1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate ‘bovine genotype’

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Research paper

Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate ‘bovine genotype’ a,∗ ˇ Victoria Morin-Adeline a , Kai Mueller a , Ana Conesa b,c , Jan Slapeta a

Faculty of Veterinary Science, McMaster Building B14, University of Sydney, New South Wales 2006, Australia Genomics of Gene Expression Lab, Prince Felipe Research Centre, Valencia, Spain c Microbiology and Cell Science Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, United States b

a r t i c l e

i n f o

Article history: Received 19 June 2015 Received in revised form 11 August 2015 Accepted 12 August 2015 Keywords: Trichomonosis Cow Pig Cat Cysteine protease RNA sequencing Transcriptome

a b s t r a c t Tritrichomonas foetus was described as a commensal of the stomach, caecum and nasal cavity of pigs before it was recognised as the cause of reproductive tract disease of cattle. T. foetus also causes chronic large bowel diarrhoea in domestic cats. Multi-locus genotyping and comparative transcriptome analysis has previously revealed that T. foetus isolated from cat and cattle hosts are genetically distinct, referred to as the ‘feline genotype’ and ‘bovine genotype’, respectively. Conversely, multi-locus genotyping has grouped porcine T. foetus with the ‘bovine genotype’. To compare the extent of the similarity between porcine T. foetus and cattle ‘bovine genotype’ isolates, RNA-sequencing (RNA-seq) was used to produce the first cell-wide transcriptome library of porcine T. foetus PIG30/1. Comparative transcriptome analysis of the PIG30/1 with the published bovine (BP-4) and feline (G10/1) transcriptomes revealed that the porcine T. foetus shares a 4.7 fold greater number of orthologous genes with the bovine T. foetus than with the feline T. foetus. Comparing transcription of the virulence factors, cysteine proteases (CP) between the three isolates, the porcine T. foetus was found to preferentially transcribe CP8 like the ‘bovine genotype’ T. foetus, compared to thehigh transcription of CP7 seen for ‘feline genotype’ T. foetus. At the cell-wide transcriptome level, the porcine T. foetus isolate (PIG30/1) groups closer with the ‘bovine genotype’ T. foetus rather than the ‘feline genotype’ T. foetus. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Isolates of the flagellate Tritrichomonas foetus that reside in the stomach, caecum and nasal cavity of pigs are not of clinical significance to their porcine hosts (Fitzgerald et al., 1958; Hammond et al., 1958; Hibler et al., 1960; Pakandl, 1994; Mostegl et al., 2011). The same species is a serious urogenital tract parasite of cattle with transmission occurring via mating of infected, asymptomatic bulls to females. Although now considered a rare disease as a result of artificial insemination, bovine trichomonosis causes abortion and infertility in infected female cattle (Riedmuller, 1928; Parsonson et al., 1976; Rhyan et al., 1988; Rae et al., 2004). More recently, T. foetus is globally recognized as the cause of chronic large bowel diarrhoea in domestic cats (Levy et al., 2003; Bell et al., 2010; Lim et al., 2010). While T. foetus is not known to have environmentally resistant cysts that facilitate transmission, cross-species infection

∗ Corresponding author: Fax: +61 2 935 17348. ˇ E-mail address: [email protected] (J. Slapeta).

has been suggested (Fitzgerald et al., 1958). Indeed, experimental infections of T. foetus in their non-respective hosts have shown that the porcine T. foetus isolate is able to cause disease in cattle (Fitzgerald et al., 1958), and similarly the feline and bovine isolates have been shown to cause disease in their non-respective hosts (Stockdale et al., 2007; Stockdale et al., 2008). Recently, however, a high prevalence of T. foetus in pigs farmed in close proximity with T. foetus-free cattle has been shown, implying that the risk of crossinfections of T. foetus from pigs to cattle is negligible (Mueller et al., 2015). Owing to the broad host range and organ tropism of T. foetus, much emphasis has been placed on identifying the difference, if any, between host-specific isolates. Various molecular methods including multi-locus genotyping have grouped the porcine T. foetus isolate with bovine T. foetus isolates, whereas isolates of T. foetus from cats are genetically distinct from bovine isolates ˇ ˇ (Tachezy et al., 2002; Slapeta et al., 2010; Slapeta et al., 2012). In an accompanying study by Mueller et al. (2015), the porcine isolate PIG30/1 was shown to be identical to the ‘bovine genotype’ of T. foetus at 9 diagnostic molecular markers. Whole genome dif-

