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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Trypanosomal infection-induced anaemia is a devastating scourge for cattle in widespread regions. Although Trypanosoma vivax is considered as one of the most important parasites regarding economic impact in Africa and South America, very few in-depth studies have been conducted due to the difficulty of manipulating this parasite. Several hypotheses were proposed to explain trypanosome induced-anaemia but mechanisms have not yet been elucidated. Here, we characterized a multigenic family of trans-sialidases in T. vivax, some of which are released into the host serum during infection. These enzymes are able to trigger erythrophagocytosis by desialylating the major surface erythrocytes sialoglycoproteins, the glycophorins. Using an ex vivo assay to quantify erythrophagocytosis throughout infection, we showed that erythrocyte desialylation alone results in significant levels of anaemia during the acute phase of the disease. Characterization of virulence factors such as the trans-sialidases is vital to develop a control strategy against the disease or parasite.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Animal trypanosomiasis is the most prevalent cattle disease in Africa and South America. It causes annual livestock production losses of several billions for the African continent and strongly hinders agricultural and economical development of emerging countries (Oliveira et al., 2009; Batista et al., 2012; Pimentel Dde et al., 2012; Sow et al., 2012). Trypanosoma vivax and T. congolense are the most prominent parasites involved in this disease that also affects sheep and goats. Antigenic variation developed by African trypanosomes to evade the immune system makes the conventional vaccination strategy unfeasible, thus, characterization of pathogenic factors and virulence mechanisms represents the only way to control the parasite and the associated pathogenesis.

Trypanosoma vivax is considered less pathogenic for cattle than T. congolense but is the most prevalent cause of animal African trypanosomiasis in West Africa (Osorio et al., 2008). T. vivax is usually cyclically transmitted by tsetse flies in Africa but (contrary to T. congolense) has spread beyond the ‘tsetse fly belt’ where it is transmitted mechanically by biting flies, tabanid and contaminated material (Hoare, 1972; Gardiner, 1989). Despite its worrying and growing pervasiveness, and its marked differences with other African trypanosomes in physiopathological features like tissue distribution and biology (life cycle), T. vivax has been neglected for sophisticated experiments, especially gene function analysis. In fact, Trypanosoma brucei brucei, which is not nearly as significant in terms of clinical impact on livestock, has drawn almost all the attention due to the fact that its axenic culture has been well established for several decades allowing the development of genetic engineering (Baltz et al., 1985). Furthermore, T. b. brucei exhibits a high genome identity with the more restrictive species responsible for human African trypanosomiasis, T. b. gambiense and T. b. rhodesiense. On the contrary, T. vivax studies were hampered by lack of reproducible long-term axenic culture, poor access to natural hosts and the inability of most isolates to infect laboratory rodents. Currently, only one recent study has reported transfection methods for T. vivax (D'Archivio et al., 2011), no reverse genetic studies are yet in progress (absence of molecular tools), and very few studies using T. vivax-infected mice have been conducted (de Gee et al., 1982; Chamond et al., 2010). Overall, knowledge of host–pathogen interactions during T. vivax infection is meagre.

Trypanosoma vivax infections are characterized by an initial acute phase with a very high parasitaemia where the immune system is not yet fully mobilized, and a second chronic phase with mild parasitaemia where a major adapted immune response occurs (Murray and Dexter, 1988). The major pathological symptom is anaemia which could rapidly lead to animal death. Widespread red blood cell (RBC) phagocytosis by macrophages has been observed during the acute phase whereas haematopoietic parameters like decreased erythropoiesis are more likely involved in the chronic phase (Gardiner, 1989; Stijlemans et al., 2008). RBC phagocytosis occurs at a high rate in spleen, liver and haemal lymph nodes (Anosa, 1988), but also takes place in the peripheral blood of T. vivax-infected cattle (Kimeto et al., 1990). Several factors have been proposed to be responsible for erythrophagocytosis: immunological mechanisms (Facer et al., 1982), macrophage hyperactivation (Anosa and Kaneko, 1983) and erythrocyte injuries via haemolysins, proteases (Lonsdale-Eccles and Grab, 1987), phospholipases (Tizard et al., 1978) and neuraminidases (Esievo et al., 1982). Recently, there has been some validation of the hypothesis that trypanosomal sialidase (SA)-mediated desialylation of erythrocytes leads to their phagocytosis (Nok and Balogun, 2003; Buratai et al., 2006; Coustou et al., 2012). In fact, it is well known that neuraminidases are able to expose galactose resulting in recognition by the phagocytic system (Bratosin et al., 1998).

Trypanosomal neuraminidases are classified in two enzyme groups: SA similar to bacterial and viral neuraminidases that hydrolyse sialic acids, and trans-sialidases (TS) which not only cleave sialic acid from host glycoconjugates but also transfer sialic acid onto surface acceptor molecules (Schenkman et al., 1994). The roles of SA/TS in parasite virulence has been very well established in Trypanosoma cruzi, the intracellular parasite responsible for Chagas disease in South America (Frasch, 2000). In African trypanosomes, it is known that a trans-sialylation process covers the parasites with a protective coat essential for parasite survival in the fly gut during the insect stages of the life cycle (Nagamune et al., 2004). But the expression and functions of trypanosome SA/TS in the mammalian hosts are much more controversial. We recently reinforced the hypothesis of the role of SA/TS in erythrocyte desialylation by demonstrating that these enzymes are essential for virulence in T. congolense, notably for the development of anaemia (Coustou et al., 2012). Currently, however, the direct correlation between trypanosomal SA/TS, erythrocyte desialylation and phagocytosis has not been established and even less so in T. vivax where molecular studies are scarce.

