Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation

Arthur G Hunt; Daniel K Howe; Ashley Brown; Michelle Yeargan

doi:10.1371/journal.pone.0259109

. 2021 Oct 28;16(10):e0259109. doi: 10.1371/journal.pone.0259109

Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation

Arthur G Hunt ^1,^*, Daniel K Howe ², Ashley Brown ², Michelle Yeargan ²

Editor: Stuart Alexander Ralph³

PMCID: PMC8553156 PMID: 34710156

Abstract

In recent years, a class of chemical compounds (benzoxaboroles) that are active against a range of parasites has been shown to target mRNA polyadenylation by inhibiting the activity of CPSF73, the endonucleolytic core of the eukaryotic polyadenylation complex. One particular compound, termed AN3661, is active against several apicomplexan parasites that cause disease in humans. In this study, we report that AN3661 is active against an apicomplexan that causes disease in horses and marine mammals (Sarcocystis neurona), with an approximate IC₅₀ value of 14.99 nM. Consistent with the reported mode of action of AN3661 against other apicomplexans, S. neurona mutants resistant to AN3661 had an alteration in CPSF73 that was identical to a mutation previously documented in AN3661-resistant Toxoplasma gondii and Plasmodium falciparum. AN3661 had a wide-ranging effect on poly(A) site choice in S. neurona, with more than half of all expressed genes showing some alteration in mRNA 3’ ends. This was accompanied by changes in the relative expression of more than 25% of S. neurona genes and an overall 5-fold reduction of S. neurona transcripts in infected cells. In contrast, AN3661 had no discernible effect on poly(A) site choice or gene expression in the host cells. These transcriptomic studies indicate that AN3661 is exceedingly specific for the parasite CPSF73 protein, and has the potential to augment other therapies for the control of apicomplexan parasites in domestic animals.

Introduction

The phylum Apicomplexa encompasses a very broad group of obligate intracellular parasites that are a significant cause of disease worldwide [1]. Several of these are important human pathogens, such as Plasmodium spp. and Toxoplasma gondii. As well, multiple members of this phylum cause disease in domestic animals and have a significant economic impact on agriculture. Both Theileria spp. and Babesia spp. are tick-borne haemoprotozoan pathogens that infect and cause disease in cattle and horses. Species of Eimeria are enteric parasites that cause diarrheal diseases in a variety of animals and are especially detrimental to the poultry industry. Neospora caninum was initially described as a neurologic pathogen of canids, but has subsequently been identified as a major cause of reproductive failure in cattle. Sarcocystis neurona is an important cause of the neurologic disease equine protozoal myeloencephalitis (EPM) and an emerging pathogen of marine mammals. While the economic impact of these pathogens is difficult to assess, the annual global cost of neosporosis alone has been estimated at greater than $1 billion dollars [2].

Although vaccination is an option for reducing coccidiosis caused by Eimeria spp. in poultry, vaccines against other coccidian parasites have generally proven ineffective. Consequently, chemotherapy has been the standard approach to try to control infection and disease caused by these parasites (reviewed in [3–5]). Drug combinations that target folate metabolism, such as pyrimethamine and a sulfonamide, have been used for decades to treat infections by multiple apicomplexan parasites, including the coccidia. However, the routine use of anti-folate drugs to treat coccidial infections has been hindered by several factors, including the duration of treatment (6 months for horses with EPM) and associated toxicities (anemia, leukopenia, teratogenicity). The benzeneacetonitrile compounds diclazuril, ponazuril, and toltrazuril are effective treatments for agricultural animals infected with coccidian parasites. However, concerns about drug residues in food products and the ease in developing resistance have limited the use of the benzeneacetonitrile drugs in agricultural animals [3–5]. Anticoccidial medications are used extensively against Eimeria and are quite commonly incorporated into poultry feeds. A variety of drugs have been employed for this purpose, but ionophore antibiotics, such as monensin, are currently the most widely used anticoccidials to control coccidiosis (reviewed in [6]). While resistance to the ionophore drugs has been slower to occur, reports now indicate that monensin-resistant isolates of Eimeria are widespread [6]. Consequently, the continued use of existing anticoccidial drugs to control these parasites might not be a viable long-term option. Taken together, these considerations reveal an ongoing need for affective alternative therapies for coccidian diseases in agricultural animals.

Recently, it was reported that a member of a class of benzoxaboroles, termed AN3661, inhibits growth of Plasmodium falciparum [7], T. gondii [8], and Cryptosporidium parvum [9]. The demonstrated mode of action of AN3661 is novel, in that it targets the 73 kDa subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF73, of the apicomplexan polyadenylation complex. In this report, we show that AN3661 similarly inhibited the coccidian parasite S. neurona. We also show that AN3661 had a decided effect on transcriptional dynamics of S. neurona, while drug treatment of the mammalian host cells had little impact on gene expression and mRNA polyadenylation in these cells. Together, these results expand the list of apicomplexans species that are susceptible to AN3661, and they reveal that the drug is an exceedingly selective inhibitor, with little discernible impact on the gene expression dynamics of the mammalian host.

Materials and methods

Parasite cultures, DNA isolation, and parasite growth assays

S. neurona strains (wild-type strain SN3.E1, YFP-expressing clone F9F, and AN3661-resistant clones were propagated by serial passage in monolayers of BT cells, as described [10]. Upon lysis of the infected monolayers, extracellular merozoites were harvested by passing through 23 and 25-gauge needles and filter-purified to remove host cell debris. Merozoites were pelleted and stored at -80°C until used for DNA or RNA isolation.

For parasite growth assays, YFP-expressing S. neurona was used according to procedures described previously [11]. Freshly-released merozoites purified from host cell debris were resuspended in culture medium without phenol red, and 96-well plates containing BT monolayers were inoculated with 4 x 10⁴ parasites per well, eight replicates (wells) per treatment. Control wells containing no parasites or no drug were included to account for background fluorescence and relative growth, respectively. Non-invaded merozoites were washed out after 2 hrs, and medium containing the appropriate dilution of AN3661 was added back to the wells and left for the duration of the growth assay. The plates were incubated at 37°C for 4 days, and fluorescence was measured using a Synergy H1 plate reader (BioTek, Winooski, VT, USA). The relative fluorescence units (RFUs) in the no-parasite wells was subtracted from the no-drug control and treatment wells, and growth of S. neurona in the presence of AN3661 was determined by comparing the RFUs in treated wells with those from non-treated control wells. The half-maximal inhibitory concentration (IC₅₀) was determined by regression analysis (GraphPad Prism v. 9).

Isolation and sequence analysis of AN3661-resistant clones of S. neurona

Sarcocystis neurona strain SN3.E1 was mutagenized with 2 mM N-ethyl-N-nitrosourea (ENU), as described previously for T. gondii [12]. The mutagenized culture was allowed to recover and expand for 3 days before addition of 90 nM AN3661 to select for mutant parasites that had become resistant to the drug. The S. neurona culture was maintained in medium containing AN3661 until the parasites disrupted the host cell monolayer (approximately 5 weeks), and single-cell clones of AN3661-resistant parasites were isolated in 96-well plates and expanded in the presence of drug for further analyses, as described [13].

The full-length coding region of SnCPSF73 was amplified in sections and sequenced using the primers listed in S1 Table. Sequences obtained from the AN3661-resistant clones were aligned with the SnCPSF73 gene from the SN3.E1 reference genome (SN3_01500330) to identify nucleotide polymorphisms. The SnCPSF73 amino acid sequence was further aligned to the human (NP_057291.1), Arabidopsis (At1g61010.1), T. gondii (TGME49_285200-t26_1), and P. falciparum (PF3D7_1438500.1) CPSF73 amino acid sequences so as to display commonalities in mutations that arise in AN3661-resistant S. neurona clones.