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Please cite this article in press as: Morin-Adeline, V., et al., Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate ‘bovine genotype’. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.012

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Table 1 Summary statistics of the Tritrichomonas foetus (PIG30/1) transcriptome. Summary statistics for T. foetus RNA sequencing Feature

Porcine PIG30/1a

Bovine BP-4b

Feline G10/1b

Total number of reads Total base pairs (nt) Total number of assembled contigs Total assembled bases (nt) Mean length of contigs(nt) %GC content in transcriptome Minimum contig length (nt) Maximum contig length (nt) Contig N50

55,506,706 5,606,177,306 43,308 47,094,268 1,087 33.90 201 17,203 1,503

64,744,882 6,539,233,082 42,363 37,882,427 895.25 34.62 201 14,314 1,259

64,009,804 6,464,990,204 36,559 29,525,551 806.61 34.87 201 17,195 1,178

a

Summary statistics of the porcine T. foetus transcriptome sequenced in this study. Summary statistics of the bovine and feline T. foetus transcriptome sequenced by Morin-Adeline et al. (2014) for comparison. Paired-end RNA sequencing of the bovine and feline T. foetus transcriptomes were performed on an Illumina HiSeq2000 platform and assembled using the same de novo approach as for the porcine T. foetus transcriptome in this study. b

ferences between the ‘feline genotype’ and the ‘bovine genotype’ are minute and analysis of 1511 orthologous protein-coding genes shared between a bovine (BP-4) and a feline (G10/1) isolate indicate little divergence, despite their vastly different origin (. at 9 diagnostic molecular markers. Morin-Adeline et al., 2014). In addition, differences in specific virulence factors have been investigated at a cell-wide level in an attempt to understand molecular mechaˇ nisms that govern host-specificity of the different isolates (Slapeta et al., 2010, 2012; Morin-Adeline et al., 2014). In particular, the cysteine protease (CP) gene family have been used as they are regarded as a crucial aspect of T. foetus virulence involved in cleavage and inactivation of host protective antibodies (Bastida-Corcuera et al., 2000). Distinct differences exist in the cysteine protease gene family between the bovine and feline genotype T. foetus and to date, 7CPs have been used to as molecular diagnostic markers to disˇ tinguish the ‘bovine genotype’ from the ‘feline genotype’ (Slapeta et al., 2012). The most divergent of the family, CP2, confirmed that T. foetus isolate (PIG30/1) is ‘bovine genotype’ (Mueller et al., 2015). The apparent identity of the porcine and bovine isolates of T. foetus compared to the feline isolate of T. foetus, together with their broad host range, presents an intriguing model to studying the factors that drive T. foetus host adaptation. Comparative analysis of transcriptomes offers an ideal method for whole-cell comparisons in the absence of a sequenced nuclear genome (Morin-Adeline et al., 2014a). In this study, RNA-sequencing (RNA-seq) was utilised to obtain whole-cell transcribed sequences of the novel PIG30/1 porcine T. foetus isolated by Mueller et al. (2015). The newly assembled transcriptome of PIG30/1 was then compared to the published bovine (BP-4) and feline (G10/1) T. foetus (Morin-Adeline et al., 2014).

2. Material and methods 2.1. Parasite culture The porcine T. foetus PIG30/1 isolate used for this study was isolated from pig faeces at The University of Sydney May Farm, Camden, New South Wales, Australia (Mueller et al., 2015). Axenic cultures of the parasite were maintained in Modified Diamond’s Medium (MDM) (200 ml) prepared with 4 g Trypticase peptone (BD211921; Becton, Dickinson and company, NSW, Australia), 2 g yeast extract (BD211929; Becton, Dickinson and company, NSW, Australia) and 1 g maltose (BD216830; Becton, Dickinson and company, NSW, Australia) at pH 7.2 (ATCC Medium 719 without agar; http://www.atcc.org/) and supplemented with 10% inactivated sheep serum (Life Sciences, Australia). The antibiotics PenStrep & Fungizone was added to the media to protect against bacterial contamination at a final concentration of 10 U/ml penicillin, 10 ␮g/ml

