cell in more depth alongside signal peptide and transmembrane domain analysis of differentially expressed genes in the secretory cell compared to other identified major cell types.
In general, a more detailed bioinformatic analysis of secretory products and more discussions of potential functions (e.g. serpins etc.) would make the paper more interesting and could stimulate more mechanistic thinking.
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eLife assessment
In this important study, the authors generate and analyse single-cell datasets for the human parasitic nematode Brugia malayi. The new resource has the potential to uncover new details of the biology of secretory systems in this filarial nematode but the main claims are only partially supported and strengthening them would require additional experimental support and new analyses. With the methodological part strengthened, the new resource would be of broad interest to parasitologists and nematode biologists and would have the potential to accelerate research in the search of new anthelmintics and vaccines.
-
Reviewer #1 (Public Review):
The authors generated a detailed single-cell RNAseq dataset for the microfilariae stage of the human nematode parasite Brugia malayi. This is an impressive and important achievement, given that it is difficult to obtain sufficient material from human parasites and the microfilariae are protected by a chitin sheath. The authors collected microfilariae from jirds and carefully worked out a protocol of digestion, dissociation and filtering, to obtain single-cell material for sequencing.
The single-cell resource was complemented with a dataset derived from FACS-sorted large secretory cells, allowing the identification of several specific proteins expressed in this unique microfilarial cell-type important for immune evasion.
The authors also generated new data for secretory cells of Caenorhabditis elegans and …
Reviewer #1 (Public Review):
The authors generated a detailed single-cell RNAseq dataset for the microfilariae stage of the human nematode parasite Brugia malayi. This is an impressive and important achievement, given that it is difficult to obtain sufficient material from human parasites and the microfilariae are protected by a chitin sheath. The authors collected microfilariae from jirds and carefully worked out a protocol of digestion, dissociation and filtering, to obtain single-cell material for sequencing.
The single-cell resource was complemented with a dataset derived from FACS-sorted large secretory cells, allowing the identification of several specific proteins expressed in this unique microfilarial cell-type important for immune evasion.
The authors also generated new data for secretory cells of Caenorhabditis elegans and concluded that there is limited similarity between the composition of Brugia and C. elegans secretory cell types.
In a further set of experiments, the authors analysed gene expression changes in dissociated Brugia cells to the commonly used anthelminthic drug ivermectin. This revealed specific gene expression changes across various cell types, providing new insights into how the drug effects the parasite.
Finally, the authors developed a method to keep dissociated Brugia cells alive in culture for two days. This method will aid cellular studies of this parasite.
The authors may want to explore the new resource in more detail to reach more specific biological conclusions. For example, the authors mention that the large secretory cells are critical to parasite survival and immune evasion. With a more complete list of genes expressed in these cells the authors could try to reach more specific conclusions or predictions. Are there newly identified secreted factors that could contribute to immune evasion? It would be important to read in more detail about such proteins (including an analysis of the sequences and phylogenies), especially if the authors could identify new candidates as potential vaccine or diagnostic targets. Likewise, can the data be used to understand in more detail the mechanism of immune evasion or ivermectin action?
The authors searched for known secreted proteins, including antigens, vaccine targets, and diagnostic markers and mapped the expression of these to the single-cell atlas. It is not clear from the paper how comprehensive previous studies to identify secretory proteins were. With the new resource in hand, the authors could look at all secreted proteins (with a signal peptide) expressed in the ES and other cells. The paper would benefit from a more comprehensive overview of the classes of secretory proteins and their expression.
The authors show that an abundance of C2H2 transcription factors is localizing almost exclusively to the secretory cells. It would be useful to see a classification of these proteins and phylogenetic analysis relating them to C2H2 from C. elegans and other animals.
In general, a more detailed bioinformatic analysis of secretory products and more discussions of potential functions (e.g. serpins etc.) would make the paper more interesting and could stimulate more mechanistic thinking.
