The diversity and expansion of the trans-sialidase gene family is a common feature in Trypanosoma cruzi clade members

Infection, Genetics and Evolution 37 (2016) 266–274

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The diversity and expansion of the trans-sialidase gene family is a common feature in Trypanosoma cruzi clade members Miguel Angel Chiurillo a,⁎, Danielle R. Cortez b, Fábio M. Lima b, Caroline Cortez b, José Luis Ramírez c, Andre G. Martins d, Myrna G. Serrano e, Marta M.G. Teixeira d, José Franco da Silveira b a

Laboratorio de Genética Molecular “Dr. Yunis-Turbay”, Decanato de Ciencias de la Salud, Universidad Centroccidental Lisandro Alvarado, Barquisimeto 3001, Estado Lara, Venezuela Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP 04023-062, Brazil Centro de Biotecnología, Fundación Instituto de Estudios Avanzados, Caracas, Venezuela d Departamento de Parasitologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil e Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA, USA b c

a r t i c l e

i n f o

Article history: Received 9 September 2015 Received in revised form 21 November 2015 Accepted 23 November 2015 Available online 02 December 2015 Keywords: Trypanosoma cruzi clade Trans-sialidase Gene superfamily Conserved motif Phylogeny DTUs

a b s t r a c t Trans-sialidase (TS) is a polymorphic protein superfamily described in members of the protozoan genus Trypanosoma. Of the eight TS groups recently described, TS group I proteins (some of which have catalytic activity) are present in the distantly related Trypanosoma brucei and Trypanosoma cruzi phylogenetic clades, whereas other TS groups have only been described in some species belonging to the T. cruzi clade. In the present study we analyzed the repertoire, distribution and phylogenetic relationships of TS genes among species of the T. cruzi clade based on sequence similarity, multiple sequence alignment and tree-reconstruction approaches using TS sequences obtained with the aid of PCR-based strategies or retrieved from genome databases. We included the following representative isolates of the T. cruzi clade from South America: T. cruzi, T. cruzi Tcbat, Trypanosoma cruzi marinkellei, Trypanosoma dionisii, Trypanosoma rangeli and Trypanosoma conorhini. The cloned sequences encoded conserved TS protein motifs Asp-box and VTVxNVxLYNR but lacked the FRIP motif (conserved in TS group I). The T. conorhini sequences were the most divergent. The hybridization patterns of TS probes with chromosomal bands confirmed the abundance of these sequences in species in the T. cruzi clade. Divergence and relationship analysis placed most of the TS sequences in the groups defined in T. cruzi. Further examination of members of TS group II, which includes T. cruzi surface glycoproteins implicated in host cell attachment and invasion, showed that sequences of T. cruzi Tcbat grouped with those of T. cruzi genotype TcI. Our analysis indicates that different members of the T. cruzi clade, with different vertebrate hosts, vectors and pathogenicity, share the extensive expansion and sequence diversification of the TS gene family. Altogether, our results are congruent with the evolutionary history of the T. cruzi clade and represent a contribution to the understanding of the molecular evolution and role of TS proteins in trypanosomes. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Trypanosomes are obligate parasitic protozoa found worldwide in all vertebrate classes (Hamilton et al., 2007; Hoare, 1972). An extensive phylogenetic analysis of trypanosomes and biogeographical and geological evidence placed the human pathogens Trypanosoma brucei and Trypanosoma cruzi unequivocally in two early diverging clades (Stevens et al., 1999a). The T. cruzi clade is a major assemblage formed by species of the subgenus Schizotrypanum, consisting of T. cruzi and its allied trypanosomes restricted to bats, T. cruzi marinkellei, Trypanosoma dionisii and Trypanosoma erneyi (Cavazzana et al., 2010; Lima et al., 2012a), together with its sister clade Trypanosoma rangeli/ ⁎ Corresponding author at: Departamento de Patologia Clínica, Universidade Estadual de Campinas, Campinas, 13083, São Paulo, Brazil. E-mail address: [email protected] (M.A. Chiurillo).

http://dx.doi.org/10.1016/j.meegid.2015.11.024 1567-1348/© 2015 Elsevier B.V. All rights reserved.

Trypanosoma conorhini and a group of trypanosomes from Australian mammals (Hamilton et al., 2007, 2012a; Stevens et al., 1999a, 1999b). It has been hypothesized that the T. cruzi clade was originated from ancestral bat trypanosomes that evolved exclusively in bats or switched into other hosts, giving rise to species that infect terrestrial mammals in the Old and New World (Hamilton et al., 2012b). T. cruzi, the etiological agent of Chagas disease, comprises a complex of genetically heterogeneous isolates distributed into six intraspecific subdivisions known as Discrete Typing Units (DTUs) TcI to TcVI (Zingales et al., 2012), to which a seventh DTU (T. cruzi Tcbat) identified in bats has recently been added (Lima et al., 2015; Marcili et al., 2009; Pinto et al., 2012; Ramírez et al., 2014a). Similarly, T. rangeli, which is not pathogenic to mammalian hosts, is also composed of phylogenetic lineages known as T. rangeli A to E (Maia da Silva et al., 2007, 2008). Schizotrypanum species have the unique feature of developing as amastigotes and differentiating into trypomastigotes inside mammalian