Poly(A) site profiling

S. neurona strain SN3.E1 was inoculated onto BT host cell monolayers grown in 6-well plates. After 48 hrs of parasite development, triplicate wells (each well representing a biological replicate) were incubated in fresh media with or without 90 nM AN3661 for an additional 24 hrs. The infected BT monolayers were then harvested from the wells using cell scrapers and centrifuged at 1100 x g for 10 min at 4°C. The cell pellets were washed 1x with ice-cold PBS and then stored at -80°C until used for RNA isolation. To conduct poly(A) site profiling in the non-infected host cells, confluent monolayers of BT cells grown in 6-well plates, triplicate wells were similarly treated with or without AN3661 for 24 hrs, and the cells harvested for RNA isolation.

Total RNA was isolated by adding 1mL Trizol to cell pellets, incubating for 5 minutes at room temperature, then adding 200 μL chloroform and incubating for an additional 3 minutes at room temperature. Samples were centrifuged at 13,200 x g for 15 minutes, and the aqueous layer was transferred to a fresh tube. 500 μL of isopropanol was added to the sample and incubated overnight at -20°C. The tubes were centrifuged at 13,200 x g for 10 minutes, and the RNA pellet was washed with 1 mL 75% EtOH. The pellet was air dried for 5–10 minutes and resuspended in RNase free water.

Libraries for genome-wide poly(A) site profiling was performed following the procedures described in Ma et al. [14] and Pati et al. [15]. So-called poly(A) tag (PAT-Seq) libraries were sequenced on an Illumina HiSeq instrument; the sequencing data are available under Bioproject PRJNA713353. Sequencing reads were trimmed, demultiplexed, and mapped to the respective genomes using CLC Genomics Workbench (latest version used was 20.0.4); the results of these analyses are summarized in S1 File. Subsequent analyses were performed following the pipelines described in Bell et al. [16] and de Lorenzo et al. [17]. This pipeline is described in detail in S2 File.

Gene expression was estimated by mapping individual PAT-Seq reads to the S. neurona or B. taurus genome annotations using the RNASeq tool in CLC Genomics Workbench. The process is described in detail in S2 File.

Host cell cytotoxicity assay

The cytotoxicity of AN3661 for the bovine host cells was determined by release of lactate dehydrogenase (LDH) from treated cells using a colorimetric assay (Pierce LDH Cytotoxicity Assay, Thermo Scientific). Briefly, BT cells seeded in a 96-well plate were treated in triplicate in increasing concentrations of AN3661. The plate was incubated for 24 hours, and LDH release from the treated cells was determined spectrophotometrically by subtracting the 680 nm absorbance (background) from the 490 nm absorbance following the protocol provided by the manufacturer. The maximum release of LDH was determined by addition of lysis buffer (provided by the manufacturer) to non-treated triplicate wells of BT cells. The LDH positive control was provided by the manufacturer.

Results

AN3661 inhibits the growth of Sarcocystis neurona and Neospora caninum

To test the hypothesis that AN3661 inhibits S. neurona, growth assays were performed using a parasite clone expressing YFP, as described previously [11,18]. Our preliminary growth assays suggested that AN3661 inhibited S. neurona at low nanomolar concentrations (data not shown). Based on these initial assays, S. neurona growth was examined in the presence of AN3661 over a range of 1 nM to 100 nM (Fig 1A). This showed that S. neurona is very sensitive to AN3661, with an estimated IC₅₀ of 10.68 nM and nearly all parasite growth inhibited at concentrations greater than 50 nM (Fig 1A). To obtain a better estimate of the AN3661 IC₅₀ for S. neurona, the growth assay was repeated using a narrower range of drug concentration (1–25 nM). This assay revealed an IC₅₀ of 14.99 nm (Fig 1B), a concentration comparable to the IC₅₀ described for two isolates of P. falciparum [7], but significantly less than the IC₅₀ reported for C. parvum (80 nM [9]) and T. gondii (900 nM [8]).

Fig 1 — An S. *neurona* clone expressing YFP was seeded into 96-well plates containing monolayers of BT host cells and incubated for 4 days (S. *neurona*) in the presence of increasing concentrations of AN3661 (eight replicates/concentration). Parasite growth inhibition was based on relative fluorescence of treated wells compared to control wells containing no drug, and the IC₅₀ was determined by regression analysis. (A) Parasite growth in AN3661 concentrations between 1 nM and 100 nM suggested an IC₅₀ of 10.68 nM, with parasite growth virtually halted at concentrations greater than 50 nM. (B) Parasite growth across a narrow range of AN3661 concentrations indicated an IC₅₀ of 14.99 nM.

AN3661-resistant S. neurona have mutations near the active site of CPSF73

To further explore the mechanism of inhibition of S. neurona by AN3661, parasites were treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and grown in the presence of 90 nM AN3661, a concentration of the drug that was found to be highly effective for inhibiting growth of the parasite (Fig 1A). Seven drug-resistant single-cell clones were isolated, and five were successfully used for further analyses. Based on the prior studies indicating that CPSF73 is the target of AN3661, the coding region of this gene (SN3_01500330) was amplified by PCR, sequenced, and the sequences aligned with the wild-type S. neurona CPSF73 coding sequence. These comparisons revealed that all five clones carried a point mutation resulting in a Y668N change in the S. neurona protein (Fig 2), a position that is also altered in AN3661-resistant T. gondii and P. falciparum [7,8]. While it is possible that each clone may possess additional mutations, the consistent alteration of Y668 in five independent clones nonetheless supports the hypothesis that AN3661 targets CPSF73 in S. neurona, much as it does in other apicomplexans.

Fig 2 — Alignments were performed using the default settings in CLC Genomics Workbench. Amino acid residues that are identical in all five sequences are shown in black, and other residues in gray. Residues that are altered in T. *gondii* AN3661-resistant mutants are denoted with a blue arrow, those altered in resistant P. *falciparum* mutants in red, and those altered in both T. *gondii* and P. *falciparum* mutants in green. The green star denotes the position (Y668 in the S. *neurona* protein, corresponding to Y483 of the T. *gondii* CPSF73) that is altered in the five AN3661-resistance S. *neurona* clones. The alignment is truncated, focusing on the conserved core of the protein. Tg–T. *gondii*; Sn–S. *neurona*; Pf–P. *falciparum*; At–*Arabidopsis thaliana;* Hs–human. Numbers on the right denote the residue numbers for the respective proteins.

Effects of AN3661 on gene expression and poly(A) site choice in S. neurona

To assess the effects of AN3661 on transcription dynamics of S. neurona, genome-wide transcription was assessed by preparing and sequencing 3’ end-directed cDNA tags (so-called PATSeq), and subsequent analyses. For this, bovine turbinate (BT) cell cultures were infected with S. neurona in triplicate and grown with or without 90 nM AN3661, a concentration of drug found to fully inhibit parasite growth (Fig 1A). Between 3 and 6 million reads were returned for each library (S1 File). After demultiplexing and trimming, the reads were mapped to the S. neurona SN3.E1 (GenBank accession GCA_000727475.1) and Bos taurus (Hereford) genomes (ARS-UCD1.2); the latter mapping was used to assess the effects of the inhibitor on transcriptional dynamics in the host cells (following section). To confirm the reproducibility of the libraries, global gene expression measurements were conducted and gene-by-gene comparisons or relative expression levels were made and presented as the Pearson correlation coefficients for each pairwise comparison. Values for these coefficients ranged from 0.96 to 0.99, with one exception (which yielded a coefficient of 0.85; S1 File).

To assess the overall effects of the CPSF73 inhibitor on transcriptional output, the relative quantities of reads that map to the two genomes were compared in control and AN3661-treated samples. As shown in Fig 3A, more than 60% of the reads mapped to the S. neurona genome in the non-treated cultures. In contrast, in AN3661-treated cultures, only about 25% of reads mapped to the parasite genome. This result indicates an almost 5-fold reduction in the overall proportion of transcripts from S. neurona that had been treated with the inhibitor, and is consistent with the growth characteristics of the parasite under these conditions (Fig 1A).