In this context, we identified and characterized T. vivax enzymes able to trigger erythrophagocytosis: a multigenic TS family. We also developed an ex vivo assay in an experimental trypanosomiasis murine model to quantify erythrophagocytosis. We clearly demonstrated the importance of erythrocyte desialylation throughout the infection process in a murine model for the first time. In order to gain better insights into the mechanism of action, we identified the glycophorins as one of the most important targets for desialylation. In this study a direct link between virulence factors released in the blood during infection and anaemia is established, and it is clearly demonstrated that desialylation alone leads to the development of significant levels of anaemia in the acute phase of animal trypanosomiasis. Also, we again validated the use of a murine model for animal trypanosomiasis to elucidate virulence mechanisms.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In silico identification and characterization of a multigenic family of sialidases in T. vivax

Trypanosoma vivax genome (Y486 strain) is partly sequenced and available on the Genome Data Base TritrypDB (http://tritrypdb.org/tritrypdb/). We found seven genes encoding proteins displaying typical features of trypanosomal SA (Fig. 1, Fig. S1, Table S1) and annotated as putative T. vivax trans-sialidases. Of these genes, five which we named TvTS1–5 encode proteins of 849–898 amino acids (aa) which is similar to the size of other described trypanosomal SA and two encode shorter products (522 and 368 aa) (Fig. 1A). The two shorter forms have high identity with TvTS1 and TvTS5 but are likely to be the result of incomplete sequencing since the sequences contain stretches of undetermined nucleotides and frameshifts.

figure

Figure 1. Multigenic trans-sialidases family of T. vivax.

A. In silico analysis of TvTS family. Protein domains were defined with InterProscan (IPR011040 neuraminidase, IPR013320 concanavalinA-like lectin, IPR008377 trypanosome SA) and ScanProsite (PS00082 extradiol ring-cleavage dioxygenases signature, PS51257 prokaryotic membrane lipoprotein lipid attachment site). The following softwares were used for prediction: SignalP 3.0 for signal peptide, TargetP 1.1 for subcellular localization (SP, secretory pathway; mTP, mitochondrial targeting peptide; Nd, not determined), GPI-SOM and PredGPI for GPI anchoring signal, DAS, HMMTOP and TopPred for transmembrane domains, CSS-Palm 2.0 for palmitoylation sites.

B. Comparison of TvTS1–5 amino acid (aa) sequences. Sequences were aligned with clustalw. The N-terminal catalytic domain and the C-terminal lectin-like domain are separated by a dotted line. FRIP box (double black line) and Asp boxes (single black line) are represented. The motifs VVDPTVVAK (A), ISRVIGNS (B), VPVMLITHP (C), LYCLHE (D), VTVxNYXLYNR (E) are underlined. Residues critical for enzyme function are marked with arrowheads and listed in Fig. 1.

C. Comparison of active sites in trypanosomal SA/TS. TbSA and TbTS: T. brucei SA and TS; TcTS: T. cruzi TS; TrSA: T. rangeli SA; TcoTS T. congolense TS. TcTS, TrSA, TbSA and TbTS analyses are based on previous studies (Tiralongo et al., 2003; Montagna et al., 2006). TvTS2 aa common to other TvTS are shown in black and differences in grey. Residues are illustrated in Fig. 1. Sia: sialic acids, SA: sialidase activity, TS: trans-sialidase activity.

See also Fig. S1 and Table S1.

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Since TvTS1–5 contained all the SA motifs including the FRIP and Asp boxes and the residues critical for catalytic function, they were likely to encode active enzymes (Roggentin et al., 1993; Tiralongo et al., 2003) (Fig. 1 and C). The transferring abilities of trypanosomal TS also appeared to be conserved (Buschiazzo et al., 2002; Tiralongo et al., 2003; Montagna et al., 2006) (Fig. 1C). TvTS1–5 enzymes exhibited relatively high identity to each other: 50.9–66.3% (Table S1). Similar to T. brucei and T. congolense families, conservation was higher in the catalytic domain (63–90.1%) than in the lectin-like domain (42.8–50.3%) (Montagna et al., 2006; Coustou et al., 2012). Nevertheless, slight differences of crucial residues were evident. For example, the variability of residue 475 of TvTS2, important for the binding specificity, could reflect enzymatic diversity particularly concerning substrate specificity (Fig. 1C).

TvTS enzymes also showed 16.9–31.2% identity to the 8 SA/TS of T. brucei, 14.1–31.9% to the 11 SA/TS of T. congolense, and 25.5–31.9% to a representative T. cruzi TS (Table S1). But contrary to T. brucei and T. congolense, the degenerate TS-like subfamily was not detected in T. vivax. Furthermore, phylogenetic analysis showed that TvTS formed a separate cluster whereas TcoTS, TbTS and TbSA were closer to their orthologues than to members of their own family (Fig. S1).

In silico characterization of TvTS enzymes indicated other differences with trypanosomal SA/TS characterized thus far: (i) none of these proteins are predicted to be GPI-anchored nor possesses a trans-membrane domain, (ii) three of the TvTS1–5 proteins displayed a mitochondrial signalling peptide and only TvTS2 is clearly predicted to follow the secretory pathway, and (iii) TvTS1 and TvTS3 possess additional motifs, i.e. an extradiol ring-cleavage dioxygenase degenerate signature (some critical residues for dioxygenase activity are not conserved) and a prokaryotic membrane lipoprotein lipid attachment site profile respectively. In fact, most of the trypanosomal SA described so far were associated with cell membranes and/or secreted, here some of the TvTS enzymes could be mitochondrial signa-anchored proteins therefore associated with the outer membrane of the mitochondria. For now, the only known example of mitochondrial SA is the long isoform of human Neu4 SA predominantly expressed in the brain (Yamaguchi et al., 2005) and also associated with the outer membrane of the mitochondria. The biological significance of these differences is not obvious but suggest that T. vivax TS play additional or different roles during infections.

Detection and characterization of SA and TS activities and identification of TvTS enzymes expressed in T. vivax BSF

To investigate SA and TS activities presence in the mammalian stage of T. vivax (BSF), we used fluorimetric assays to quantify enzyme-mediated hydrolysis of sialic acid (SA assay) and transfer of sialic acid from a donor, α2,3-sialyllactose, to an acceptor, MU-β-d-galactopyranoside (TS assay). We previously characterized these activities in T. congolense IL3000 and demonstrated their absence in T. brucei 427 BSF (Coustou et al., 2012). Here, we compared SA and TS activities in crude BSF extracts of two T. congolense strains, two T. brucei strains and T. vivax Y486 (Fig. 2 and B). The results clearly demonstrated the presence of both activities in T. vivax BSF. While SA activity has previously been reported in literature, TS activity has never been described (Esievo et al., 1982; Buratai et al., 2006). The level of SA activity was comparable in T. congolense and T. vivax (∼ 12 μU per 109 cells versus ∼ 16) whereas TS activity was approximately four times higher in T. vivax (∼ 620 μU per 109 cells versus ∼ 2143). T. brucei displayed neither of these activities.

figure

Figure 2. SA and TS activities in T. vivax Y486 BSF.