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cells. T. cruzi and T. rangeli are generalist species capable of infecting virtually all terrestrial mammalian orders, while other species and sub-species of the subgenus Schizotrypanum infect only bats, and T. conorhini is a tropicopolitan parasite of Rattus species. All these species are transmitted by triatomine bugs, except T. dionisii and other bat trypanosomes from Europe and Africa, which are transmitted by cimicids, bugs closely related to triatomines (Cavazzana et al., 2010; Hamilton et al., 2012a, 2012b; Hoare, 1972; Lima et al., 2012a). Trans-sialidase/trans-sialidase-like (TS) proteins constitute a large polymorphic superfamily that has been identified in several species of the genus Trypanosoma. TS is the largest T. cruzi gene family, comprising more than 1400 genes, half of which are apparently functional (ElSayed et al., 2005). Recently, cluster analysis of T. cruzi (clone CL Brener) TS sequences (Freitas et al., 2011) added four groups to the four previously identified (Colli, 1993; Cross and Takle, 1993; Frasch, 2000), demonstrating that this family is even more heterogeneous than previously thought. The first group includes active trans-sialidases and sialidases found in T. cruzi and T. rangeli, respectively (Buschiazzo et al., 1997; Oliveira et al., 2014). TS group II includes a set of heterogeneous GPI-anchored surface glycoproteins that are expressed in T. cruzi trypomastigote and intracellular amastigote stages (Correa et al., 2013; Mattos et al., 2014) and have been implicated in adhesion and/or internalization of T. cruzi into host cells (Colli, 1993; Schenkman et al., 1994; Yoshida, 2006; Alves and Colli, 2008; Mattos et al., 2014). TS genes from group II have also been identified in T. rangeli (Añez-Rojas et al., 2005; Grisard et al., 2010; Peña et al., 2009; Stoco et al., 2014), while conversely, T. brucei and phylogenetically related species have TS group I genes only. The identification of TS genes and proteins in several trypanosomes raises the question as to whether the TS repertoires evolved differently in the various species of the genus. Hence, comparative analysis of T. cruzi and related trypanosomes, whether they express TS activity or not, can be valuable to clarify the role played by proteins encoded by this multigene family. Therefore, our aim was to investigate whether the expansion and diversity of the TS gene family is a common characteristic of trypanosome genomes, with special emphasis on TS group II genes, to finally provide new insights into the evolutionary histories of the TS multigene family and the Trypanosoma group. Here, we characterized TS genes from phylogenetically related trypanosome species, including those of the T. cruzi clade, with important differences in their life cycles and infective/pathogenic capacities.

2. Methods 2.1. Cloning of TS sequences from different species of the T. cruzi clade The characteristics of the trypanosome isolates used in this work are shown in Table 1. Genomic DNAs from T. cruzi Tcbat, T. c. marinkellei, T. dionisii, T. rangeli saimiri isolate (from squirrel monkey), T. conorhini, T. brucei, Trypanosoma evansi and Trypanosoma vivax were used in PCR reactions with degenerate primers based on the sequences of the signal peptide (forward: ASRKGCCCAACATGTCCCGG) and VTVxNVxLYNR (reverse: CAGYGGGCGGTTGTACARAAAGAC) motifs. Primers were designed using conserved sequences from TS groups of T. cruzi and T. rangeli to exclude amplification of sequences belonging to TS group I, which are present in both T. cruzi and T. brucei clades. PCR products ranging from 1.8 to 2.2 kb were then cloned into pGEM-T Easy vector (Promega), and one to two clones from each species were chosen for further analysis. To obtain TS probes, we designed primers to amplify an internal region (~680 bp) of TS group II members (TSA_forward: TTGTGATGGRGRATGGCAC and TSA_reverse: TCCAACCA GACCRGCCGTGGG). The following DNA fragments used as probes were also cloned and sequenced: TcTS (T. cruzi), TcmTS (T. c. marinkellei), Tconorh_35/TcoTS (T. conorhini), Tdio_45/TdTS (T. dionisii) and TrTS (T. rangeli).

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Table 1 TS sequences of different isolates of T. cruzi clade determined in this study and used in phylogenetic analysis. Isolate

T. cruzi Tcbat T. c. marinkellei

T. dionisii T. rangeli (saimiri) T. conorhini

TS ID

GenBank accession number

Size (bp)

1923 2037 1944 1953

AA sequence identity with TS members deposited in GenBank %

TS Trypanosomea group

52–56 62–81 52–78 53–83

II II II II

Tcbat_22 Tcbat_23 Tcmar_14 Tcmar_16

KF650717 KF650718 KF650719 KF650720

Tdio_35 Tdio_66 Tsai_4

KF650724 680 54–59 II KF650725 1977 47–51 II KF650726 1914 62–74 II

Tconorh_8 KF650721 2205 36–45 I Tconorh_14 KF650722 1992 62–74 V Tconorh_45 KF650723 674 48–61 II

T. cruzi T. cruzi T. cruzi T. c. marinkellei T. cruzi T. cruzi T. rangeli T. rangeli T. cruzi T. rangeli

TS: Trans-sialidase; AA: amino acids; bp: base pairs. TCC: Trypanosomatid Culture Collection of the University of São Paulo, SP, Brazil. a Origin of homologous sequence with best match.