This analysis was augmented by a genome-wide analysis of parasite gene expression in control and inhibitor-treated cultures. As shown in Fig 3B and S3 File, in inhibitor-treated cells, 1253 genes showed a minimum of a 2-fold change in gene expression with false discovery rate adjusted p-values of 0.05 or less. This represents 18% of all annotated genes in the S. neurona SN3.E1 genome version used for this analysis, and 22% of all genes that showed some degree of gene expression. Genes encoding enzymes and proteins associated with protein biosynthesis were over-represented in the set of differentially-expressed genes (S3 File). This probably reflects a general effect of the inhibitor on transcriptional output and protein synthesis, and concomitant adjustments by the parasite.

Given the target of AN3661 (namely, CPSF73), these global transcriptomics analyses were supplemented with a study of the drug’s effects on poly(A) site choice in the parasite. For this, the analysis pipeline described earlier [16,17,19] was adapted to identify individual poly(A) sites whose relative usage was affected by the inhibitor. In this analysis, relative usage is defined as the fraction of all reads mapping to a given gene that also mapped to a given poly(A) site. This approach is biased towards identifying changes in sites whose usages may be low, and does not apply to genes that have only one poly(A) site. Nonetheless, it serves as a useful metric to assess effects on poly(A) site choice, the rationale being that changes that affect the core machinery will result in increased usage of minor, non-canonical poly(A) sites. The results of this analysis (S4 File) revealed that 1363 individual sites in 990 genes showed significant changes in poly(A) site usage using an false discovery rate (FDR)-adjusted p-value cutoff of 0.05. A more lenient cutoff (raw p-value < 0.05) yielded 5730 poly(A) sites in 2763 genes whose usage was altered in AN3661-treated cells. This is a substantial fraction of all genes (5955) with detectable levels of expression, and indicates a wide-ranging impact of the inhibitor on mRNA 3’ end formation.

To assess the possible contribution of this altered poly(A) site choice to overall gene expression levels, the sets of genes whose expression change were compared with those affected by changes in poly(A) site choice. The results (Fig 4) show that between 24 and 27% of genes whose poly(A) site profiles change also showed significant changes in overall transcript levels. These results suggest that alternative polyadenylation (APA) may contribute to the overall pattern of gene expression in inhibitor-treated cells.

Fig 4 — Venn diagrams showing the overlaps between genes affected by APA (using the padj or p-value cutoffs described in the text, from S4 File; blue circles) and differentially expressed genes (from S3 File; yellow circles). Venn diagrams were made using Venny [20].

An inhibitor of mRNA polyadenylation is expected to alter transcription termination and increase the production of readthrough transcripts that might be “rescued” (and manifest as polyadenylated transcripts) through the use of cryptic or non-canonical polyadenylation sites. The latter would be apparent as an increased number of PATSeq reads mapping to unannotated parts of the genome or that are antisense in orientation to annotated genes. When these reads were measured, it was apparent that AN3661 increased by almost two-fold the overall levels of PATSeq reads that mapped outside of annotated regions (Fig 5A) or antisense to annotated genes (Fig 5B). The summed impact was that almost 10% of the total transcriptional output appeared to be associated with non-canonical poly(A) sites (Fig 5C). (Examples that illustrate these changes are shown in S1 Fig). This result corroborates those shown in S4 File and together support the hypothesis that AN3661 has a sizeable impact on transcription by altering mRNA polyadenylation.

Fig 5 — Fraction (represented as percentages) of all mapped reads that map outside of or antisense to annotated genes. SN3 –untreated control cells. A90 –cells treated with 90 nM AN3661. Error bars denote standard deviations calculated from the values in each replicate of the respective sample. A. Reads that map outside of annotated regions. B. Reads that map antisense to annotated genes. C. Sum of data shown in panels A and B.

Effects of AN3661 on mRNA polyadenylation and gene expression in the BT host cells

In other systems, AN3661 has a high degree of specificity, with strong inhibition of the growth of P. falciparum, T. gondii, and C. parvum but little discernible effects on their respective hosts. To confirm that this is the case with the host cells used here for growth of S. neurona, cytotoxicity and gene expression was assessed in BT cells that were treated with AN3661. In a standard LDH release assay that measures cytotoxicity (Pierce LDH Cytotoxicity Assay), the LDH levels in media prepared from BT cells treated with AN3661 concentrations ranging from 1 nM to 1 mM were indistinguishable from LDH released from non-treated cells (0 nM), and were only slightly more than 10% of the maximum release (lysed cells) control (Fig 6). Therefore, AN3661 was not cytotoxic to the BT host cell line, even at concentrations approaching 100,000-fold greater than the IC₅₀ for S. neurona (14.99 nM; Fig 1B).

Fig 6 — Triplicate wells of BT cultures in a 96-well plate were treated for 24 hours with drug concentrations from 1 nM to 1 mM, and the level of LDH released into the medium was compared to the LDH positive control provided by the manufacturer (Thermo Scientific), the maximum LDH release (i.e., lysed cells), and the spontaneous LDH release (non-treated cells). The LDH activity present in the samples was determined spectrophotometrically by subtracting the absorbance at 680 nm (background) from absorbance at 490 nm. Thus, ΔA = A490 nm−A_{680 nm}, where A is absorbance.

To assess the effects of AN3661 on gene expression in the BT host cells, PATSeq libraries were prepared from uninfected cells grown in the absence or presence of 90 nM AN3661 (the same concentration as was used to study gene expression in S. neurona-infected cells). These libraries were sequenced using the Illumina platform and mapped to the B. taurus (Hereford) genome. Consistency between replicates was assessed by comparisons of genome-wide expression in individual replicates. These pairwise comparisons of libraries prepared from uninfected host cells yielded consistently high Pearson Correlation coefficients (uniformly greater than 0.96; S1 File). Given this, several additional analyses were conducted to assess the impacts of AN3661 on gene expression and polyadenylation.

As was done for the analysis of S. neurona gene expression, gene-by-gene expression in treated and control host cells was measured. The results showed that only 78 genes (of 14,785 whose expression could be measured in this study) had significantly-different expression (2-fold or greater change in expression, with an FDR-adjusted p-value less than 0.05) in AN3661-treated BT host cells (Fig 7A, S5 File). By way of comparison, S. neurona incited changes in the expression of 1489 genes in the host cells (Fig 7B, S6 File). Together, these results indicate that AN3661 has a minimal impact on overall gene expression in BT host cells.

Fig 7 — A. Comparison of host cell gene expression in untreated and AN3661-treated cells. B. Comparison of host cell gene expression in uninfected and S. *neurona*-infected cells.

In parallel, the effect of AN3661 on poly(A) site choice in uninfected BT cells was assessed using the analysis pipeline described in S2 File. A site-by-site analysis of poly(A) site usage in control and treated cells showed that none of the >35,000 sites for which results could be returned showed changes that satisfied the FDR-adjusted p-value cut-off of 0.05 (S7 File). Using the more lenient raw p-value cutoff of 0.05 (as was done for the analysis of S. neurona poly(A) site choice described above), 32 sites from 31 genes were returned. These results indicate minimal effect of AN3661 on mRNA polyadenylation in BT cells.

To further explore possible effects on transcription, the numbers of PATSeq reads that map outside of, or antisense to, annotated BT genes was tabulated. The result of this exercise showed that there were no appreciable differences in the quantities of such reads between control and treated BT cells (Fig 8). Together, these results indicate that AN3661 has almost no impact on gene expression, mRNA polyadenylation, and transcriptional readthrough in BT cells.