streptomycin and 0.25 ␮g/ml amphotericin B (Gibco 15,240,062, Life Sciences, Australia). All cultures were maintained in sterile 15 ml glass bijou bottles and sub-cultured daily. 2.2. Total RNA isolation Trophozoites of PIG30/1 T. foetus at mid-exponential phase in culture were collected and 107 cells were pelleted at 3220 × g for 5 min and prepared for RNA-seq (Morin-Adeline et al., 2014). RNA isolation was carried out using the RNeasy Micro kit (Qiagen, New South Wales, Australia) according to the manufacturer’s instructions. Homogenisation was carried out in a FastPrep® -24 high-speed homogeniser (MP Biomedicals, USA) for 30 s at 4 m/s. An in-column DNAase (Sigma–Aldrich) treatment step was included, which was incubated at room temperature for 15 min. RNA was eluted in 30 ␮l of sterile water, assessed for quality and quantity using a 2100 Bioanalyzer (Agilent Technologies, Inc) and preserved in an RNAstable® tube (Biometrica) by drying in a Savant SpeedVac (ThermoFisher, Australia) concentrator connected to a vapor trap for 1 h. Paired-end RNA sequencing on the Illumina HiSeq2000 platform was performed by Macrogen (Seoul, Korea). 2.3. Transcriptome assembly, annotation and identification of shared transcripts between T. foetus isolates The quality of raw sequence reads was confirmed using FASTQC (Babraham Bioinfomatics, http://www.bioinformatics.babraham. ac.uk/projects/fastqc/) prior to assembly. A de novo transcriptome assembly approach was adopted using the default parameters of Trinity within the Galaxy suite platform to assemble right and left reads, resulting in the porcine T. foetus transcriptome (PIG30/1) (Giardine et al., 2005; Blankenberg et al., 2010; Goecks et al., 2010). Sequences were initially annotated using BLASTX against the NCBI non-redundant (nr) database with an e-value cut-off of 1 × 10-3 implemented in the Galaxy Suite (Camacho et al., 2009; Cock et al., 2013). Functional annotation of sequences at the gene ontology (GO) level was carried out using the Blast2GO platform (version 3.0) abiding to default parameters (Conesa et al., 2005). Combined graphs were generated for cellular component, biological processes and molecular function with a level 3 cut-off and filtered to a minimum of 100 sequences per GO category. The GO-enzyme code mapping function in Blast2GO, which maps GO terms to enzyme codes, as well as a search of enzyme pathways from the online Kyoto Encyclopedia of Genes and Genomes (KEGG), was implemented using default settings. To identify putative shared orthologue transcripts between the bovine, feline and porcine T. foetus isolates, a reciprocal BLAST method was adopted in the Galaxy Suite platform (Giardine et al.,

Please cite this article in press as: Morin-Adeline, V., et al., Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate ‘bovine genotype’. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.012