2.2. Separation of trypanosome chromosomal DNA by pulsed-field gel electrophoresis Chromosomal-sized DNA from epimastigotes of T. cruzi CL Brener, T. rangeli (Choachi strain), T. c. marinkellei, T. dionisii and T. conorhini was separated by PFGE as previously described (Cano et al., 1995). The membranes were hybridized with 32P-labeled TS probes overnight, washed at high stringency after hybridization (Souza et al., 2011) and exposed to X-ray film.

2.3. DNA sequence and phylogenetic analysis Nucleotide sequence assembly, translation into amino acids and primer design were performed using DNAMAN v. 5.2.2 software (Lynnon BioSoft). BLAST algorithms were used to search for homologous nucleic acid or protein sequences in GenBank and TriTryp databases at http://www.ncbi.nlm.nih.gov and http://tritrypdb.org, respectively. The signal peptide sequence was predicted using SignalP 4.1 Server software (http://www.cbs.dtu.dk/services/SignalP/). The GenBank/EMBL/DDBJ accession numbers for the sequences from T. cruzi Tcbat, T. c. marinkellei, T. dionisii, T. rangeli (saimiri) and T. conorhini identified in this study are KF650717 to KF650726 (Table 1). These sequences were aligned with those retrieved by BLAST searches against draft and annotated genomes of trypanosomatids freely available in the TriTrypDB and NCBI databases (Table 2). We also searched for TS genes in genomes generated as part of the ongoing ATOL (Assembling the Tree of Life, NSF-USA) and TCC-USP (Trypanosomatid Culture Collection of the University of São Paulo, SP, Brazil) programs, as well as in genomes of T. cruzi strains sequenced by the Kinetoplastid Genome Sequencing and Analysis Consortium NIH/ NHGRI/NIAID (Table 2). Multiple nucleotide and amino acid alignments were carried out using ClustalW and then manually adjusted. Phylogenetic trees were constructed with MEGA 5.2 software. TS phylogeny was inferred from protein sequence alignments using the maximum likelihood (ML) method and Jones–Taylor–Thornton substitution model and assuming a 4 gamma-distributed rate heterogeneity from the data. Robustness was evaluated with 100 bootstrap replicates, and the log-likelihood ratio test was used to assess the accuracy of each branch. A neighborjoining (NJ) tree with nucleotide sequences from TS group II was constructed using the bootstrap method with 1000 replicates and the Kimura 2-parameter model.

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Table 2 Trypanosome species and isolates with their respective TS sequences obtained in this study or retrieved from data banks. Species/subespecies Trypanosome strain/isolate

Host species

Geographic Accession number origin

Origin of sequences

EKG01323; TCSYLVIO_005401; TCSYLVIO_007741 KB222878; KB223076 ESS55615; ESS55626 EF154827; AF426132; AAA21303; ABR19835 X57235 X70948; ABO28970; AF128843 XM_799078; XM_800086; XM_800788; XM_802406; XM_802711; XM_803072; XM_803086; XM_803518; XM_803864; XM_805296; XM_805583; XM_806976; XM_807627; XM_808522; XM_808523; XM_809532; XM_810613; XM_812072; XM_811278; XM_811657; XM_814626; XP_803096; TcCLB.510643.100 AN0X01004973; KB205211 KB851306; KB851307; KB851998; KB8512232 contig_4654; contig_5033; contig_5693 Scaffold_231; Scaffold_317; Scaffold_630 D50686 KF650717; KF650718 T1994_5_c00300; T1994_5_c00421; T1994_5_c00452; T1994_5_c00448; T1994_5_c00900; T1994_5_c00911; T1994_5_c00961; T1994_5_c01076; T1994_5_c01157; T1994_5_c01159; T1994_5_c01229; T1994_5_c01266; T1994_5_c01472; T1994_5_c01906; T1994_5_c02006; T1994_5_c02246; T1994_5_c02338

GenBank; TriTrypDB TriTrypDB GenBank GenBank GenBank

PCR Genome draft (ATOL) GenBank

T. cruzi Sylvio X10.6 JR cl4 Dm28c G/TCC30 CA-I CL CL Brener

Human Human Opossum Opossum Human Triatomine Triatomine

H. sapiens H. sapiens D. marsupialis D. marsupialis H. sapiens T. infestans T. infestans

Brazil Venezuela Venezuela Brazil Argentina Brazil Brazil

Esmeraldo cl3 Tula cl2 M6241 Can III Y/TCC34 TcBat/TCC793 TcBat/1994

Human Human Human Human Human Bat Bat

H. sapiens H. sapiens H. sapiens H. sapiens H. sapiens M. levis M. levis

Brazil Chile Brazil Brazil Brazil Brazil Brazil

TCC426

Bat

L. silvicolum

Brazil

KF650719; KF650720

B7

Bat

P. discolor

Brazil

EKF26571; EKF28504; EKF29441; EKF29466; EKF29577; EKF29623; EKF29815; EKF30763; EKF31219; EKF32191; EKF33447; EKF38277; EKF38900; EKF38904

TCC211

Bat

E. brasiliensis

Brazil

KF650724; KF650725 Tdio_scaffold00279; Tdio_scaffold00341; Tdio_scaffold00345; Tdio_scaffold05154

PCR Genome draft (ATOL)