A and B. SA (A) and TS (B) activities were measured on crude extracts of T. brucei 427 and Antat 1.1, T. congolense IL3000 and STIB910 and T. vivax Y486 BSF. BSF were cultured in vitro or purified from mouse blood, results were the same regardless of origin. Each bar represents the SA or TS activity ± standard deviation (SD) of at least three independent experiments.

C. The proportion of TS activity, expressed as a percentage of total activity, was measured in the supernatant (soluble) or pellet (insoluble), following centrifugation of osmotically lysed cells (T. vivax Y486 BSF).

D. SA and TS activity in T. vivax Y486 BSF culture was monitored over 2 days. BSF were obtained from infected mice and cultivated for 2 days; during this short period cell death is minimal. Data are expressed as the mean of two independent experiments.

See also Figs S2 and S3.

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Although T. brucei and T. congolense insect forms (procyclic, PCF and epimastigote, EMF) displayed very high SA/TS activities (Engstler et al., 1993; 1995; Coustou et al., 2012), neither of these activities were detected in T. vivax EMF. This could be explained by the fact that T. vivax BSF ingested during fly blood meal differentiate into EMF in the proboscis, pharynx and oesophagus of the fly (Hoare, 1972), whereas T. congolense and T. brucei differentiate into PCF which travel to the fly midgut. Therefore, protection from the digestive enzymes by trans-sialylation (Nagamune et al., 2004) may not be necessary for T. vivax EMF.

The TS/SA activities ratio suggested that TvTS enzymes expressed in BSF were predominantly TS and not strict hydrolases. Generally, TS exhibit low SA activity rather than high TS activity due to the catalytic properties of these enzymes (Demir and Roitberg, 2009). Furthermore we saw above that critical residues for transferring activity are conserved in TvTS1–5. The same situation is observed in T. congolense and could illustrate a common strategy for the virulence of these two parasites species.

To further characterize these activities, we determined the proportion of TS activity associated with insoluble material after hypotonic lysis in T. vivax BSF (Fig. 2C). We observed, as previously described for T. congolense (Coustou et al., 2012), that the major part of the activity was membrane-associated. We also monitored the culture medium of T. vivax BSF for released SA and TS activity and observed that significant activity accumulated in the medium during growth, a property also described for T. congolense (Coustou et al., 2012). Taken together, these data showed that part of the activity remained membrane-bound while part is secreted which correlates quite well with the in silico prediction as some TvTS enzymes were predicted to be secreted while others seem to be anchored in the outer membrane of the mitochondria.

Then to determine the expression pattern of TvTS enzymes, we produced recombinant proteins in heterologous systems to raise anti-TvTS-sera. Only the soluble TvTS2 was successfully produced in the yeast Pichia pastoris, while the others were produced as insoluble material in Escherichia coli. TvTS2 was active and its biochemical properties were analysed. TvTS2 hydrolysed MU-Neu5Ac, 3′sialyl-lactose, ganglioside GD3 or fetuin and was also able to transfer sialic acid to an acceptor, indicating TS activity. The basic kinetic parameters of TvTS2 were determined (Fig. S2). The results were very similar to those obtained from T. brucei or T. congolense TS (Tiralongo et al., 2003; Montagna et al., 2006; Koliwer-Brandl et al., 2011; Coustou et al., 2012).

Recombinant proteins were used to immunize rabbits or mice. A Western blot analysis with anti-sera gave a positive signal on T. vivax BSF crude extracts around 100 kDa, the expected size of TvTS enzymes (Fig. S3). But no significant signal was detected in EMF (data not shown). Immunofluorescence analyses were also realized and different kind of signals were obtained (Fig. 2E). Anti-TvTS2 serum showed typical mitochondrial labelling which was corroborated by colocalization with a mitochondrial marker, the heat-shock protein HSP60 (Bringaud et al., 1995), and also a less intense labelling of the whole cell, typical for cytosolic compartment. Anti-TvTS3 serum showed labelling of the whole cell surface with a dotted line along the cell body, which is typical for membrane protein (Field et al., 2010). Again these data are correlated with in silico prediction and also with activity as part of it is soluble and could be in the cytosol while another part is secreted or membrane-bound and could be either in the mitochondrial or in cell surface membrane.

The anti-sera were also used in immunoprecipitation assays on T. vivax BSF and EMF followed by mass spectrometry analysis but no TvTS peptides were obtained. As TS activity is mostly associated with insoluble material, membrane extracts of T. vivax Y486 EMF and BSF were analysed in mass spectrometry. Peptides from TvTS1, 3, 4 and 5 were detected in BSF (Table S2). The presence of TvTS1, 3 and 5-specific peptides allowed us to affirm that at least these three TvTS enzymes were expressed in T. vivax Y486 BSF. No TvTS peptides were identified in EMF. We also analysed the culture medium of T. vivax Y486 BSF as we showed that TS activity accumulated and we confirmed TvTS presence by Western blot analysis (data not shown). However, no TvTS peptides were detected by mass spectrometry analysis which could be explained by a quantity of TvTS enzymes too low to be detected or presence of other abundant proteins at this molecular weight masking TvTS (similar results were obtained with T. congolense).

In conclusion, we identified, for the first time, at least three TvTS family members expressed in T. vivax Y486 BSF, which are likely to be responsible for the SA/TS activities in the infected mammalian hosts. Furthermore, we confirmed that the absence of SA/TS activities in EMF insect stage is due to the absence of TvTS proteins.