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2005; Blankenberg et al., 2010; Goecks et al., 2010). First, the NCBI BLAST + makeblastdb tool was used to create nucleotide databases of the current porcine T. foetus (PIG30/1) transcriptome and of the previously published bovine (BP-4; SRA accession: SRX540117) and feline T. foetus (G10/1; SRA accession: SRX540971) transcriptomes available at LabArchives [http://dx.doi.org/10.6070/H4GH9FWD] (Morin-Adeline et al., 2014). To identify similar sequences between the three T. foetus isolates, combinations of megaBLASTS between the assembled transcriptome of one isolate against the newly created database of another and vice versa were carried out with an e-value cut off of 1 × 10−5 , while filtering out low-complexity regions. To increase the stringency of orthologue identification for inclusion in the reciprocal BLAST, megaBLAST results were subsequently filtered to obtained alignments that covered a minimum of 70% of the query sequence. The selected sequence pairs were subjected to a reciprocal best similarity BLAST between pairs of isolates in which the best hits were identified by the lowest e-value. Top hits of the reciprocal BLAST were considered putative orthologues between each pair of T. foetus isolates. Bash scripts on a linux cluster were written to select putative three-way orthologue sequences on the basis of being a putative best-hit in all three isolates. 2.4. Protease and protease inhibitor discovery Mining the pig T. foetus transcriptome for putative proteases was carried out as previously described (Morin-Adeline et al., 2014). Briefly, transcripts that associated with a BLASTX descriptor containing the synonymous terms “protease”, “peptidase” and “proteinase” were selected to create a non-redundant list of PIG30/1 T. foetus proteases. The list was further refined by removing all transcripts containing the BLASTX descriptor term “inhibitor” and these transcripts were used to create a separate list of protease inhibitors. Sequences corresponding to the newly obtained list of protease transcript IDs were obtained from the assembled porcine T. foetus transcriptome and submitted to an online batch BLAST against the MEROPS peptidase database (http://merops.sanger.ac. uk/) (Rawlings et al., 2014). Similarly, the list of protease inhibitors was blasted against the MEROPS protease inhibitor database. A transcript with a positive hit to a protease active site in the MEROPS database was considered a putative protease and used in further analysis. The putative porcine T. foetus protease inhibitors were compared to previously identified protease inhibitors from the transcriptome of the bovine and feline T. foetus (Morin-Adeline et al., 2014) by a 2-sequence BLASTN pairwise nucleotide alignment. Expression counts of putative proteases was carried out initially by mapping raw sequencing reads onto the list of putative proteases using a combination of bowtie (V2.1.0) and tophat (V2.0.8) (Trapnell et al., 2009; Langmead and Salzberg, 2012). Qualimap (V0.7.1) was used to count the number of mapped sequencing reads using the proportional algorithm (Garcia-Alcalde et al., 2012). Counts were normalized as reads per kilobase per million (RPKM) and transcripts producing an RPKM of >500 were considered as highly expressed proteases. A 2-sequence BLASTN pairwise nucleotide alignment (Altschul et al., 1990) between the highly expressed MEROPS-confirmed porcine T. foetus proteases and published bovine T. foetus cysteine protease (TFCP) sequences from ˇ Huang et al. (2013) and Slapeta et al. (2012) was carried out to identify putative transcripts of known CPs. 2.5. Sequence data and data accessibility All left and right paired transcriptome sequence reads has been submitted to the sequence read data (SRA) repository under the BioProject accession: PRJNA279911 [http://www.ncbi.nlm.nih. gov/bioproject/PRJNA279911]. Transcriptome assembly of T. foe-

3

tus (PIG30/1) is available at LabArchives [http://dx.doi.org/10.6070/ H4F18WQ1]. 3. Results 3.1. Bovine isolate (BP-4) and porcine isolate (PIG30/1) belong to the T. foetus ‘bovine genotype’ A transcriptome library consisting of 43,308 transcripts was obtained upon assembly of the 55,506,706 sequencing reads belonging to the PIG30/1 isolate (Table 1). The assembled library has a GC-content of 33.90% with a mean transcript length of 1087 nt (nucleotides) and a minimum and maximum transcript length of 201 nt and 17,203 nt, respectively. Annotation of the assembled library revealed that of the 30,146 transcripts that received a positive BLASTX hit from the NCBI non-redundant database, 25,393 transcripts produced a top hit to Trichomonas vaginalis, while 132 were to T. foetus. With the exception of several new functional categories, annotation at the gene ontology (GO) level revealed highly similar GO annotation and functional category distribution to the bovine (BP-4) and feline (G10/1) isolates of T. foetus (Morin-Adeline et al., 2014) (Fig. 1A). Gene ontology terms relating to cellular component were identical between the porcine, bovine and feline T. foetus transcriptome, while a few categories were present or absent in the molecular function and biological process categories of the porcine isolate. From the molecular function GO category; chromatin binding, ligase activity, nucleoside-triphosphatase regulator activity, enzyme activator activity, phosphatase regulator activity were not observed in the porcine T. foetus transcriptome. Similarly, from the biological process GO category; regulation of biological process, multicellular organism development, cellular development process, response to abiotic stimulus, reproductive process, cellular component biogenesis, cellular process involved in reproduction, cell proliferation, and death were not observed in the porcine T. foetus. Mapping the porcine T. foetus transcriptome to the enzyme codes revealed that hydrolases were the largest represented enzyme class (4281/5,801), followed by the transferase class of enzymes (1214/5,801) and the oxidoreductase class of enzymes (130/5,801) (Fig. 1B). Mapping enzymes to KEGG pathways produced 87 putative metabolic pathways occurring in the porcine T. foetus (Supplementary File 1). Mapping the PIG30/1 transcriptome to the KEGG database confirmed that the porcine T. foetus transcribes the enzymes pyruvate, phosphate dikinase (PPDK) (7 identified homologues) (KEGG accession EC 2.7.9.1) and pyruvate kinase (PK) (4 identified homologues) (KEGG accession EC 2.7.1.40). Reciprocal BLAST between the bovine and porcine isolate, the bovine and feline isolate, and the porcine and feline isolate yielded 22,110, 15,269 and 14,345 putative orthologue pairs, respectively. From these orthologue pairs, 12,279 sequences were identified as shared orthologues between all three T. foetus isolates (Fig. 1C, Supplementary File 2). Both the bovine isolate (BP-4) and porcine isolate (PIG30/1) belonging to the T. foetus ‘bovine genotype’ shared approximately 10,000 orthologous transcripts, while each shared less than 3000 independent transcripts with the feline isolate (G10/1) belonging to the T. foetus ‘feline genotype’ (Fig. 1C). 3.2. Cysteine protease 8 is the highest transcribed protease in both the porcine and bovine isolates of T. foetus From the BLASTX analysis of the whole porcine T. foetus transcriptome, 735 transcripts had the descriptor “protease”, “peptidase” or “proteinase” and 12 transcripts were protease inhibitors. A total of 510 transcripts were confirmed as putative proteases containing 1189 protease active sites from the MEROPS database. Of the inhibitors, 9 of the 13 inhibitor transcripts returned as puta-