LDG DOG82 Choachi

Human Dog Triatomine

H. sapiens C. l. familiaris R. prolixus

Colombia Venezuela Colombia

GenBank GenBank GenBank

SC58 Rodent saimiri/TCC012 Monkey

P. dasythrix S. sciureus

Brazil Brazil

L14943; U83180 FJ404802; FJ404803; AF426022 KC54907; KC54922; KC54951; KC54953; KC544946; KC544956 ESL05185; ESL05199; ESL05472 KF650726

TCC025

Rodent

R. rattus

Brazil

KF650721; KF650722; KF650723 Tco_7.2_s595; Tco_7.2_s1621; Tco_7.2_s204; Tco_7.2_s2418; Tco_7.2_s2835; Tco_7.2_s3012; Tco_7.2_s3269; Tco_7.2_s754;

PCR Genome draft (ATOL)

TREU927 EATRO 427

Tsetse fly Sheep

Glossina sp

Kenya Uganda

XM_842470 AF310232

GenBank GenBank

DAL972

Human

H. sapiens

Ivory Coast

FN554970

GenBank

STIB 805

Buffalo

B. bubalis

China

TevSTIB805.7.7540

TriTrypDB

Y486

Cattle

B. primigenius

Nigeria

CCD21087; CCD19737

GenBank

IL3000

cattle

B. primigenius

Kenya

CCD12514; CCC91739

GenBank

AY142111

GenBank

XM_009317479

GenBank

GenBank; TriTrypDB

TriTrypDB GenBank Genome draft (WU) Genome draft (WU) TriTrypDB PCR Genome draft (USP)

T. cruzi marinkellei

T. dionisii

T. rangeli

GenBank PCR

T. conorhini

T. b. brucei

T. b. gambiensi T. evansi T. vivax T. congolense T. carassii cyprinid fish T. grayi ANR4

tsetse fly

G. palpalis gambiensis Gambia

TCC: Trypanosomatid Culture Collection of the University of São Paulo, SP, Brazil. TritrypDB (http://tritrypdb.org). WU: Washington University (USA) — Kinetoplastid Genome Sequencing and Analysis Consortium (NIH/NHGRI/NIAID). ATOL: Assembling the Tree of Life (NSF-USA); USP: Department of Parasitology, University of São Paulo, USP. PCR: Polymerase Chain Reaction, sequences obtained in this work with GenBank accession number.

3. Results 3.1. Cloning and analysis of TS genes from different trypanosome species Functional members of the TS superfamily have the signature motif VTVxNVxLYNR (VTV) and the signal sequence for cleavage/ addition of the GPI anchor at the C-terminal region. VTV is the

most conserved TS motif and is considered to be associated with virulence (Alves and Colli, 2008; Tonelli et al., 2010, 2011). Two other typical motifs, ASP box (SxDxGxTW) and FRIP (xRxP), can be found in many groups in the TS family, whereas C-terminal repeats are present in only a few. Members of TS group II usually lack the FRIP motif at the N-terminal end (Frasch, 2000; Freitas et al., 2011).

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By using a set of primers based on conserved TS sequences (the signal peptide and VTV motifs), we were able to amplify homologous genes in the following different species of the T. cruzi clade: T. cruzi Tcbat, T. c. marinkellei, T. dionisii, T. conorhini and T. rangeli (saimiri) (Table 1). The primers were designed to amplify TS variants except those belonging to group I, which are probably present in all species of the genus Trypanosoma. Since no stop codons interrupting putative open reading frames were found, the sequences obtained appear to correspond to whole genes. No amplification was observed when genomic DNAs from T. brucei, T. evansi and T. vivax were used in the PCR reactions. Sequences amplified with primers TSA_F/R from T. conorhini (Tconorh_45) and T. dionisii (Tdio_35) were also included in the phylogenetic analysis (Table 1). Inspection of the multiple sequence alignments of the TS genes, including T. cruzi and T. rangeli reference sequences, showed a number of features that are shared with members of the TS superfamily, such as the presence of a signal peptide sequence in the region coding for the N-terminus of the protein, two conserved copies of the bacterial neuraminidase motif SxDxGxTW, one copy of the subterminal motif VTVxNVxLYNR and the absence of the FRIP motif (Fig.1 and Supplementary Fig. 1). Sequences from T. c. marinkellei, T. cruzi Tcbat, T. dionisii and T. conorh_45 from T. conorhini showed 48–80% identity at amino acid level with T. cruzi TS group II sequences deposited in genome databases, whereas amino acid sequences deduced for T. rangeli (saimiri) Tsai_4 shared higher identity (~74%) with T. rangeli (DOG82 strain) TS group II sequences. T. conorh_14 was more closely related to sequences of the T. cruzi TS group V (62–74%), while T. conorh_8 proved to be the most divergent sequence and in the BLAST analysis showed similarity mainly with members of TS group I from T. rangeli, T. c. marinkellei and T. cruzi (36–45%). 3.2. Genomic organization of TS genes across trypanosome species Since the genomes of several species used in this study were not fully sequenced, the hybridization of chromosomal bands separated by PFGE with specific probes gave us useful information on the distribution and relative copy number variation of TS genes across the species of the T. cruzi clade. We used probes derived from each organism included in these analyses (T. cruzi CL Brener, TcTS; T. c. marinkellei, TcmTS; T. rangeli Choachi strain, TrTS; T. dionisii, TdTS and T. conorhini, TcoTS) to avoid lack of detection or weak signal when hybridized with the respective organism (Supplementary Fig. 2). Nucleotide sequence of TS fragments used as probes has an overall identity of 65% among them, and each one of them exhibited ≥90% identity with TS sequences of the organism from which they were derived. Under high-stringency conditions TS probes recognized many chromosomal bands, mainly when homologous probes for each species were used, confirming the widespread presence of these sequences in the genome of species and