SA and TS activities are present in the sera of T. vivax-infected mice

To investigate the potential erythrophagocytosis process, we defined a suitable experimental trypanosomiasis murine model. We chose CD1 mice where T. vivax infection is more subchronic than acute, which allowed a more thorough study of the development of anaemia and, almost significantly, closely resembles natural T. vivax infection in cattle (Chamond et al., 2010). We also defined the parasite inoculum and chose to inject 2·106 parasites of the rodent-adapted Y486 strain intraperitoneally. We obtained a subchronic infection profile with waves of parasitaemia and significant development of anaemia (Fig. 3A). Mice survival was ∼ 2–4 weeks. This type of infection mimicked the initial phase of T. vivax or T. congolense bovine infection with a rapid drop of PCV (up to ∼ 20%) and a high parasite load (up to ∼ 1·109 parasites per ml of blood). We monitored the reticulocyte level (erythropoietic precursor of erythrocytes) and observed a high level of reticulocyte production (up to nine times the basal level, ∼ 18% versus ∼ 2% of the RBC) in response to anaemia (Fig. 3A). This reticulogenesis followed the waves of parasitaemia and anaemia and clearly showed that haematopoiesis is not impaired during infection. This physiological compensation of anaemia had to be taken into account since the intact, freshly produced reticulocytes were included in the measures of RBC sialylation state at some points of the experiment.

figure

Figure 3. Analysis of parasitaemia, anaemia and SA activity during experimental T. vivax infection in mice.

A. Parasitaemia, haematocrit and reticulocytes were monitored during T. vivax Y486 infection in CD1 mice. Haematocrit was expressed in PCV and reticulocytes as the percentage of RBC.

B. SA activity was monitored during infection and plotted with haematocrit and parasitaemia.

Two representative mice out of 10 are shown.

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To determine the potential role of TvTS in anaemia, we also determined the level of SA activity in the serum and observed a significant SA activity that fluctuated with parasitaemia (Fig. 3B). Monitoring of TS activity gave identical profiles (data not shown). SA/TS activities probably resulted from active secretion with a correlation with parasite load in the blood but also from passive release after immune-mediated lysis which would explain the relatively high SA activity even in the absence of parasites between each wave of parasitaemia. Release of virulence factors after immune-mediated trypanosomes lysis is a well-known phenomenon (Antoine-Moussiaux et al., 2009). Moreover, these enzymes seem to be quite stable and could accumulate, the observed profiles have, therefore, resulted from a combination of all these infection traits. These results are comparable with T. congolense mouse infection (Coustou et al., 2012), and could again highlight a common mechanism of anaemia development.

Development of an ex vivo erythrophagocytosis assay

Bratosin et al. developed an elegant flow cytofluorimetric procedure using fluorescent erythrocytes to measure erythrophagocytosis of senescent RBC (Bratosin et al., 2002). This method is quantitative, sensitive and avoids the use of radioisotopes. To study the propensity of erythrocytes to be recognized and phagocytosed by host cells during T. vivax murine infection, we adopted a similar method to develop an ex vivo erythrophagocytosis assay. This assay is based on the use of erythrocytes, macrophages and serum from the same mouse to evaluate erythrophagocytosis by flow cytometry analysis. We improved erythrocyte labelling by using calcein AM (cell-permeant, converted by intracellular esterases into green-fluorescent calcein) instead of PKH-26 (irreversibly anchored into cell plasma membrane even in damaged cells) to decrease the background.

The assay procedure starts with collection of peritoneal macrophages from T. vivax Y486-infected mice or non-infected control mice that were put into culture plates to adhere (1–5·105 per well). At the same time, blood was collected from these mice, erythrocytes and serum were separated and erythrocytes were labelled with calcein AM. After 3 h, non-attached macrophages were eliminated, and erythrocytes (3·106) and mouse serum (10%) were added to the wells. Eight combinations of biological material were obtained depending on the source (infected, Y, or control, C) of the three components: macrophages, serum and erythrocytes (first, second and third letter of the combination respectively). After 4 and 24 h, erythrophagocytosis was measured by flow cytometry (Fig. 4). To differentiate between macrophages and residual external erythrocytes, macrophages were labelled with anti-CD16/32 (Fig. 4B), and the proportion of macrophages containing phagocytosed erythrocytes (calcein labelled) was determined (Fig. 4 and D).

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Figure 4. Flow cytometry analysis of an ex vivo erythrophagocytosis assay.

A–D. Analytical method of flow cytometry data from an ex vivo erythrophagocytosis assay (CCY combination).

A. Dot plot representation of the mixed erythrocyte and macrophage populations. Delimitation of the two populations is shown and the macrophage population is marked as P1.

B. APC fluorescence histogram of population P1. APC is conjugated to anti-CD16/32 antibodies (macrophage antigen marker). The positive population is marked as P2.

C. FITC fluorescence histogram of population P2. FITC fluorescence reflects calcein-labelled erythrocytes. Positive population (P3) represents macrophages containing phagocytosed erythrocytes.

D. Dot plot representation of population P2. The negative APC population is not plotted. The proportions of APC-positive, FITC-negative macrophages (lower right quadrant) and APC-positive FITC-positive macrophages (upper right quadrant = P3) were determined.

E. Results of an ex vivo erythrophagocytosis assay. In infected mice, parasitaemia reached its maximum (5·108 parasites per ml) and haematocrit began to drop (35%).

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An example was reported in Fig. 4 with a Y486-infected CD1 mouse 7 days after infection (top of the first peak of parasitaemia). The proportion of macrophages containing phagocytosed erythrocytes was dependent on the combination of the three serum components. The base levels of erythrophagocytosis were ∼ 2% (4 h of incubation) and ∼ 11% (24 h) under control conditions (CCC) and the highest level were ∼ 10% (3 h) and 47% (24 h) when all the components came from infected mice (YYY). To compare the results, the ratio of erythrophagocytosis between one combination and the base level (CCC) was the most relevant parameter i.e. erythrophagocytosis increased around five times from CCC compared with YYY. In this example, the ratio of erythrophagocytosis was high when erythrocytes from infected mice were used. This illustrates an increased sensitivity of infected-mouse erythrocytes to phagocytosis.

In conclusion, with this method, we were able to quantify ex vivo erythrophagocytosis which allowed extrapolation of the propensity of erythrocytes to be phagocytosed in vivo. Moreover, we evaluated the influence of each of the three components of the biological system. We therefore conducted a complete analysis of the different combinations throughout infection.