Fig 8 — Fraction of all mapped reads that map outside of or antisense to annotated genes in control and AN3661-treated host cells was measured and plotted as shown. BT–untreated control cells. BTI–cells treated with 90 nM AN3661. For this plot, the fractions of reads that map outside of and antisense to annotated genes were summed and plotted as shown. Note the scale of the y-axis.

Discussion

AN3661 targets CPSF73 and mRNA polyadenylation in S. neurona

In recent years, a variety of benzoxaboroles have been found to target mRNA processing in a number of unicellular parasites, including several that can cause disease in humans [7–9,21–23]. The benzoxaborole AN3661, the subject of this study, inhibits P. falciparum, T. gondii, and C. parvum by targeting CPSF73, a subunit of the parasite polyadenylation complex [7–9]. The results presented in this study extend the range of apicomplexan parasites that are inhibited by AN3661 to include two species that have importance in agriculture. Moreover, the finding that AN3661-resistant S. neurona clones carry mutations in the gene that encodes CPSF73 corroborates earlier studies indicating that this drug targets the mRNA polyadenylation in apicomplexan parasites.

While the target of AN3661 and the physiological consequences of treatment with the drug have been well-established [7–9], a detailed understanding of the consequences of AN3661 treatment on genome-wide transcription dynamics in apicomplexans parasites is lacking. The results presented in this report provide added insight into the effects of the drug on 3’ end processing and transcription termination. Treatment with the drug results in a general diminution of parasite transcript levels in infected cells (Fig 3A). This is consistent with the growth inhibition attendant with drug treatment (Fig 1). Interestingly, some genes are more affected by drug treatment than others, resulting in a wide-ranging change in relative gene expression in the two conditions (Fig 3B). These changes in all likelihood reflect the differential responses of gene expression to the physiological changes brought about by the alterations in mRNA polyadenylation incited by the drug.

Along with the overall transcript-level response, treatment with AN3661 also leads to a global alteration in poly(A) site choice in S. neurona, affecting almost half of all expressed genes in the parasite. This is very likely a consequence of a general inhibition of 3’ end processing by the drug. AN3661 binds in the active site of the apicomplexan CPSF73 in a manner predicted to interfere with the metal ion-mediated catalytic mechanism of the endonuclease [9]. Inhibition of processing at the primary (preferred) poly(A) site is expected to result in a population of extended primary transcripts, the collection of which would present numerous alternate poly(A) signals to the processing apparatus. Any of these sites might be recognized and processed, guided by the dynamics of the interactions between the nascent RNA and the poly(A) complex. Weak sites that are usually skipped might be utilized at greater frequencies in drug-treated cells. There is increased read-through transcription in drug-treated S. neurona cells (Fig 5), and additional potential sites might be present in extended transcripts. Taken together, the global shifts in poly(A) site choice upon AN3661 treatment are consistent with the reported mode of action of the drug [7–9].

In these regards, the effects of AN3661 on polyadenylation in S. neurona are similar to the consequences of reduction of CPSF73 levels or activity in mammalian cells and in yeast. The mammalian CPSF73 is a target of a compound (JTE-607) that is active against acute myeloid leukemia and Ewings sarcoma cell lines, and acts by mimicking RNA binding at the CPSF73 active site [24,25]. In JTE-607 treated cells, a substantial increase in read-through transcription is seen [25], much as is seen in AN3661-treated S. neurona cells (Fig 5). This parallel is supportive of the model for the proposed mechanism of action of AN3661 in S. neurona, namely that the compound inhibits CPSF73 and alters efficient mRNA polyadenylation and transcription termination.

YSH1 is the yeast counterpart of CPSF73 and resides in a complex that includes other CPSF subunit orthologs [26]. Like the mammalian CPSF73, YSH1 is the endonuclease that cleaves the pre-mRNA prior to polyadenylation. YSH1 levels are maintained in part by the action of the IPA1 gene product, such that ipa1 mutants have substantially-reduced levels of YSH1 [27]. ipa1 mutants exhibit substantial degrees of transcriptional readthrough, and also considerable changes in the usage of annotated poly(A) sites [28]. These are features also seen in AN3661-treated S. neurona cells. This parallel provides further support for the proposed mechanism of action of AN3661 in S. neurona.

AN3661 has a minimal impact on host cell transcription dynamics

A remarkable feature of AN3661 is its selectivity as far as cellular toxicity is concerned. Previously, it was shown that AN3661 had little effect on the growth of cells or animals that serve as hosts for P. falciparum, T. gondii, and C. parvum [7–9]. The results presented in this study show that the drug does not affect the growth of the BT cells used for propagation of S. neurona. This reinforces the theme that AN3661 has high specificity for its target in the parasites, and does not affect the host (mammalian) ortholog, CPSF73.

This specificity implies that the drug has no impact on mRNA polyadenylation in host cells or animals. However, the compound JTE-607 (mentioned above) is an inhibitor of CPSF73 function that selectively inhibits the growth of acute myeloid leukemia and Ewing’s sarcoma cell lines [24,25], and has been shown to prolong life in a mouse leukemia model [29]. This compound causes wide-ranging changes in poly(A) site usage and transcriptional readthrough in mammalian cells [25]. That a selective compound can alter mRNA polyadenylation raises the possibility that AN3661 may alter 3’ end processing, but in ways that have minimal or no impact on host cell physiology. It is thus important that AN3661 had no discernible effect on mRNA polyadenylation and transcription dynamics in BT host cells. In particular, there was no discernible impact of the drug on poly(A) site usage in BT cells (S7 File). Moreover, there were no indications of increased usage of distal poly(A) sites in AN3661-treated BT cells (Fig 8), nor was there evidence for the usage of novel sites downstream of annotated transcription units (S7 File). These parameters (altered poly(A) site choice, increased distal poly(A) site usage, and transcription downstream of annotated genes) are hallmarks of the inhibition of CPSF73 in mammals and yeast [24,25,27,28]. That AN3661 does not alter these features of gene expression in BT cells is strong evidence that the compound in fact does not interfere with CPSF73 functioning in these cells.

The results presented in this report strengthen the case for AN3661 (and related compounds that also target CPSF73 in parasites) as therapeutic agents. However, enthusiasm for such a use is tempered by the scope of mutations in the parasite CPSF73 that can reduce sensitivity to the drug. In S. neurona, single mutations near the CPSF73 active site were sufficient to provide resistance to the compound (Fig 2). Resistance also could be achieved by single base changes in P. falciparum [7] and T. gondii [8]. The reported frequency of spontaneous resistance clones in vitro in P. falciparum (with resistant clones arising readily in initial populations of as few as 10⁶ cells) [7] is especially notable, as this frequency indicates that resistance would likely arise quickly during a treatment regimen. However, when used in concert with drugs that target different processes, compounds that selectively target CPSF73 in apicomplexans may find use. The absence of effects of the drug on host cells makes such uses attractive, as AN3661 would likely not add any side effects when used in a combination therapy.

Conclusions

The results reported in this study show that the benzoxaborole AN3661 inhibits the growth of S. neurona, an apicomplexan parasite of horses and marine mammals. This inhibition can be attributed to the targeting by the drug of CPSF73. AN3661 had a minimal impact on the bovine host cells used to propagate the parasites; importantly, there was no discernible effect of the drug on poly(A) site choice or overall gene expression in the host cells. Collectively, these results reveal AN3661 to be an exceedingly selective inhibitor of apicomplexan mRNA polyadenylation.

Supporting information

S1 Fig. Changes in transcriptional dynamics in S. neurona treated with AN3661.

Browser tracks showing examples of changes in poly(A) site usage in AN3661-treated cells. The order for each representation is (top to bottom): chromosomal location (in bp), gene annotation, coding region (CDS) annotation, reads from un-treated cells (Sn3 mapping), and reads from AN3661-treated cells (A90-1 mapping). Reads colored green are oriented in the sense (5’->3’ left to right) direction, and reads colored green are oriented in the antisense direction. Tracks were created using CLC Genomics Workbench.