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subspecies of the T. cruzi clade (Supplementary Fig. 2). However, the specificity of the signal resulting from hybridization of the chromosomal DNA to each heterologous probe varied considerably, which was in general consistent with the identity observed at the nucleotide level. Moreover, differences in intensity and distribution of the hybridization signals indicate a variation in the copy number and distribution of TS genes among members of the T. cruzi clade. The probe derived from the T. cruzi TS gene (TcTS) recognized several chromosomal bands in all the trypanosomes included in this analysis, although the hybridization signal was much more intense and widely distributed in T. cruzi and T. c. marinkellei (Supplementary Fig. 2). TS probes from T. c. marinkellei (TcmTS) and T. dionisii (TdTS) hybridized with many chromosomal bands of T. cruzi, T. c. marinkellei and T. dionisii, but little or no hybridization was detected for T. rangeli and T. conorhini. The opposite result was observed with T. conorhini (TcoTS) and T. rangeli (TrTS) TS probes, which revealed some strong signals with chromosomal bands of these species but weak signals with bands from other trypanosomes. We also studied the presence and distribution of gp82, gp85 and gp90 genes in chromosomal bands separated by PFGE in species of the T. cruzi clade. These genes are well known members of the TS group II of T. cruzi, encoding proteins involved in the invasion of mammalian cells by trypomastigotes (Correa et al., 2013; Málaga and Yoshida, 2001; Ramirez et al., 1993). When the entire ORF of representative members of gp82, gp90 and gp85 genes were used as probes (GenBank accession numbers L14824, AF426132 and AF085686.1, respectively) we observed hybridization with most of the chromosomal bands of T. cruzi clone CL Brener and T. c. marinkellei (Supplementary Fig. 3) indicating that these TS genes are spread throughout the genome of these parasites, but they are apparently absent in T. dionisii, T. rangeli and T. conorhini. We also identified orthologs of T. cruzi gp82 and gp90 genes in T. cruzi Tcbat and T. c. marinkellei genomes, and alignment of predicted amino acid sequences indicated an overall percentage identity of 63% and 73%, respectively (Supplementary Figs. 4 and 5). These results suggest that the TS superfamily participates in host–parasite interactions in trypanosomes exhibiting different life cycles, vertebrate host species and vectors from those of T. cruzi.

3.3. Phylogenetic analysis The sequences used in the phylogenetic analysis are listed in Table 2. For the analysis we mainly used full-length sequences, however; in order to increase the representation of TS sequences of some species, we also included a few fragments or partial genes that display conserved TS motifs, exhibit high identity and overlap with homologous sequences. Although phylogenetic trees could be affected by having partial sequences included in the alignment, we consider that the overall

Fig. 1. Modular architecture of the TS gene family in trypanosomes of the T. cruzi clade. Schematic illustration of conserved motifs in the TS superfamily. Predicted amino acid sequences for the signal peptide, Asp-box and VTV (VTVxNVxLYNR) terminal motif from sequences obtained in this work and from representative members of TS group II are shown. The signal peptide and VTV motifs are not shown in Tdio_35 and Tconorh_45 sequences because these domains are located outside the cloned region with the PCR strategy used here. GPI anchor sequences are not shown because this region was not used in the cloning procedure. The top panel shows conserved motifs found in the TS superfamily. GenBank accession numbers of sequences from T. cruzi and T. rangeli used as references are shown. Graphical representations are not to scale.

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topology is informative and also it is still possible to get the evolutionary distance between sequences. Interestingly, the reconstructed phylogeny revealed that the variability among the sequences appears to be consistent with the grouping of T. cruzi TS genes into eight groups proposed by Freitas et al. (2011). PCR sequences from T. cruzi Tcbat, T. c. marinkellei, T. dionisii and T. rangeli (saimiri) fell into TS group II. Sequences retrieved from T. c. marinkellei and T. cruzi Tcbat genomes were assigned to eight and seven TS groups previously described in T. cruzi, respectively (Fig. 2). T. rangeli sequences available from genome databases and the ongoing T. dionisii and T. conorhini genome projects also showed diversification in this gene family. The most divergent sequences were from T. conorhini, which were assigned to TS groups II and V, and one of them did not fall into any group despite having conserved TS motifs in the predicted amino acid sequences. We were able to identify sequences that clustered in TS group I in all the trypanosome genomes analyzed, and these clearly separated into

two groups corresponding to the T. cruzi and T. brucei clades in the phylogenetic tree. Similarly, TS group II sequences segregated into two subclusters, with sequences of T. cruzi, T. cruzi Tcbat, T. c. marinkellei and T. dionisii in one and of T. rangeli and T. conorhini in the other (Fig. 2). It is noteworthy that the TS group I sequences of the African crocodilian trypanosome Trypanosoma grayi clustered close to T. cruzi sequences. Although phylogenomic approaches with the draft genome of T. grayi suggested that this trypanosome is more closely related to T. cruzi than to T. brucei (Kelly et al., 2014), a phylogenetic analysis of trypanosomes including two species very closely related to South American crocodilians (Fermino et al., 2013) supported the distant phylogenetic position of T. grayi in relation to clades T. cruzi and T. brucei. The highly divergent TS sequence of T. carassii, a freshwater-fish trypanosome (Hamilton et al., 2007), also clustered in TS group I close to T. grayi. To further characterize the TS group II sequences, we constructed a phylogenetic tree based on nucleotide sequences using an NJ algorithm