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Reviewer #2 (Public Review):
The overall objective of this paper is to characterize the cells that are responsible for producing the secretions of the parasitic larvae, Brugia malayi. This parasite is a human pathogen that is one of three responsible for lymphatic filariasis/elephantiasis a disease that threatens half of the world's population. The specific focus of this work is protein secretions made by the parasites. In general, it is well-known that parasitic worms can manipulate and evade host immune immunity via secreted products. Studies have focused on the activities of these secretions and specific molecules. What is lacking is a detailed description of the identity and anatomical location of the cells that produce them. This is especially important as these cells are the target of different classes of anthelminthic drugs. This …
Reviewer #2 (Public Review):
The overall objective of this paper is to characterize the cells that are responsible for producing the secretions of the parasitic larvae, Brugia malayi. This parasite is a human pathogen that is one of three responsible for lymphatic filariasis/elephantiasis a disease that threatens half of the world's population. The specific focus of this work is protein secretions made by the parasites. In general, it is well-known that parasitic worms can manipulate and evade host immune immunity via secreted products. Studies have focused on the activities of these secretions and specific molecules. What is lacking is a detailed description of the identity and anatomical location of the cells that produce them. This is especially important as these cells are the target of different classes of anthelminthic drugs. This knowledge could allow new strategies to target these pathogens and to better understand the mechanism of actions.
To better understand this important topic, this manuscript describes a method to dissociate cells of the pre-larval stage (microfilariae) of the human parasitic filarial nematode, Brugia malayi. This method is then used to create an atlas of cells based on the expression profiles of individual dissociated cells. Cells are grouped into clusters with similar patterns of expression using single-cell mRNA sequencing analysis pipelines and the clusters are defined by using a combination of known functionalities based on the well-established, free living, soil nematode, C. elegans, and different functional classifications based on genes of interest. These include known antigens as well as targets of 3 classes of anthelmintic molecules. Using the scRNA-seq data, clear hypotheses can be made about ion channel and structural protein composition, the putative targets of the anthelminthics. Finally, it is proposed that the dissociated cells can be cultured which can facilitate future studies since cell lines or primary cultures of cells from filarial worms are not available.
This paper represents a huge undertaking on an important and understudied area. The authors have taken on a major challenge to gain novel insights, and to provide data and protocols for the field to use. The data are well-presented and support the conclusions of the work. The authors have broadly achieved their goals and the data generated and methodology will be important for the community.
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Reviewer #3 (Public Review):
Henthorn and coworkers obtain a single cell atlas of the parasite nematode Brugia malayi to search for excretory secretory products. These are involved in therapeutic responses but it is unknown what are the cell types that express them. In fact this seems as an ideal question to be addressed with single cell transcriptomics. The authors analyse their dataset, coming to the conclusion that many of these ES products are expressed broadly throughout the parasite, including secretory and non secretory types. This would be a nice conclusion if supported by the data. Then they go on to compare responses to exposure of ivermectin at the single cell level.
I must praise the attempt of using single cell transcriptomics to examine this question. These relatively novel methods have been so far used to collect …
Reviewer #3 (Public Review):
Henthorn and coworkers obtain a single cell atlas of the parasite nematode Brugia malayi to search for excretory secretory products. These are involved in therapeutic responses but it is unknown what are the cell types that express them. In fact this seems as an ideal question to be addressed with single cell transcriptomics. The authors analyse their dataset, coming to the conclusion that many of these ES products are expressed broadly throughout the parasite, including secretory and non secretory types. This would be a nice conclusion if supported by the data. Then they go on to compare responses to exposure of ivermectin at the single cell level.
I must praise the attempt of using single cell transcriptomics to examine this question. These relatively novel methods have been so far used to collect information of cell types, but have immense potential for the investigation of important questions in neglected diseases like this. Fundamental knowledge about the biology of Brugia malayi and the tissue and cell types present are key to understanding their pathogenesis and advancing new therapeutic options. The authors start this research project with the right model and right technique.
My major concern is the quality of their single cell data. The authors perform no FACS or other methods to clear their suspension from cellular debris. This arises from all cell types, and then gets encapsulated with single cells in the droplet-based single cell transcriptomic process. Then, all cell barcodes receive genes from a single cell but also from a collection of cellular debris particles (arising from all other cell types). This, and only this, can in principle explain one of the major findings in the abstract: secreted antigens are expressed broadly in many cell types. This same caveat might explain their finding of pan neuronal markers broadly expressed - conceptually very similar to what the authors hold for secreted antigens, but that the authors only mention briefly and do not explain.
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