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Click here for additional data file.^{(634.9KB, pdf)}

S1 Table. Primers used in this study.

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Click here for additional data file.^{(9.9KB, xlsx)}

S1 File. Reads and mapping statistics.

(XLSX)

Click here for additional data file.^{(13.6KB, xlsx)}

S2 File. Analysis pipeline for poly(A) site determinations.

(DOCX)

Click here for additional data file.^{(26.6KB, docx)}

S3 File. Gene expression analysis in AN3661-treated S. neurona.

S. neurona-infected BT cells were treated with 90 nM AN3661, RNA isolated, and PATSeq libraries constructed and sequenced. PATSeq reads were used to assess gene expression in drug-treated S. neurona cells.

(XLSX)

Click here for additional data file.^{(1.5MB, xlsx)}

S4 File. Poly(A) site analysis in AN3661-treated S. neurona.

S. neurona-infected BT cells were treated with 90 nM AN3661, RNA isolated, and PATSeq libraries constructed and sequenced. The data were used to assess poly(A) site choice in the parasite in the two conditions.

(XLSX)

Click here for additional data file.^{(10.4MB, xlsx)}

S5 File. Gene expression in control and AN3661-treated BT cells.

RNA was isolated from control and AN3661-treated BT cells and PATSeq libraries constructed and sequenced. PATSeq reads were used to assess host gene expression.

(XLSX)

Click here for additional data file.^{(3MB, xlsx)}

S6 File. Host gene expression in uninfected and S. neurona- infected BT cells.

RNA was isolated from control and S. neurona-infected BT cells and PATSeq libraries constructed and sequenced. PATSeq reads were used to assess host gene expression.

(XLSX)

Click here for additional data file.^{(3.5MB, xlsx)}

S7 File. Poly(A) site analysis in AN3661-treated BT host cells.

RNA was isolated from control and AN3661-treated BT cells and PATSeq libraries constructed and sequenced. The data were used to assess poly(A) site choice in the host in the two conditions.

(XLSX)

Click here for additional data file.^{(5.3MB, xlsx)}

Acknowledgments

The authors acknowledge the excellent technical assistance of Carol Von Lanken.

Data Availability

The high throughput sequencing data associated with this project are available under Bioproject PRJNA713353. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA713353.

Funding Statement

This research was supported by the USDA National Institute of Food and Agriculture (AGH - Hatch project accession #1020849 and DKH – Hatch project accession 1012262; https://nifa.usda.gov/) and the Amerman Family Equine Research Fund (DKH). The sponsors played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0259109.r001

Decision Letter 0

Stuart Alexander Ralph

24 Jun 2021

PONE-D-21-14533

Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation

PLOS ONE

Dear Dr. Hunt,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Three expert reviewers have now commented on the manuscript. All found that the manuscript reported data that were worthy of publication. The referees reported issues with the description of data, in particular the treatment of the drug response data, and the description of replicates in these experiments, which should be addressed before publication

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Reviewers' comments:

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Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Review Comments to the Author

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Reviewer #1: The study by Hunt et al. shows the effects of treating Sarcocystis neurona and Neospora caninum with a known inhibitor of the apicomplexan polyadenylation complex. The study is technically sound and underlying sequencing data have been deposited to a public repository. Results of bioinformatics analyses are mostly included in well-documented Supplementary Tables. Before publication, the authors should appropriately analyse the IC50 data and include replicate number. The differential gene expression analysis (as presented in Figure 3B) should also be explained in the Materials and Methods - it was not clear from the methods in Supplementary File S2 how this was performed after read mapping.

I would also suggest moving some of Fig S1 to the main Figures, to complement visualization of the data represented in Figure 5.

Major comments

Fig1A and 1B: The IC50 curves have inconsistent curved lines between data points. The authors should fit an IC50 regression and calculate the IC50 from that.

Minor comments

Lines 101 and 103: Should consistently use g instead of RPM (typo RMP on line 103)

Line 108: Specify that the data are available at the NCBI Sequencing Read Archive under the Bioproject accession PRJNA713353

Line 116: The Supplemental File 2 docx has the heading “Supplemental File 3”

Line 133: Were biological replicates performed for the growth assays? This should be specified (eg n = 1, or n = 3) in the Figure 1 legend. If n >1 error bars should be added or individual datapoints plotted.

Line 143: convention to denote mutation as (for example) Y668N, but using residue numbering for the S. neurona CPSF73.

Line 150: There are some residues in black that are not identical between all 5 sequences, eg residue 519. Change the Figure 2 legend to reflect this.

Figure 7: The tick labels are too blurry to read

Line 518: Italicise species names

Line 539: remove COI disclosure

S2 File: To enable reproducibility, add the code for “tagtrim” to the S2 File or a public repository such as GitHub.

Reviewer #2: This manuscript describes results of S. neurona and N. caninum treated with AN3661. Similar to T. gondii and P. falciparum, AN3661-resistant S. neurona had mutations in the cpsf gene. The study examines the effect of AN3661 on poly(A) sites and transcriptional changes in S. neurona. There are a few suggestions to improve the manuscript, below:

Major points:

1. Figure 1: How many times was this experiment done? There are no error bars here.

2. Fig 1A: % inhibition is calculated compared to control wells containing no drug, which I assume would be 0% inhibition. Why does the curve in panel A (S. neurona) not reach 0% inhibition?

3. Fig 1B: There are no data points around the 50% inhibition range. The data points around the 50% inhibition mark is at 5% and then it jumps to ~98% inhibition. This does not allow for accurate IC50 determination. It looks like there are only 5 concentrations examined. This data would be improved if you added more concentrations, especially around the 50% range.

4. For growth inhibition, parasites were only exposed for 2 h before washing out, and 90 nM AN3661 inhibited 100% when examined 4 days later, but measurements were taken daily? This is confusing.

5. It is unclear how AN3661-R S. neurona parasites were obtained. They were selected with 90 nM AN36661 – from Fig 1 this inhibits 100%? How long was 90nM AN3661 put on AN3661? Not described in materials and methods section.

6. Growth inhibition studies parasites were exposed to 90nM AN3661 for 2h and 4 days later there was no growth. But for poly(A) site profiling parasites were exposed to 90 nM AN3661 for 24hrs. Parasites are then harvested and examined for poly(A) site profiling. Why this concentration and this time frame? It seems that the parasites would be “in the process of dying” – i.e. if you let it go for a couple more days (without drug) the parasites would die (as you show for growth inhibition).

7. How many biological replicates were performed for poly(A) site profiling?

8. Fig 3 legend: how long were S. neurona and B. taurus treated for with 90 nM AN3661? Why was this drug concentration chosen – this inhibits Sn at 100% (Fig 1)

9. Fig 5: what do the error bars represent? Not described in the legend

10. Fig 6: why is LDH positive control twice as much as “max LDH release from lysed cells”? Why are the bar graphs of different width?

11. Fig 8: what do the error bars represent? Not described in the legend

12. Whole genome seqencing was not performed on AN3661-resistant parasites, but rather the researchers amplified the cpsf gene and then sequenced the gene. Therefore would be good to mention in discussion that there could be other genetic mutations underlying AN3661-R.

Minor points:

1. Lines 34-35: this sentence is confusing. If vaccines are ineffective against coccidian parasite how is this an option to reduce coccidiosis?

2. Line 40: EPM not defined

3. Line 103: RPM (typo)

4. The figure legends are peppered in the body of the manuscript.

5. After the first mention of Sarcocystis neurona, future mentions should be S. neurona

6. Line 162: should be bovine turbinate (BT) not bovine (BT) turbinate

7. Line 192: FDR is 0.05 while Line 271 refers to false discovery rate adjusted p-value (i.e. q-value) less than 0.05. BT analysis refers to analysis done on Sn. Was the same analyses performed? Please clarify.