Fig. 2. Phylogenetic relationships between sequences of members of the TS superfamily. Deduced amino acid sequences of the TS superfamily were aligned using ClustalW. A maximum likelihood tree was generated using MEGA 5.2 software and is represented as a circle. For each sequence used in this analysis, the species from which it was obtained and its database accession number are shown. Clusters are highlighted in color and identified using Roman numerals for each of the T. cruzi TS superfamily groups proposed by Freitas et al., 2011. Sequences with the * symbol were those obtained in this work. The TS group I sequences from organisms belonging to T. cruzi or T. brucei clades are identified by a red or blue curved bar, respectively. Neuraminidase sequences from Clostridium perfringens and Salmonella typhimurium were used as an outgroup.

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(Fig. 3). In this analysis we included TS group II sequences from isolates of T. cruzi lineages TcI (Sylvio X10/1, DM28c and JR cl. 4), TcII (Esmeraldo), TcIII (M6241), TcIV (Can III), TcVI (CL Brener and Tula cl2) and Tcbat. The tree showed two main branches supported by high bootstrap values (Fig. 3). As expected, sequences from T. rangeli and T. conorhini clustered in one of the branches, while sequences from T. cruzi, T. cruzi Tcbat, T. c. marinkellei and T. dionisii clustered in the

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other branch; some of these had lower bootstrap values and were in groups consistent with the accepted phylogeny of the T. cruzi clade. The branch in which T. cruzi sequences clustered formed five groups consistent with sequences from T. cruzi TcI, TcII, TcIII, TcIV and TcVI strains, the most distant being the branch corresponding to sequences from T. cruzi Can III (TcIV). Of note was the fact that sequences from T. cruzi Tcbat grouped closest to TcI sequences, and this branch clustered

Fig. 3. Phylogenetic analysis based on multiple nucleotide sequence alignments of TS II group members. A neighbor-joining tree was generated using MEGA 5.2 software and the bootstrap method with 1000 replicates. Evolutionary distances were computed using the Kimura 2-parameter method. Sequences marked with an asterisk are those obtained in this work. Accession numbers of sequences retrieved from genome databases are shown. TcChr: chromosome-sized scaffolds of clone CL Brener derived from the Esmeraldo-like (S) and non-Esmeraldo-like (P) parental haplotypes. The scale bar corresponds to the number of substitutions per site. Sequences from organisms belonging to T. cruzi discrete typing units (DTUs) are highlighted in colored boxes as follows: TcI, blue; TcII/VI, green; TcIII, red; TcVI, orange; and TcVI, pink. TS group I sequences from T. cruzi were used as outgroup (enclosed in a gray box).

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with TcII and TcVI. Sequences from T. cruzi CL Brener belonging to Esmeraldo-like and non Esmeraldo-like haplotypes clustered with Esmeraldo strain (TcII) and Tula cl2 (TcVI), respectively.

4. Discussion Our analysis shows that the TS gene family is much more abundant and diverse in T. cruzi than in representative members of the T. brucei clade and T. grayi. These species have about 10 TS genes in their genomes (tritrypdb.org), most of which can be placed in TS group I, and none in the TS groups II to VIII described in T. cruzi. Furthermore, our work not only supports previous reports and recently published genome information on T. c. marinkellei and T. rangeli (Añez-Rojas et al., 2005; Franzén et al., 2012; Grisard et al., 2010; Peña et al., 2009; Stoco et al., 2014; Wagner et al., 2013) but also provides new evidence that other trypanosomes of the T. cruzi clade also have a large and diverse number of TS sequences. Additionally, according to genome data and the hybridization results of this study, the TS gene repertoire in T. cruzi Tcbat and T. c. marinkellei, which are phylogenetically close to T. cruzi, is not only larger but also more divergent than in other members of the clade. The repertoire, degree of sequence identity and phylogeny of TS group II genes inferred in this work agree with the phylogenetic relationships, biogeographical patterns and evolutionary history of species, subspecies and isolates of trypanosomes nested in the T. cruzi clade (Caballero et al., 2015; Hamilton et al., 2007, 2012a, 2012b; Lima et al., 2012a, 2015; Stevens et al., 1999a, 1999b). Similar to what was observed with the analysis of cruzipain and proline racemase (PRAC) gene family repertoires (Caballero et al., 2015; Lima et al., 2012b), by using TS II groups sequences we corroborated the strongly supported clade formed by T. c. cruzi, T. c. marinkeleii and T. dionisii and its sister clade (in other branch) formed by T. rangeli isolates and T. conorhini. As expected, TS group II sequences of T. c. marinkellei, regarded as a T. cruzi subspecies, formed an outgroup for the clade composed exclusively of sequences from T. cruzi, as demonstrated before with other genetic markers (Cavazzana et al., 2010; Franzén et al., 2012; Lima et al., 2012a, 2012b, 2015; Marcili et al., 2009). The network composed of TS group II sequences clearly evidenced subclades corresponding to each DTU in the T. cruzi branch. In concordance with previous studies, our analysis suggests a closer relationship between Tcbat and TcI. Tcbat is a trypanosome that infects bats and, although not virulent to mice, has been found in humans in mixed infection with T. cruzi TcI (Marcili et al., 2009; Ramírez et al., 2014b). Analysis of several nuclear and mitochondrial genes of trypanosomes isolated from bats in Brazil, Panama and Colombia supports the hypothesis that Tcbat should be considered an additional DTU of T. cruzi with a close relationship with TcI (Caballero et al., 2015; Lima et al., 2012b, 2015; Marcili et al., 2009; Pinto et al., 2012; Ramírez et al., 2014a). However, in disagreement with our results, recent reports of multilocus phylogenetic analyses showed that TcI clustered with TcIII, while TcII was the most basal DTU (Diosque et al., 2014; Lima et al., 2015). Consistently with the postulated hybrid origin of T. cruzi CL Brener (TcVI), i.e., that the recent emergence of TcV/TcVI DTUs were originated by TcII and TcIII hybridization events (Flores-López and Machado, 2011; Westenberger et al., 2005), we observed clustering of TcII together with T. cruzi CL Brener Esmeraldo-like. Nevertheless, non-Esmeraldolike sequences grouped with TcVI (Tula cl2) rather than TcIII sequences. Discrepancies with previous reports about clustering of T. cruzi sequences from different DTUs using other genes or multilocus analysis might be caused by the relative low number of sequences included to construct the phylogenetic tree. However, our goal in this particular analysis was to show that the examination of TS group II genes, which are the best-studied TSs with non-catalytic activity, could also help to study phylogenetic relationships in the T. cruzi clade. In fact, our work raised the possibility that TS group II genes might be employed as