Erythrophagocytosis is a major cause of anaemia during T. vivax infection

To understand the significance of erythrophagocytosis during T. vivax infection, we defined four critical stages in Y486 experimental infection and examined the rate of erythrophagocytosis in each stage (Fig. 5A). Stages were defined as follows: stage 1, increase of parasites in the first wave of parasitaemia associated with slight anaemia; stage 2, peak of the first wave of parasitaemia and the beginning of the decrease associated with strong anaemia, and therefore the most interesting stage; stage 3, absence of parasites in the blood associated with haematocrit recovery; stage 4, high level of parasites in the blood causing the second wave of parasitaemia associated with strong anaemia.

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Figure 5. Erythrophagocytosis throughout T. vivax experimental infection in mice.

A. Typical parasitaemia and haematocrit profiles of T. vivax Y486 experimental infection. Four important and representative stages of the infection were defined to serve as references.

B. Results of erythrophagocytosis assays expressed as ratio of macrophages containing phagocytosed erythrocytes in the indicated condition over the macrophages containing phagocytosed erythrocytes in control condition (CCC). Mean values and SD are shown when at least three assays were performed (assay number is indicated by n). A ratio lower than 1 (marked as 1) indicates that erythrophagocytosis was not different from the basal level. Mice collected for assays were classified into infection stages depending on infection parameters.

C–F. Histograms of erythrophagocytosis ratios through T. vivax infection for a given combination.

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The ratio of erythrophagocytosis (Fig. 5B) clearly showed that erythrophagocytosis is greatly enhanced in stage 2 and 4 when erythrocytes from infected mice were used (YYY, CCY, CYY and YCY), whereas it is only slightly enhanced or equal in other conditions. On detailed examination of the results, the YYY combination (Fig. 5C) showed the most significant ratio of erythrophagocytosis, ∼ 8 (4 h) and ∼ 5 (24 h) in stage 2, and ∼ 3 in stage 4. In stage 1 and 3 the ratio is ∼ 2. This indicated that high levels of erythrophagocytosis occurred in infected mice particularly at the beginning of anaemia, which suggested that erythrophagocytosis is a major trigger for the development of anaemia during T. vivax infection. To analyse the role of each of the components, we analysed the combinations CCY (Fig. 4D), YCC (Fig. 4E) and CYC (Fig. 4F) which allowed examination of the involvement of erythrocytes, macrophages and serum respectively. Significantly, the ratio of erythrophagocytosis in CCY conditions showed approximately the same profile as YYY conditions with a lower ratio increase in stage 2 (∼ 4) and 1 (∼ 1). This confirmed the importance of erythrocytes for the stimulation of phagocytosis since even in the presence of control macrophages and serum, phagocytosis is four times higher at a time of infection where the level of anaemia is high. There was only a slight increase in erythrophagocytosis when infected macrophages and/or serum are used especially in stage 2 and their effect appeared to be synergistic. This demonstrated that even if macrophages and serum elements such as antibodies participate and amplify the phenomenon of erythrophagocytosis, their contribution is not definitive compared with erythrocytes.

We showed that this ex vivo erythrophagocytosis assay gave significant and reproducible results. We observed that infection-associated erythrophagocytosis was visible throughout infection and varied with parasitaemia and also established a direct correlation between the presence of parasites in the blood, the development of anaemia and ex vivo erythrophagocytosis. We propose that this reflects on many aspects the anaemia process encountered in vivo which highlighted the existence of erythrocyte modifications during infection which triggered their phagocytosis by monocytes and macrophages.

Erythrocyte sialylation profiles are modified during T. vivax infection

We hypothesized that TvTS enzymes released during infection desialylate erythrocytes rendering them prone to phagocytosis. To test our hypothesis, we used different techniques to examine erythrocyte glycosylation and sialylation profiles during infection.

The majority of erythrocyte glycoproteins are sialoglycoproteins named glycophorins. They constitute more than 80% of sialic acids bound to erythrocyte membrane glycoconjugates (Furthmayr et al., 1975). In mice, two glycophorin isoforms, gp2 and gp3, were identified (Sarris and Palade, 1982). Glycophorins tend to form multimers in SDS-PAGE resulting in complex migration profiles: gp2 and gp3 monomers are observed around 44 and 29 kDa respectively, gp3 dimers named gp1 around 66 kDa and other multimers around 94 kDa (Sarris and Palade, 1982). Moreover, experimental conditions (protein concentration, buffer composition) can modify this profile. Therefore, we determined glycosylation profiles in control mice with defined experimental conditions to map glycophorin bands which are likely to be preferential targets for erythrocyte desialylation.

Erythrocyte ghosts were submitted to SDS-PAGE and gels were silver stained directly or after periodic acid oxidation to enhance staining sensitivity which is typically greatly reduced by glycosylations (Fig. 6A) (Van-Seuningen and Davril, 1992). We obtained a typical profile and defined five glycoproteic bands (A–E), we assumed that band C corresponds to gp2, bands D and/or E to gp3, band B to gp1 and bands > 90 kDa named A to glycophorin multimers. Subsequently, Western blotting analysis with antibodies commonly used to detect glycophorins were performed (Fig. 6A). TER-119, a monoclonal antibody which recognizes a molecule associated with glycophorins and allows detection of all the glycophorin bands (Kina et al., 2000), as expected, recognized the glycoproteic bands A to E. Monoclonal antibodies anti-glycophorin A (GlyA, gp2 orthologue) and anti-glycophorin C (GlyC, gp3 orthologue) recognized band C and bands B and E respectively. Finally, peptides matching glycophorins were abundant and preferentially found in the bands A to E by mass spectrometry analyses (data not shown). Subsequently, we examined modifications of this glycophorin profile during infection with several tools. The results are presented for a single mouse which developed a rapid infection with a substantial decrease of haematocrit and a survival of 16 days to facilitate comprehension (Fig. 6B). However, it must be noted that the same results were obtained for three mice which developed similar infection patterns and for mice with more chronic infections, where glycophorin profile modification correlated with the degree of PCV variation (data not shown). First, we analysed general changes of the glycoprotein profile and observed significant modifications especially for the bands B, D and E which clearly increased as seen on the gel on the right showing only glycoproteins (Fig. 6C). Modifications were also visible on a total protein gel by decrease in size and transition from blurry regions to distinct bands, which is typical for glycosylation modification (stars in Fig. 6C). The increase of bands D and E signifies that glycophorin migration as monomers was more important (change in the ratio between multimer/monomer migrating forms) maybe as a consequence of glycophorins desialylation known to facilitate monomers on SDS-PAGE (Sarris and Palade, 1982).

figure

Figure 6. Modification profiles of the mouse erythrocyte surface glycoproteins throughout T. vivax infection.