8. Would be good to define “Adjusted p-value” vs :p-value”

Reviewer #3: The manuscript is exceedingly well-written and well-organized with sufficient detail to support scientific claims.

I have only a few comments/questions/suggested edits:

1. The information provided in the introduction could be better supported with citations.

2. Line 52 - affective should be effective

3. Sequencing of the SnCPSF73 gene of the AN3661-resistant clones was performed. Did the authors consider sequencing the full genome of the clones to ensure no other mutant genes were contributing to the phenotype?

4. Line 294 - Typo "BY" host cells, should be "BT"

5. Line 351 - typo "t="

6. Figure 2 - What does the blue star indicate?

7. Figure 3B. x/y axis text not legible, poor resolution.

8. figure 8. (Line 437) Should this be BT cells instead of BY?

9. Figure 8. - Does the "I" in BTI stand for anything? Inhibitor? Because there aren't any parasites in this population of cells consider changing for clarity (e.g., BT-A90).

**********

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Reviewer #1: No

Reviewer #2: Yes: Caroline Ng

Reviewer #3: No

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Attachment

Submitted filename: Plos one Review.docx

Click here for additional data file.^{(15.7KB, docx)}

PLoS One. 2021 Oct 28;16(10):e0259109. doi: 10.1371/journal.pone.0259109.r002

Author response to Decision Letter 0

8 Aug 2021

To the Editor,

The following summarizes changes and other discussion regarding the review of manuscript PONE-D-21-14533, “Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation”, that we have submitted for publication in PLOS ONE. This document is organized using the reviewing outline and format.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

As indicated in the detailed response, we have made changes to satisfy the concerns regarding statistical analyses and presentation of some data items.

Reviewer #3: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

As indicated in the detailed response, we have made changes to satisfy the concerns regarding statistical analyses and presentation of some data items.

Reviewer #2: No

As indicated in the detailed response, we have made changes to satisfy the concerns regarding statistical analyses and presentation of some data items.

Reviewer #3: Yes

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

5. Review Comments to the Author

We have added the requested information to the Supplementary File S2, as an additional section (Section III). This addition should satisfy the request. Additional text was not added to the Materials and Methods, since this would be redundant. (File S2 has been further edited to fix some other issues and hopefully make it easier to follow.)

The assessment of growth inhibition, including replicate numbers, has been clarified in the Materials and Methods, Results, and the Figure legend, and IC50 values for S. neurona have now been more appropriately determined by regression analysis. Since it was a minor component of the study and the data were not strong, the Neospora growth assay and IC50 determination have been removed from the manuscript.

I would also suggest moving some of Fig S1 to the main Figures, to complement visualization of the data represented in Figure 5.

After trying to add some browser tracks to Fig. 5, we find that the resulting revised figure is very difficult to read, because of the different formats and styles for the charts and browser tracks. Thus, we have decided to leave Fig. 5 and Fig. S1 unchanged.

Major comments

Fig1A and 1B: The IC50 curves have inconsistent curved lines between data points. The authors should fit an IC50 regression and calculate the IC50 from that.

Our presentation of the growth assay results (Fig. 1) has been revised, and the IC50 for S. neurona has been calculated by regression analysis. We have decided that the N. caninum growth assays were not pursued adequately. Since these data were a minor component of the study, we have elected to remove the old Figure 1B. The new Figure 1B shows results of a S. neurona growth assay using a more narrow range of AN3661 concentrations to better estimate the IC50.

Minor comments

Lines 101 and 103: Should consistently use g instead of RPM (typo RMP on line 103)

RPM has been replaced with x g.

Line 108: Specify that the data are available at the NCBI Sequencing Read Archive under the Bioproject accession PRJNA713353

This change has been made as requested (lines 119-120).

Line 116: The Supplemental File 2 docx has the heading “Supplemental File 3”

This error has been corrected.

The number of assays and replicates has been clarified in the figure legend (lines 154-155) and the Materials and Methods (lines 74-79), and the figure has been revised as recommended.

Line 143: convention to denote mutation as (for example) Y668N, but using residue numbering for the S. neurona CPSF73.

Figure 2 has been revised to account for this, and to add residue numbers appropriate for each sequence that was analyzed.

Line 150: There are some residues in black that are not identical between all 5 sequences, eg residue 519. Change the Figure 2 legend to reflect this.

The legend to Fig. 2 has been revised to better explain the residue shading that was used to depict the alignment.

Figure 7: The tick labels are too blurry to read

The font size for the tick legends have been increased to allow for easier reading.

Line 518: Italicise species names

This correction has been made.

Line 539: remove COI disclosure

This correction has been made.

S2 File: To enable reproducibility, add the code for “tagtrim” to the S2 File or a public repository such as GitHub.

The “tagtrim” program has been deposited on Github and links provided.

Major points:

1. Figure 1: How many times was this experiment done? There are no error bars here.

The number of assays and replicates has been clarified in the text (lines 140-150), figure legend (lines 154-155) and the Materials and Methods (lines 74-79), and the figure has been revised as recommended.

2. Fig 1A: % inhibition is calculated compared to control wells containing no drug, which I assume would be 0% inhibition. Why does the curve in panel A (S. neurona) not reach 0% inhibition?

Although these data have been re-plotted and the figure revised, the principle is the same. The reviewer is correct that the % inhibition (now % growth) in the treatment wells is relative to the no-drug control wells. Consequently, the control wells are not plotted in the graph; only the treatment data are plotted. Since some inhibition was observed even at the lowest concentration, the curve does not reach 0% inhibition (now 100% growth).

The reviewer is correct that the growth assay data for Neospora (Fig.1B) was not sufficient. Since Sarcocystis is the primary focus of the study, we have removed the Neospora results. A new Figure 1B is now presented, which shows the growth curve for S. neurona over a more narrow range of drug concentrations.

4. For growth inhibition, parasites were only exposed for 2 h before washing out, and 90 nM AN3661 inhibited 100% when examined 4 days later, but measurements were taken daily? This is confusing.

We apologize for the lack of clarity. The growth assays were conducted with the AN3661 drug present for the duration of the 4-day growth period, not just during the initial 2-hour incubation when parasites were allowed to invade the host cells. This has been clarified in the Materials and Methods (lines 74-87).

Although the plates were monitored daily, only the data from day 4 are presented in Figure 1 and used for the IC50 calculations. This has been clarified in the Materials and Methods (lines 74-87).

The mutagenized population was grown in the presence of AN3661 for approximately 5 weeks, followed by continued selection during the single-cell cloning. This has been clarified in the Materials and Methods (lines 90-96). As mentioned by the reviewer, 90nM was chosen because our growth assays (e.g., Fig. 1) showed that this fully inhibited parasite growth (now mentioned in the Results section, lines 164-167).

As clarified above for item #4, the AN3661 drug was present for the entire 4-day growth assay, not just during the initial 2-hour incubation. It is believed that AN3661 is not a parasiticidal drug, but rather just inhibits parasite growth via disruption of normal RNA polyadenylation (the intent of the transcriptome experiment). As we mention above for item #5, 90 nM was chosen because it fully inhibited parasite growth and we wanted to ensure large differences in poly(A) profiling between the treatment sample and the non-treated control sample.

7. How many biological replicates were performed for poly(A) site profiling?

The Materials and Methods (lines 102-109) have been edited to make it clear that three biological replicates were prepared for each condition in the poly(A) site profiling.