markers for T. cruzi genotyping as diverse genes have been used before (Caballero et al., 2015; Lima et al., 2012b; Zingales et al., 2012). In the present work, the sequence obtained from T. rangeli (saimiri) isolate, belonging to the most basal lineage (TrB), clustered together with TS group II genes from T. rangeli strains of the divergent DOG82, SC58 (TrD) and Choachi (TrA) lineages. Several molecular markers, such as SSU rRNA, ITS rDNA, SL RNA, cathepsin L-like and PRAC genes have indicated that T. rangeli comprises at least five phylogenetic lineages associated with their respective vectors, which are different Rhodnius species. (Caballero et al., 2015; Maia da Silva et al., 2004, 2007; Ortiz et al., 2009); so it would be interesting to evaluate, using a higher number of isolates, if the TS sequences could also be useful for genotyping T. rangeli. Moreover, our hybridization results together with the fact that the most divergent TS sequences were those from T. conorhini, are consistent with its phylogenetic position, far from those of T. cruzi and its allied bat trypanosome species and close to T. rangeli (Caballero et al., 2015; Hamilton et al., 2007, 2012a; Lima et al., 2012a). Differences in the TS repertoire of T. conorhini may be related to its little-understood life cycle in vertebrate hosts (Rattus and monkeys) and the digestive tract of its exclusive vector Triatoma rubrofasciata (Caballero et al., 2015). Besides demonstrating that TS is a diverse multigene family in species of the T. cruzi clade, evidence of expression of TS superfamily members and their role in mammalian cell infection has been observed in other trypanosomatids different to T. cruzi, such as in metacyclic forms of T. dionisii and T. cruzi Tcbat, that express functional transsialidase and gp82 proteins, respectively (Maeda et al., 2011; Oliveira et al., 2009). In addition, while T. rangeli epimastigotes are known to express an active sialidase, both epimastigotes and trypomastigotes of this protozoan parasite also express other members of the transsialidase family without known enzymatic activity (Añez-Rojas et al., 2005; Buschiazzo et al., 1997; Grisard et al., 2010; Peña et al., 2009; Wagner et al., 2013). In T. cruzi, TS group II proteins have no transsialidase activity but participate in parasite–host cell adhesion and invasion and tissue tropism by interacting with multiple ligands, such as laminin, fibronectin, collagen and intermediate filaments (Ramirez et al., 1993; Yoshida, 2006; Tonelli et al., 2010). However, as T. rangeli does not develop within mammalian cells, it has been suggested that TS group II proteins may be required for infection to become established in triatomine bugs when the parasite invades their hemocele and salivary glands and is then transmitted by the salivary route, exclusive features of T. rangeli in the T. cruzi clade (Añez-Rojas et al., 2005; Peña et al., 2009). As the genus Trypanosoma is monophyletic, it has been hypothesized that trans-sialidase was present in the common ancestor of both T. cruzi and T. brucei phylogenetic lineages (Briones et al., 1995). Briones et al. (1995) further speculated that an ancestor gene encoding an active trans-sialidase in insect forms of the genus Trypanosoma underwent successive gene duplication and diversification events that resulted in the current trans-sialidase repertoires expressed in mammalian forms. The evidence in the present study that an extensive expansion and major sequence divergence of the TS family is shared by members of the T. cruzi clade but not by African trypanosomes of the T. brucei clade indicates that the duplication and diversification events in the TS family may have occurred after the separation of T. cruzi and T. brucei clades in a common ancestor of the T. cruzi clade. In this clade, the new TS variants may have acquired new roles in the various trypanosome species and developmental stages, such as intracellular and blood forms as well as forms developing in the digestive tract, hemolymph and salivary glands of triatomines, to enable these to adapt to different vertebrate hosts, vectors and environments. Moreover, TS group I genes have only been identified in species belonging to different clades of genus Trypanosoma and are apparently absent in all the other trypanosomatids. Therefore, according to the hypothesis of extensive horizontal gene transfer to support the occurrence of sialidase/transsialidase enzymes in phylogenetically distant organisms (virus, bacteria,