A. Identification of CD1 mouse erythrocyte membrane glycoproteins. After SDS-PAGE gels were silver stained directly (1) or after periodic acid oxidation to also reveal glycoproteins (2) or were analysed by Western blotting with monoclonal TER-119 (TER119), anti-glycophorin A (GlyA) and anti-glycophorin C (GlyC) antibodies.

B. Monitoring of parasitaemia and haematocrit in the Y486 T. vivax-infected CD1 mouse used to analyse erythrocyte membranes profiles (C–F).

C. Comparison of erythrocyte membranes proteins (left) and glycoproteins (right) throughout T. vivax infection on SDS-PAGE. Gels were silver stained directly (left) or after brief periodic acid oxidation to reveal only the glycoproteins (right).

D. Lectin blot analysis of erythrocyte membrane glycoproteins throughout T. vivax infection. Blots were probed with biotin-conjugated Sambucus nigra agglutinin (SNA). Black arrows (1–6) indicate lectin signals, grey arrows (7–9) indicate sialylated proteins appearing on day 15 post infection. Some arrows were replicated on related blots.

E. Western blot analysis on erythrocytes ghosts with TER119 throughout Y486 T. vivax infection.

F. Western blot analysis of glycophorins expression with GlyA or GlyC antibodies and of integrin β1 (β1) expression throughout Y486 T. vivax infection.

Molecular weights (kDa) are indicated on the left. A total of 107 erythrocytes ghosts were loaded per well. Days post infection are indicated at the top. See also Fig. S4.

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Then, we used the Sambucus nigra agglutinin (SNA) to specifically see sialylation modifications. SNA binds preferentially to sialic acid attached to terminal galactose with α-2,6 linkage (highly represented in glycophorins), and to a lesser degree with α-2,3 linkage (Fig. 6D). The sialylation profile was strongly affected during the course of infection (signals 1–9). Signals 1–4 decreased but signal 4, which completely disappeared on day 8, reappeared from day 11–15. Bands of small molecular weights (signal 6) and signal 5 (∼ 130 kDa) appeared on day 8. Finally, signals 7–9 appeared at the end of the infection. These changes could correspond to desialylation, modification of the amount of sialylated proteins or modification of their migration as sialylation influences migration. Production of reticulocytes, observed during infection (Fig. 3A), is likely to participate to these changes as reticulocytes are strongly glycosylated and bear intact fresh glycosylations (that could explain some signal increases) and also possess membrane proteins absent from mature erythrocytes such as the transferrin receptor, or integrin β1 (Liu et al., 2009).

So, to explore the different possibilities, we used several antibodies: TER-119, GlyA and GlyC to examine the amount of the different glycophorin forms and anti-integrin β1 antibodies (β1), since this heavily sialylated protein is predicted to migrate at 130 kDa and constituted a good marker for reticulocytes (Fig. 6 and F). First, no decrease of glycophorins were observed showing that the decrease of SNA signal corresponded to desialylation and not to reduction of the amount of protein. In fact, as expected, the apparition of galactose residues on the erythrocyte surface in parallel with desialylation was observed using a specific lectin (Fig. S4). On the contrary, most of the glycophorin signals increased during infection and some new glycophorin bands appeared on TER-119 (corresponding to signals 7 and 9) and on GlyA (corresponding to signal 8) blots. β1 Western blot clearly demonstrated the increasing number of reticulocytes throughout the infection which is in accordance with the increase in the amount of glycophorins. To confirm these data, we used a bleeding induced-anaemia murine model where reticulogenesis increased as anaemia progressed (Fig. S4). We observed that gp2 (signal 4, GlyA) slightly increased with reticulogenesis and that integrin β1 is expressed concomitantly with reticulogenesis, demonstrating that some signal increase (like signal 8 or 5) results from reticulocytes production while others (like signals 1–4 decrease) are exclusively due to trypanosome-mediated erythrocyte desialylation. Again, all these data were corroborated by mass spectrometry analyses (data not shown).

Profiles of Wheat Germ Agglutinin and Maackia amurensis lectin which bind sialic acid in addition to other glycosidic residues, were more complex and, therefore, more difficult to interpret. However, all profiles showed a common trait: modifications appeared during infections and seemed to result either from desialylation or from increased reticulogenesis (data not shown).

To summarize, some glycoproteins, most of them being glycophorins, underwent desialylation resulting in decreased signals (1–4) or possibly migration as monomers (6), while others corresponded to reticulocyte production (5–9 and re-apparition of signal 4). But most importantly, the phenomenon of desialylation throughout T. vivax infection, as described in the ageing process of erythrocytes (Bratosin et al., 1998), leads to galactose exposition recognized as a signal for phagocytosis by macrophages.

Trypanosomal TS are clearly involved in erythrophagocytosis and anaemia through erythrocytes desialylation

To establish a direct link between trypanosomal TS, desialylation and erythrophagocytosis and given the fact that inhibition of expression of TvTS enzymes with genetic tools is not possible in T. vivax, we used two methods. First, recombinant TS were injected into mice to observe potential effects on haematocrit and were used to treat erythrocytes in vitro to analyse their effect on erythrocyte sialylation and subsequent propensity to be phagocytosed (Fig. 7). We observed that injections of T. congolense or T. vivax TS resulted in haematocrit depression visible 24 h after the first injection and until 2 days after the final third injection (Fig. 7A). The most significant decrease (up to 43% of haematocrit) was observed 5 days after the first injection. These results implied that the presence of trypanosomal TS in the blood had a direct impact on haematocrit. Incubation of erythrocytes with recombinant TS in vitro resulted in modifications of SNA profiles which resembled those observed during infection especially when TvTS2 is used instead of bacterial neuraminidase (Fig. 7B). In fact, desialylation resulted in decreased SNA signals as observed for the gp2 signal 4 and increased presence of signal 6 (small molecular weights bands) and signal 7 that probably corresponded to glycophorin-modified migration as desialylation may facilitate their migration as monomers and may prevent multimer aggregation.

figure

Figure 7. Effects of recombinant trypanosomal TS on the sialylation profile of erythrocytes and phagocytosis.