8. Fig 3 legend: how long were S. neurona and B. taurus treated for with 90 nM AN3661? Why was this drug concentration chosen – this inhibits Sn at 100% (Fig 1)

As indicated in the Materials and Methods, the AN3661 treatments were done for 24 hr. 90 nM was chosen for the AN3661 concentration for the poly(A) site profiling so as to assure that the two populations (treated and control) were distinct. Use of a concentration nearer the LD50 could have resulted in a mixed population of cells exhibiting different extents of inhibition and transcriptome alterations.

9. Fig 5: what do the error bars represent? Not described in the legend

The error bars represent standard deviations calculated from values in each of the replicates. The figure legend has been edited to make this clear.

10. Fig 6: why is LDH positive control twice as much as “max LDH release from lysed cells”? Why are the bar graphs of different width?

The LDH positive control is an enzyme control provided by the manufacturer to ensure that the assay has been conducted properly. The maximum release sample represents the same number of BT cells as those that have been treated and, therefore, shows the level of LDH release that would be seen if a drug was highly cytotoxic. We neglected to include a description of the LDH release assay in the Materials and Methods section. This has been added (lines 128-135). We have also revised the results and Fig. 6 legend to better explain the results of this experiment.

11. Fig 8: what do the error bars represent? Not described in the legend

The error bars represent standard deviations calculated from values in each of the replicates. The figure legend has been edited to make this clear.

12. Whole genome sequencing was not performed on AN3661-resistant parasites, but rather the researchers amplified the cpsf gene and then sequenced the gene. Therefore would be good to mention in discussion that there could be other genetic mutations underlying AN3661-R.

This point has been raised in the revision (lines 172-175).

Minor points:

1. Lines 34-35: this sentence is confusing. If vaccines are ineffective against coccidian parasite how is this an option to reduce coccidiosis?

This sentence has been revised to specify that vaccination is effective against poultry coccidiosis caused by Eimeria, but has not been effective against other species of coccidia.

2. Line 40: EPM not defined

This has been changed to Equine Protozoal Myeloencephalitis.

3. Line 103: RPM (typo)

This error has been corrected.

4. The figure legends are peppered in the body of the manuscript.

This is the format required for submissions to PLoS ONE.

5. After the first mention of Sarcocystis neurona, future mentions should be S. neurona

This correction has been made.

6. Line 162: should be bovine turbinate (BT) not bovine (BT) turbinate

This correction has been made.

This statement has been corrected, as suggested. The parameter used in these analyses was indeed the q-value.

8. Would be good to define “Adjusted p-value” vs :p-value”

Assuming that this refers to the description of the poly(A) site analyses (lines 237-239 and 303-306), the terms “FDR-adjusted p-value” and “raw p-value” have been inserted where needed.

Reviewer #3: The manuscript is exceedingly well-written and well-organized with sufficient detail to support scientific claims.

I have only a few comments/questions/suggested edits:

1. The information provided in the introduction could be better supported with citations.

An additional reference that succinctly reviews the field had been added.

2. Line 52 - affective should be effective

This correction has been made.

We have not done whole-genome sequencing, largely because of cost considerations. Were this the first study of AN3661-resistant apicomplexans, we would concur that whole-genome sequencing and a more exhaustive ruling out of other changes would be called for. However, it is well-established that the changes we report are responsible for AN3661 resistance in other apicomplexans; the consistency we see in multiple mutant isolates suffices to confirm the hypothesis, in our opinion.

4. Line 294 - Typo "BY" host cells, should be "BT"

This error has been corrected.

5. Line 351 - typo "t="

This error has been corrected.

6. Figure 2 - What does the blue star indicate?

These were inadvertent additions to the figure and do not belong. They are carryovers from a different version of the figure, intended to highlight features not discussed in this study.

7. Figure 3B. x/y axis text not legible, poor resolution.

The figure has been modified to improve the legibility and resolution.

8. figure 8. (Line 437) Should this be BT cells instead of BY?

This error has been corrected.

9. Figure 8. - Does the "I" in BTI stand for anything? Inhibitor? Because there aren't any parasites in this population of cells consider changing for clarity (e.g., BT-A90).

We believe the term “BTI” is adequately defined in the legend, so this has not been changed.

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Caroline Ng

Reviewer #3: No

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PLoS One. doi: 10.1371/journal.pone.0259109.r003

Decision Letter 1

Stuart Alexander Ralph

31 Aug 2021

PONE-D-21-14533R1

Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation

PLOS ONE

Dear Dr. Hunt,

The referees agree that the revisions made have substantially addressed their concerns, but in the new material there are several areas where minor additional details should be provided for clarity and completeness of the manuscript, as well as some minor typographical and other areas throughout the manuscript that should be addressed.

Please submit your revised manuscript by Oct 15 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Stuart Alexander Ralph

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have satisfactorily addressed my comments. Prior to publication, the authors should ensure that the "tagtrim" GitHub repository is set to public as the address https://github.com/ArthurGHunt/tagtrim currently leads to a 404 page.

Reviewer #2: The revised manuscript is much improved, especially in regards to statistical analyses, graphical representations, and clarification of methods. Here are some suggestions to further improve the manuscript, detailed below:

Major Comments:

1. Define abbreviations at first use. Some examples (please go through to identify others):

a. CPSF73 in the abstract Line 4

b. BT cells Line 70 Methods

c. FDR Line 240

d. YSH1 Line 381

e. IPA1 Line 383

2. Reference 1 – can’t find this in PubMed. Would be good to include doi for all articles not in PubMed.

3. Statements in the introduction are largely unsupported by the necessary references. Please include a reference for each statement made, e.g. for statements in lines 24-32, lines 38-48, 50-51

4. Methods: Please include more details on the LDH assay – eg what wavelength was this assay read at? What is the composition of the lysis buffer?

5. How many total drug-resistant single-cell clones were obtained? (in reference to lines 167-168)

6. Fig. 4: what does DEGs stand for? What does the blue circle and the yellow circle indicate? Unclear from the figure and legend.

7. “The results (Fig. 4) show that between 24 and 27% of genes 247 whose poly(A) site profiles change also showed significant changes in overall transcript levels.” – I don’t see these percentages in the figure.

8. Fig. 6: What is the measurement that was taken? As written it looks like a measurement was taken at Abs 490 through Abs 680 nm. A quick look into the manufacturer’s protocol suggests that this is not the case. Please correct the figure itself to more accurately reflect what was done, and also describe this in the figure legend.

9. Statement on Lines 418-420 incorrect: studies were performed with various initial populations of P. falciparum; the least amount of parasites was 2 x 107

Minor Comments:

1. Line 70: Space between cloneswere

2. Line 81: 37oC

3. Be consistent: hours denoted as “h” in line 80 but “hrs” on lines 103,104 and elsewhere

4. Fig 1 legend: Line 158: nM; line 160: IC50, and needs units after 14.99.

5. Fig 2: “Hs” is not aligned with the other names. Suggest writing Tg, Sn, Pf, At, Hs for consistency. Information about strains that this sequence comes from should be addressed in the legend. – i.e. please explain TGME49, SN3, Pf3D7

6. Line 193 – (BT) after “bovine turbinate” and not “bovine”

7. Line 340: benzoxaborole (typo)

8. Line 427: no studies on N. caninum anymore in this report

9. Fig 3 legend: S. neurona and B. taurus need to be italicized

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Caroline Ng

Reviewer #3: No

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PLoS One. 2021 Oct 28;16(10):e0259109. doi: 10.1371/journal.pone.0259109.r004

Author response to Decision Letter 1

29 Sep 2021

To the Editor,

The following summarizes changes and other discussion regarding the second review of manuscript PONE-D-21-14533, “Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation”, that we have submitted for publication in PLOS ONE. This document is organized using the reviewing outline and format. In so doing, our changes, responses, and other discussion are set apart after each section of comments and suggestions.