Please cite this article in press as: Morin-Adeline, V., et al., Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate ‘bovine genotype’. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.012

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Cellular component

A

6000

Molecular function

Biological process

5,791 5,429

5,335

Number of transcripts

4000

4,994

4,702 4,702 4,418

5000

4,277

3,867 3,682 3,293

3000

3,251

2,635

1,676

1,521 891

1000

2,126

1,767

1,721

2000

1,615

1,507

836 655

4000

241 213

676 337

120

186

PIG

C 4,281

T. foetus porcine isolate PIG30/1

3500 Number of transcripts

226

Single organism cellular process Primary metabolic process Cellular metabolic process Organic sustance metabolic process Cellular response to stimulus Single organism signalling Single organism development process Anatomical structure development Establishment of localisation Single organism metabolic process Cellular component organisation Biosynthetic process Response to stress Macromolecule localisation Cellular localisation Nitrogen compound metabolic process Response to chemical Catabolic process Regulation of biological quality Regulation of molecular function Single organism localisation Single multicellular organism process

Protein binding Heterocyclic compound binding Organic cyclic compound binding Ion binding Transferase activity Hydrolase activity Small molecule binding Carbohydrate derivative binding Single transducer activity Structural consituent of ribosome Oxidoreductase activity Lyase activity Transmembrane transporter activity Co-factor binding Substrate-specific transporter activity Enzyme regulator activity Guanyl-nucleotide exchange factor activity Macromolecular complex binding

Cell part Protein complex Membrane-bounded organelle Organelle part Membrane part Non-membrane bounded organelle Coated membrane Vesicle

4500

512

438

256 322 294 331 276 172 245 280 118

141 105

B

1,429

817 550

0

2,168

2,120

3000

19,132

2500 2000

9,831

2,066

1500 1000

12,279

1,214

17,263

19,224

500

130

75

58

43

0

CAT

T. foetus feline isolate G10/1

2,990

T. foetus bovine isolate BP-4

C OW

Fig. 1. Summary of statistics for the PIG30/1 transcriptome following annotation. A. Annotation of the PIG30/1 transcriptome at the Gene Ontology (GO) level using a minimum filter level of 100 sequences per category. B. Distribution of enzyme classes within the porcine T. foetus transcriptome. C. Venn diagram representing the number of shared orthologues between the porcine T. foetus (PIG30/1) and the bovine (BP-4), and feline (G10/1) T. foetus isolates.

Table 2 Tritrichomonas foetus (PIG30/1) protease inhibitors. Tritrichomonas foetus isolate (host)

Sequence identity

PIG30/1 (pig)

BP-4 (cow)

G10/1 (cat)

G10/1 vs PIG30/1

BP-4 vs PIG30/1

Pig Pig Pig Pig Pig Pig Pig Pig

Cow Cow Cow Cow Cow Cow Cow Cow

Cat Cat Cat Cat Cat Cat Cat Cat

296/300(99%) 329/330 (99%) 311/314 (99%) 936/943 (99%) 1138/1144 (99%) 394/401 (98%) 331/334 (99%) 898/903 (99%)