A. In vivo effect of trypanosomal TS injection on mouse haematocrit. Five to eight CD1 mice were injected with 650 μU of T. congolense TcoTS-A1 and TcoTS-D2, T. vivax TvTS2, commercial bacterial neuraminidase (NA) or BSA (control) via the retro-orbital sinus for 3 consecutive days (0, 1, 2) and haematocrit measured during 11 days. Each bar represents the mean of haematocrit values ± SD.

B. SNA lectin blot analysis of erythrocytes membranes after SA treatment. A total of 5·107 RBC from CD1 mice were treated or not (Control) with 140 μU TvTS2 or NA, for 6 h and 24 h before SDS-PAGE analysis on 10% (left) or 15% (right) acrylamide gels. Molecular weights (kDa) are indicated on the left.

C. The effect of recombinant trypanosomal TS erythrocytes treatment on erythrophagocytosis ratios. A total of 3·106 RBC were treated or not (Control) with different quantities of TvTS2, TcoTS-A1 or NA (indicated in μU) before the ex vivo erythrophagocytosis assay. Assay numbers are indicated by n.

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After treatment with TS, erythrophagocytosis increased compared with non-treated erythrocytes (Fig. 7C). The increase was comparable between trypanosomal or bacterial enzyme. For TcoTS-A1 and bacterial neuraminidase we observed that the ratio of erythrophagocytosis increased in direct proportion to the quantity of enzyme (the specific activity of TvTS2 was too low to compare), highlighting the fact that increased erythrocyte desialylation is correlated with increased phagocytosis. Therefore, the concentration of TS in the host blood during infection may be critical to aggravate anaemia.

Second, we used known TS inhibitors to evaluate the impact of TS activity inhibition on erythrophagocytosis and anaemia during T. vivax infection. Arioka and colleagues described flavonoid and anthraquinone derivatives that inhibit T. cruzi TS with relative high specificity compared with human neuraminidase 2 and low IC50 value (Arioka et al., 2010). Therefore, we tested myricetin (flavonoid derivative) and rhein (anthraquinone derivative) in vitro on recombinant TvTS2 and obtained an equivalent inhibitory effect (11 μM and 24 μM for IC50 values of myricetin and rhein respectively). These compounds were subsequently injected (1 mg kg−1) into non-infected or T. vivax Y486-infected CD1 mice 1 day after infection for 4 consecutive days and parasitaemia and haematocrit were monitored and compared with non-treated T. vivax Y486-infected CD1 mice (Fig. 8 and B). Approximately the same results were obtained with the two compounds and no effect on haematocrit was observed when mice were not infected but injection of rhein seemed to be a slightly unpleasant for mice so we preferred to focus on myricetin. In infected mice, treatment decreased the anaemia but had no effect on the parasitaemia. Non-treated mice died after 6 days with a very low value of haematocrit (∼ 30%) whereas treated mice survived and developed a chronic infection (mice died after 3 weeks of infection). The prolonged survival of treated mice probably resulted from a slight recovery of haematocrit upon treatment. To evaluate the effect of myricetin on TvTS in vivo, we measured SA activity in the serum of treated infected mice versus non-treated infected mice and showed a strong decrease of SA activity upon treatment (Fig. S5A). In the same way, inhibition of TvTS2 by incubation with inhibitor prior to injection resulted only in a very slight haematocrit decrease compared with previously described decrease in Fig. 7 after injection of active TvTS2 (Fig. S5B). We subsequently measured ex vivo erythrophagocytosis with biological material from treated infected mice compared with non-treated infected mice. We observed that the erythrophagocytosis ratio with control mice (YYY/CCC) is 2.3 and 2.5 times lower when mice were treated (Fig. 8C). Levels of erythrophagocytosis are, therefore, strongly correlated with levels of anaemia and we proposed that inhibition of TS activity resulted in decreased erythrophagocytosis and consequently, in decreased anaemia. To corroborate these data we analysed sialylation profiles of erythrocytes from treated and non-treated mice and showed that desialylation is less pronounced in treated mice which correlated perfectly with the lower level of erythrophagocytosis and reinforced the role of erythrocyte desialylation by trypanosomal TS in anaemia during T. vivax infection (Fig. 8D).

figure

Figure 8. Effect of sialidase inhibitors on anaemia and ex vivo erythrophagocytosis during experimental infection.

A and B. Effects of myricetin (Sigma) injection on parasitaemia (A) and haematocrit (B) during T. vivax Y486 infection in CD1 mice. Myricetin (1 mg kg−1) was injected intraperitoneally or in the retro-orbital sinus 1 day post infection (infection with 3–4·106 BSF of T. vivax Y486) for 4 consecutive days in five CD1 mice (treated mice). Results are shown for 7 days, after this period, treated mice survived and developed classical subchronic infections as described in Fig. 3. Non-treated CD1 mice (infected at the same time) died on day 5 or 6 post infection.

C. Comparison of ex vivo erythrophagocytosis ratio YYY/CCC of treated mice versus non-treated mice. Erythrocytes, serum and macrophages were collected 5 days post infection from four different CD1-infected mice.

D. Comparison of erythrocyte sialylation profiles on SNA blot for erythrocyte ghosts from control mice (non-treated and non-infected lane 1), infected and treated mice (lane 2 and 3) and infected and non-treated mice (lane 4). Erythrocytes were collected 5 days post infection. Treatment had no effect on the sialylation profile of non-infected mice compared with non-treated control mice (data not shown).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Currently, trypanosomiasis control is based on vector control, mainly through spraying of insecticide, which has a far from negligible environmental impact. At the level of infection, control is limited to a handful of chemicals developed more than five decades ago, which have severe side-effects, and for which several strains have developed resistance (Pepin and Milord, 1994). Due to the ability of the parasite to change its protein coat through antigenic variation, conventional vaccination has remained elusive. In this context, the search for novel trypanocidal molecules or new therapeutic targets is a priority. However, host–pathogen interactions vary greatly between the different species of trypanosomes at both the level of pathogenesis and the level of virulence, resulting in a variety of clinical symptoms. For this reason, we investigated the role of T. vivax SA/TS genes in erythrophagocytosis to determine the causative factors of the major physiopathological trait of animal trypanosomiasis, the anaemia.