Major Comments:

1. Define abbreviations at first use. Some examples (please go through to identify others):

a. CPSF73 in the abstract Line 4

b. BT cells Line 70 Methods

c. FDR Line 240

d. YSH1 Line 381 – no need to change, since this is a gene name

e. IPA1 Line 383 - no need to change, since this is a gene name

The changes in lines 4, 70, and 240 of the first revision have been made as suggested. USH1 and IPA1 are formal gene designations and not abbreviations. Accordingly, these have not been changed or further defined.

2. Reference 1 – can’t find this in PubMed. Would be good to include doi for all articles not in PubMed.

The doi for this book has been added. We believe the other references are in Pubmed.

3. Statements in the introduction are largely unsupported by the necessary references. Please include a reference for each statement made, e.g. for statements in lines 24-32, lines 38-48, 50-51

We appreciate this remark. However, these statements are all general parasitology textbook information (as could be found in the first reference). We would argue that references to the literature, or other textbooks, are appropriate for this paragraph. Accordingly, we have not added any.

4. Methods: Please include more details on the LDH assay – eg what wavelength was this assay read at? What is the composition of the lysis buffer?

This section of the Methods has been updated as requested. We would note that the lysis buffer was provided with the commercial assay. The composition of the buffer is not provided in the documentation by the manufacturer.

5. How many total drug-resistant single-cell clones were obtained? (in reference to lines 167-168)

This statement has been updated as requested.

6. Fig. 4: what does DEGs stand for? What does the blue circle and the yellow circle indicate? Unclear from the figure and legend.

The legend to Figure 4 has been edited so as to clarify these questions. In particular, in the legend we now note that the yellow circles (identifiable as “DEG” in the figure) corresponds to differentially-expressed genes.

7. “The results (Fig. 4) show that between 24 and 27% of genes 247 whose poly(A) site profiles change also showed significant changes in overall transcript levels.”

We have not altered the figure, out of the belief that to add percentages to the figure in addition to or instead of the total gene numbers would be confusing, and would not allow readers to appreciate the scope of the changes we describe. It is relatively simple for a reader to divide the numbers in the overlap by the numbers of total genes in each class, to come to the percentages we mention.

We have corrected the Y axis label and defined the terms in the revised figure legend..

9. Statement on Lines 418-420 incorrect: studies were performed with various initial populations of P. falciparum; the least amount of parasites was 2 x 107 .

From Ref 7: “We quantified the ease of in vitro selection of resistance to AN3661 by subjecting 106–108 Dd2 strain parasites to 60 nM (2 × IC90) AN3661. Regrowth was seen in two of three cultures with initial inocula of 106 parasites at days 45 and 56, one of three cultures with initial inocula of 107 parasites at day 23, and three of three cultures with initial inocula of 108 parasites, all on day 19 (Supplementary Table 4). These rates of resistance selection were similar to those observed for atovaquone.”

Minor Comments:

1. Line 70: Space between cloneswere

This correction has been made.

2. Line 81: 37oC

This correction has been made.

3. Be consistent: hours denoted as “h” in line 80 but “hrs” on lines 103,104 and elsewhere

We now use hrs throughout..

4. Fig 1 legend: Line 158: nM; line 160: IC50, and needs units after 14.99.

These corrections have been made.

The figure has been slightly modified, and the legend edited, to address this issue. We would note that the gene identifiers for the proteins have been added to the Methods.

6. Line 193 – (BT) after “bovine turbinate” and not “bovine”

This correction has been made.

7. Line 340: benzoxaborole (typo).

This correction has been made.

8. Line 427: no studies on N. caninum anymore in this report

This correction has been made.

9. Fig 3 legend: S. neurona and B. taurus need to be italicized

This correction has been made.

Attachment

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Click here for additional data file.^{(19.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0259109.r005

Decision Letter 2

Stuart Alexander Ralph

13 Oct 2021

Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation

PONE-D-21-14533R2

Dear Dr. Hunt,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Stuart Alexander Ralph

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0259109.r006

Acceptance letter

Stuart Alexander Ralph

19 Oct 2021

PONE-D-21-14533R2

Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation

Dear Dr. Hunt:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Stuart Alexander Ralph

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Changes in transcriptional dynamics in S. neurona treated with AN3661.

(PDF)

Click here for additional data file.^{(634.9KB, pdf)}

S1 Table. Primers used in this study.

(XLSX)

Click here for additional data file.^{(9.9KB, xlsx)}

S1 File. Reads and mapping statistics.

(XLSX)

Click here for additional data file.^{(13.6KB, xlsx)}

S2 File. Analysis pipeline for poly(A) site determinations.

(DOCX)

Click here for additional data file.^{(26.6KB, docx)}

S3 File. Gene expression analysis in AN3661-treated S. neurona.

(XLSX)

Click here for additional data file.^{(1.5MB, xlsx)}

S4 File. Poly(A) site analysis in AN3661-treated S. neurona.

(XLSX)

Click here for additional data file.^{(10.4MB, xlsx)}

S5 File. Gene expression in control and AN3661-treated BT cells.

RNA was isolated from control and AN3661-treated BT cells and PATSeq libraries constructed and sequenced. PATSeq reads were used to assess host gene expression.

(XLSX)

Click here for additional data file.^{(3MB, xlsx)}

S6 File. Host gene expression in uninfected and S. neurona- infected BT cells.

RNA was isolated from control and S. neurona-infected BT cells and PATSeq libraries constructed and sequenced. PATSeq reads were used to assess host gene expression.

(XLSX)

Click here for additional data file.^{(3.5MB, xlsx)}

S7 File. Poly(A) site analysis in AN3661-treated BT host cells.

RNA was isolated from control and AN3661-treated BT cells and PATSeq libraries constructed and sequenced. The data were used to assess poly(A) site choice in the host in the two conditions.

(XLSX)

Click here for additional data file.^{(5.3MB, xlsx)}

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Data Availability Statement

The high throughput sequencing data associated with this project are available under Bioproject PRJNA713353. https://www.ncbi.nlm.nih.gov/bioproject/PRJNA713353.

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PERMALINK

Transcriptional dynamics in the protozoan parasite Sarcocystis neurona and mammalian host cells after treatment with a specific inhibitor of apicomplexan mRNA polyadenylation

Arthur G Hunt

Daniel K Howe

Ashley Brown

Michelle Yeargan

Roles

Abstract

Introduction

Materials and methods

Parasite cultures, DNA isolation, and parasite growth assays

Isolation and sequence analysis of AN3661-resistant clones of S. neurona

Poly(A) site profiling

Host cell cytotoxicity assay

Results

AN3661 inhibits the growth of Sarcocystis neurona and Neospora caninum

Fig 1. Growth inhibition of S. neurona by AN3661.

AN3661-resistant S. neurona have mutations near the active site of CPSF73

Fig 2. Amino acid sequence alignments of orthologs of CPSF73.

Effects of AN3661 on gene expression and poly(A) site choice in S. neurona

Fig 3. Gene expression in control and AN3661-treated cells.

Fig 4. Genes that show differential poly(A) site usage and overall expression.

Fig 5. Fraction of all mapped reads that map outside of or antisense to annotated genes.

Effects of AN3661 on mRNA polyadenylation and gene expression in the BT host cells

Fig 6. Cytotoxicity of AN3661 for bovine turbinate BT cells determined by LDH release.

Fig 7. Volcano plots showing the extent of differential host gene expression in infected and AN3661-treated cells.

Fig 8. Transcriptional readthrough in BT host cells.

Discussion

AN3661 targets CPSF73 and mRNA polyadenylation in S. neurona

AN3661 has a minimal impact on host cell transcription dynamics

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Stuart Alexander Ralph

Roles

Author response to Decision Letter 0

Decision Letter 1

Stuart Alexander Ralph

Roles

Author response to Decision Letter 1

Decision Letter 2

Stuart Alexander Ralph

Roles

Acceptance letter

Stuart Alexander Ralph

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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