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fungus, trypanosome, etc.) (Roggentin et al., 1993), the event(s) could have occurred in an ancestral member of the genus Trypanosoma. 5. Conclusions In conclusion, we have carried out a comparative analysis of the TS superfamily in a comprehensive set of trypanosome species that cluster within the T. cruzi clade but diverge in several molecular and biological features. Our findings show that in distinct species of this clade the TS multigene family can be large and diverse as in T. cruzi. Furthermore, we demonstrated that the TS group II gene repertoire of T. cruzi allows the genotyping of DTUs, including hybrid genotypes, and could also be useful to establish inter-DTU relationships. It is important to keep in mind that even though it was not possible to identify TS genes belonging to several groups described for T. cruzi in some species of the T. cruzi clade (e.g., T. dionisii), it does not necessarily indicate the absence of TS in the genome of these parasites but a possible limitation of the TS gene identification in the analyzed genome drafts. Further studies are required to better understand the large repertoire of the TS multigene family in these species, and evaluate the expression, activity and TS functions, to elucidate the evolutionary history of trypanosomes in the T. cruzi clade and their biological differences. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2015.11.024. Acknowledgments This work is supported by grants from CDCHT-UCLA 007-ME-2007, FAPESP 11/51693-0 (MAC) and FAPESP 11/51475-3 (JFDS). We are grateful to The Wellcome Trust (TriTrypDB) and the Kinetoplastid Genome Sequencing and Analysis Consortium NIH/NHGRI/NIAID (project ID 59941) for making T. cruzi genomes available. Additional draft and ongoing genomes were obtained from the ATOL (Assembling the Tree of Life - Phylum Euglenozoa) program sponsored by the National Science Foundation, USA (PI Gregory Buck: DEB-0830056). We thank Dr. João Marcelo Pereira Alves assistance with questions regarding the ongoing Trypanosoma genomes. References Alves, M.J., Colli, W., 2008. Role of the gp85/trans-sialidase superfamily of glycoproteins in the interaction of Trypanosoma cruzi with host structures. Subcell. Biochem. 47, 58–69. Añez-Rojas, N., Peralta, A., Crisante, G., Rojas, A., Añez, N., Ramírez, J.L., Chiurillo, M.A., 2005. Trypanosoma rangeli expresses a gene of the group II trans-sialidase superfamily. Mol. Biochem. Parasitol. 142, 133–136. Briones, M.R., Egima, C.M., Eichinger, D., Schenkman, S., 1995. Trans-sialidase genes expressed in mammalian forms of Trypanosoma cruzi evolved from ancestor genes expressed in insect forms of the parasite. J. Mol. Evol. 41, 120–131. Buschiazzo, A., Campetella, O., Frasch, A.C., 1997. Trypanosoma rangeli sialidase: cloning, expression and similarity to T. cruzi trans-sialidase. Glycobiology 7, 1167–1173. Caballero, Z.C., Costa-Martins, A.G., Ferreira, R.C., Alves, J.M.P., Serrano, M.G., Camargo, E.P., Buck, G.A., Minoprio, P., Teixeira, M.M.G., 2015. Phylogenetic and syntenic data support a single horizontal transference to a Trypanosoma ancestor of a prokaryotic proline racemase implicated in parasite evasion from host defences. Parasites Vectors 8, 222. Cano, M.I., Gruber, A., Vazquez, M., Cortes, A., Levin, M.J., Gonzalez, A., Degrave, W., Rondinelli, E., Ramirez, J.L., Alonso, C., Requena, J.M., Franco da Silveira, J., 1995. Molecular karyotype of clone CL Brener chosen for the Trypanosoma cruzi genome project. Mol. Biochem. Parasitol. 71, 273–278. Cavazzana Jr., M., Marcili, A., Lima, L., da Silva, F.M., Junqueira, A.C., Veludo, H.H., Viola, L.B., Campaner, M., Nunes, V.L., Paiva, F., Coura, J.R., Camargo, E.P., Teixeira, M.M.G., 2010. Phylogeographical, ecological and biological patterns shown by nuclear (ssrRNA and gGAPDH) and mitochondrial (Cyt b) genes of trypanosomes of the subgenus Schizotrypanum parasitic in Brazilian bats. Int. J. Parasitol. 40, 345–355. Colli, W., 1993. Trans-sialidase: a unique enzyme activity discovered in the protozoan Trypanosoma cruzi. FASEB J. 7, 1257–1264. Correa, P.R., Cordero, E.M., Gentil, L.G., Bayer-Santos, E., da Silveira, J.F., 2013. Genetic structure and expression of the surface glycoprotein GP82, the main adhesin of Trypanosoma cruzi metacyclic trypomastigotes. Sci. World J. 2013, 156734. Cross, G.A., Takle, G.B., 1993. The surface trans-sialidase family of Trypanosoma cruzi. Annu. Rev. Microbiol. 47, 385–411.

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