311/311 (100%) 330/330 (100%) 341/341 (100%) 1161/1162 (99%) 1145/1145 (100%) 1363/1366 (99%) 334/334 (100%) 1991/1993 (99%)

comp11088 c0 se1 comp11281 c0 seq1 comp4073 c0 seq1 comp7519 c0 seq1 comp7519 c0 seq2 comp10095 c0 seq1 comp14337 c0 seq1 comp18269 c0 seq1

comp9915 c0 seq1 comp9941 c0 seq1 comp3242 c0 seq1 comp7451 c0 seq2 comp7451 c0 seq1 comp5569 c0 seq1 comp13520 c0 seq1 comp4109 c0 seq1

comp7405 c0 seq1 comp7804 c0 seq1 comp3687 c0 seq1 comp2876 c0 seq1 comp7790 c0 seq1 comp18864 c0 seq1 comp12847 c0 seq1 comp17054 c0 seq1

Note: Each row represents successful pairwise alignments of the porcine T. foetus transcript with a feline and bovine protease inhibitor transcript from Morin-Adeline et al. (2014), transcriptome assembly of G10/1 and BP-4 available at LabArchives [http://dx.doi.org/10.6070/H4GH9FWD]. Transcriptome assembly of T. foetus (PIG30/1) is available at LabArchives [http://dx.doi.org/10.6070/H4F18WQ1].

Please cite this article in press as: Morin-Adeline, V., et al., Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate ‘bovine genotype’. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.012

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V. Morin-Adeline et al. / Veterinary Parasitology xxx (2015) xxx–xxx Table 3 Frequency of transcripts with protease active sites present in the Tritrichomonas foetus (PIG30/1) transcriptome. Active site functional type

Cysteine Serine Glutamic Metallo Threonine Aspartic Inhibitors Asparagine Mixed Unknown Overall

Number of protease active sites Entire transcriptomea

Highly expressed proteasesb

763 189 0 151 30 16 40 0 0 0 1189

73 38 0 50 21 4 33 0 0 0 219

a Entire transcriptome: Number of each type of protease active sites detected from all MEROPS confirmed proteases (510 proteases) found in the porcine T. foetus transcriptome. b Highly expressed proteases: Number of each type of protease active sites detected from a subset of highly expressed proteases (RPKM > 500) (158 proteases) when all raw reads were mapped onto MEROPS-confirmed proteases in the porcine T. foetus transcriptome.

tive protease inhibitors (Supplementary File 3). Pairwise sequence comparisons between the porcine T. foetus protease inhibitors and the feline and bovine T. foetus protease inhibitors revealed that 8 of the MEROPS-confirmed protease inhibitors successfully produce alignments between all three isolates (Table 2). The porcine T. foetus protease inhibitors aligned with higher identities to the bovine T. foetus protease inhibitors (5/8; 100% identity) than to the feline T. foetus protease inhibitors (0/8; 100% identity). From the putative proteases, the cysteine active site obtained the majority of hits (763/1,189), followed by the serine active site (189/1,189), while no glutamic active sites were detected (Table 3). Expression counts of the proteases revealed that 158 of the MEROPS confirmed proteases were highly expressed with an RPKM of 500 or greater (Supplementary File 3). Of the 158 highly expressed putative proteases, 219 unique active sites were found, 33% of which were cysteine active sites (Table 3). Sequence alignment of porcine T. foetus proteases to 21 published bovine proteases revealed that 13 transcripts matched with identities above 98% to a published TFCP (data not shown). The highest expressed protease in the porcine T. foetus transcriptome corresponds to the known sequence of bovine T. foetus CP8 with an RPKM value of 237,918 followed by CP13 with an RPKM value of 60,001 (Supplementary File 3). The bovine or feline T. foetus TFCP1 was not found in the porcine T. foetus transcriptome, even when all reads were mapped with no mismatches onto the published CP1 sequences. Absence of TFCP1 transcripts agrees with our inability to PCR amplify TFCP1 from the T. foetus PIG30/1 isolate genomic DNA (Mueller et al., 2015). 4. Discussion The transcriptome library generated for PIG30/1 represents the first porcine T. foetus transcriptome and complements the published bovine (BP-4) and feline (G10/1) T. foetus RNA-seq data (Morin-Adeline et al., 2014). The cell-wide transcriptomic analysis confirms the closer similarity of the porcine isolate (PIG30/1) to the ‘bovine genotype’ T. foetus (PB-4) compared to the ‘feline genotype’ T. foetus (G10/1). Since the three-way reciprocal best hit (RBH) BLAST results were filtered to transcripts with at least 70% identity between the three T. foetus isolates in the current analysis, the 5-fold greater number of shared transcripts between the bovine and porcine T. foetus confirms the closeness of these two isolates compared with the feline T. foetus isolate.

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The ability of T. foetus to adapt to the varied nutrient environment of its niche within the three hosts (cow, pig, cat) hints at the highly versatile metabolism this parasite possesses. In trichomonads, energy in the form of ATP is produced by fermentative glycolysis that occurs in the cytoplasm, as well as phosphatelevel phosphorylation that occurs in the mitochondria-derived organelle, the hydrogenosome (Cerkasov et al., 1978; Lindmark et al., 1989). The end product of glycolysis is pyruvate which is the substrate for hydrogenosomal metabolism. Hydrogenosomes lack the necessary components for high energy yield by the Kreb’s cycle (Cerkasov et al., 1978; Lindmark et al., 1989), therefore, anaerobic protozoa must have efficient ATP yield from cytoplasmic fermentative metabolism. One way of achieving this is by the replacement of ATP-dependent glycolytic enzymes with pyrophosphate-dependent (PPi) enzymes as phosphate donors (Reeves et al., 1974; Mertens et al., 1989; Hrdy et al., 1993). For instance, the ubiquitous ATP-dependent pyruvate kinase (PK), an important enzyme responsible for catalysing the production of pyruvate from phosphoenoipyruvate (PEP), can also exist as the PPi phosphate dikinase (PPDK) (Mertens, 1993). Although PPKD catalyses the same reaction as PK, it offers biochemical versatility by permitting the reverse reaction to occur for the use of alternative metabolic pathways depending on nutrient availability (Mertens, 1993). In addition, a 1.5-fold increase in fermentative ATP yield is attained directly through the use of PPi-dependent glycolytic enzyme compared to the ATP-dependent enzyme (Mertens, 1993). Curiously in T. foetus, enzymatic activity of PK or PPDK has not been observed to date (Mertens et al., 1989; Muller, 1992; Hrdy et al., 1993; Slamovits and Keeling, 2006). Mapping the T. foetus PIG30/1 transcriptome with KEGG metabolic enzymes have revealed evidence of homologues to both PK and PPDK in T. foetus. The presence of both versions of this enzyme in anaerobic protozoa is not uncommon. In Entamoeba histolytica, only the activity of PPDK was originally detected and it was assumed to have replaced PK (Reeves et al., 1974). Later, identification of a PK homologue in the genome of E. histolytica led to work that confirmed PK activity similar in magnitude to PPDK activity in this parasite (Saavedra et al., 2004). In the human T. vaginalis, a similar situation exists where only the activity of PK has been detected to date, but genomic research demonstrated evidence of PPDK as well (Mertens et al., 1989; Carlton et al., 2007). PPi-dependent enzymes have previously been suggested as a drug target in protozoans as they are absent in mammalian eukaryotes (Verlinde et al., 2001). Possession of both PK and PPDK suggests that this strategy in T. foetus is not ideal as the parasite has the capacity to utilize PK as a substitute for PPDK. Furthermore, finding transcripts that correspond to both PK and PPDK not only close the gap in knowledge of T. foetus glycolytic metabolism, but implies that alternation between the two enzymes depending on the nutrient environment may facilitate the parasites survival within its three hosts. Experimental studies have shown that porcine T. foetus produced infections in cattle which more closely resembled the clinical symptoms of a natural case of bovine trichomonosis compared to cattle infected with feline isolate (Fitzgerald et al., 1958; Stockdale et al., 2007). This finding may be attributed to the differences in the pathogenesis-related genes of the two T. foetus genotypes ˇ et al., such as cysteine proteases (TFCP) (Lucas et al., 2008; Slapeta 2012). Cysteine proteases (CP) are hydrolases that are released into the host milieu and irreversibly cleave host proteinaceous substrates and defence peptides (Bastida-Corcuera et al., 2000; Sajid and McKerrow, 2002; Singh et al., 2004). Testament to their significance, hydrolases are the most abundant class of enzymes in the porcine T. foetus. The current porcine T. foetus (PIG30/1) has a CP repertoire as extensive as previously observed in the bovine and feline T. foetus transcriptomes (Morin-Adeline et al., 2014). As the porcine T. foetus resides within the dynamic, mucus-covered lin-

Please cite this article in press as: Morin-Adeline, V., et al., Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate ‘bovine genotype’. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.012

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ing of the digestive and upper respiratory tract of their host, the vast number of highly expressed CPs is likely necessary to maintain presence in their host. Identical to the bovine T. foetus, TFCP8 is the highest transcribed protease in the porcine T. foetus. This is unlike the ‘feline genotype’, in which TFCP7 was the most highly transcribed CP (Morin-Adeline et al., 2014). Differential expression of CPs between isolates of the same species of anaerobic flagellate phenotypically distinguishes the strain as high or low virulence (Biller et al., 2009; De Jesus et al., 2009). In the bovine T. foetus isolates, TFCP8 is considered a critical virulence factor associated with inducing host-specific cytopathic effects on bovine vaginal epithelial cells in vitro (Singh et al., 2004). This likely explains the pathogenic potential of the porcine T. foetus observed in cattle hosts. Four porcine T. foetus strains were previously genotyped at ˇ eight CP genes, malate dehydrogenase and ITS rDNA (Slapeta et al., 2012). The strain 1/N (ATCC 30,167), 11/S (ATCC 30,168), C19F (ATCC 30,169) and SUI-H3B were genotyped and matched 100% the bovine T. foetus isolates, except for SUI-H3B, which exhibited a single derived substitution in T. foetus cysteine protease 9 (TFCP9). All four strains (1/N, 11/S, C19F, SUI-H3B) were successfully genoˇ et al., 2012), though in PIG30/1, typed at the TFCP1 locus (Slapeta CP1 could not be detected by PCR or RNA-seq (Mueller et al., 2015; current study). In the bovine T. foetus transcriptome (BP-4), TFCP1 was identified in a sub-set of highly transcribed proteases, while in the feline T. foetus (G10/1), TFCP1 was identified just outside of the stringent established high transcription threshold (Morin-Adeline et al., 2014). In other pathogenic excavates such as T. vaginalis and Entamoeba sp., reduced expression of certain CPs is observed in strains phenotypically described with lower cytotoxicity (Biller et al., 2009; De Jesus et al., 2009). Even in these cases, CP1 expression is only decreased and not, as in the apparent case of the porcine T. foetus, completely absent as a putative phenotypic characteristic. This suggests that the porcine T. foetus isolates (n = 5) exhibit a level of diversity not previously found in genotyped bovine (n = 8) or feline (n = 7) T. foetus isolates which were all identical at all loci studied. While it may be the case that TFCP8 is central to the pathogenesis of T. foetus infections to bovine host cells, the diversity found at some CPs in the porcine isolates suggests that research focus should be directed to the interaction between TFCP8/TFCP7 and other CPs in T. foetus to explain the virulence in cattle and cats. 5. Conclusion The T. foetus PIG30/1 transcriptome sequenced and assembled is the first for a T. foetus isolated from a porcine host. Mining the transcriptome reveals that T. foetus transcribes the metabolic enzymes, pyruvate kinase and PPi phosphate dikinase which were previously not identified in this parasite species. Comparative analysis of the assembled PIG30/1 transcriptome with the published transcriptomes of the bovine (BP-4) and feline (G10/1) isolates show that the porcine transcriptome is more similar in size to the bovine T. foetus rather than the feline T. foetus. The PIG30/1 T. foetus shares approximately 5-fold greater number of orthologous protein-coding genes with the bovine T. foetus isolate than with the feline T. foetus isolate. The similarity between the porcine (PIG30/1) and bovine (BP-4) transcriptome extends to the CP virulence factors, where CP8 is the most highly transcribed CP of the ‘bovine genotype’, compared to CP7 for the ‘feline genotype’. The porcine T. foetus isolate (PIG30/1) groups with the ‘bovine genotype’ T. foetus rather than the ‘feline genotype’ T. foetus. Acknowledgements VMA is supported by the International Postgraduate Research Scholarship (IPRS) and an Australian Postgraduate Award (APA)

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Please cite this article in press as: Morin-Adeline, V., et al., Comparative RNA-seq analysis of the Tritrichomonas foetus PIG30/1 isolate from pigs reveals close association with Tritrichomonas foetus BP-4 isolate ‘bovine genotype’. Vet. Parasitol. (2015), http://dx.doi.org/10.1016/j.vetpar.2015.08.012