We found at least five genes encoding potential active TS in the T. vivax genome. At least some of them are expressed in the BSF resulting in significant SA and TS activities in the blood of the infected host. This is the first time that TS activity has been identified in T. vivax BSF and that genes potentially encoding this activity have been characterized. Similar to T. congolense, we also detected SA and TS activity in the blood during infection in murine model (Coustou et al., 2012). Enzymes released into the serum probably resulted from active secretion as seen with direct secretion of some enzymes in the culture medium and also from passive release by immune-mediated lysis of the parasites making at least the soluble part of the activity available as shown after hypotonic lysis. However, contrary to other released virulence factors like the cathepsin B protease (Mendoza-Palomares et al., 2008), no antibodies against TS enzymes were detected either in T. vivax or in T. congolense-cattle infected sera (data not shown). This lack of specific antibodies may allow the TS to accumulate and be active in the blood without being trapped by antibodies which may increase their half-life in the blood.

The unique features displayed by TvTS enzymes compared with other trypanosomal SA/TS (mitochondrial membrane localization for some, prokaryotic domains signature) can be explained by different evolutionary processes leading to adaptation. For example, T. vivax differentiates directly into EMF without passing through the intestinal tract, a property that may explain the rapid adaptation of the parasite to mechanical transmission. Such changes in the mode of transmission imply adaptation to new vectors and new geographical zones. Moreover, T. vivax in contrast to the exclusively intravascular T. congolense, may cross the blood brain barrier and invade the central nervous system (Batista et al., 2007). There, the parasite could target other substrates such as gangliosides which are highly enriched in neurones and are vital components of the cells and this could be the cause of gangliosidosis similar to Trypanosoma evansi responsible for surra in animals (Nok et al., 2003). It is interesting to note that the only other case of mitochondrial neuraminidase reported so far (human Neu4L) is also bound to the outer mitochondrial membrane and is expressed in the human brain (Bigi et al., 2010). So, diversity of substrates and ecological niches implied a different evolution of such a multigenic family which is often associated with virulence.

We demonstrated clearly that erythrophagocytosis is responsible for anaemia in the acute phase of infection. This is in accordance with previous studies showing that immunological competence is not essential for the development of anaemia, at least in the initial phase of animal trypanosomiasis (Murray and Dexter, 1988; Naessens, 2006). We also showed for the first time that the state of the erythrocyte surface determines this process. Immune complexes or auto-antibodies that could be generated in this kind of infection are not essential for this part of anaemia. Similarly, macrophage activation is not necessary to trigger erythrophagocytosis. However, these factors may certainly amplify the phenomenon as the infection progresses in cattle.

Desialylation of erythrocytes is known to lead to their phagocytosis; this phenomenon is mostly observed in erythrocyte senescence whereby old erythrocytes are cleared from the bloodstream (Bratosin et al., 1998). It has been proposed that the sialylation state of erythrocytes may be involved in trypanotolerance. Trypanotolerance is the ability of certain breeds of West-African cattle, existing in small numbers and of low productivity, to resist the effects of trypanosome infection through better control of anaemia leading eventually to the control of parasitaemia, and ultimately self-cure (Naessens, 2006). Here we identify for the first time, the most important changes in sialylation state of erythrocytes during T. vivax infections, and showed that these modifications could be reproduced in vitro by TvTS but also TcoTS proteins. As the anaemia development in T. congolense infections is similar and TcoTS are essential virulence factors, we proposed that erythrocyte desialylation via trypanosomal TS also lead to phagocytosis and, subsequently, anaemia in T. congolense. On the other hand, we were not surprised to find that glycophorins are preferential targets of desialylation as they constitute the vast majority of sialic acids bound to the surface of erythrocytes. Moreover, they are also targeted as receptors by other pathogens like Encephalomyocarditis virus and Plasmodium falciparum (Jungery et al., 1983; Tavakkol and Burness, 1990).

Trypanosoma vivax infection profiles may vary from mild to very acute forms, and the severity of the disease depends on parasites strains, endemicity and host species (Njiokou et al., 2004; Chamond et al., 2010). One of the most acute forms of infection is associated with haemorrhagic syndrome which regularly emerges as infection foci, especially in East Africa (Gardiner et al., 1989; Magona et al., 2008). To explain this physiopathological feature, several hypothesis are proposed but mechanisms are still largely unknown. Haemolysins and immune-mediated mechanisms are probably involved. However, the potential involvement of virulence factors such as SA/TS has not been investigated. Thus, it would be interesting to examine TvTS genes in terms of genes numbers, sequence variability and expression patterns in haemorrhagic strains. One could imagine that increased TS expression, substrate affinity or catalytic efficiency could be responsible for erythrocyte injuries leading to haemolysis. Typically, the use of a murine model to compare East and West African T. vivax strains in order to perform similar analysis (erythrocytes sialylation state and erythrophagocytosis) seems very appropriate. On the other hand, different factors could be investigated with our erythrophagocytosis assay including the role of macrophages and seric elements, since it was suggested that TNFα secretion is greater in East T. vivax stocks which could lead to macrophage hyperactivation among other things (Naessens, 2006).

Until now, only partial data were available from field studies to explain anaemia in T. vivax infections. Here we show that erythrophagocytosis is responsible for the first severe drop of haematocrit in the acute phase of infections. TS enzymes which are released early in infection cause desialylation of erythrocytes leading to their subsequent phagocytosis and resulting in anaemia. This study has reinforced the proposed role for trypanosomal SA/TS as essential virulence factors and their use as therapeutic targets against disease.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Parasites cultivation

The T. vivax Y486 strain was first isolated in a Zebu in West Africa (Nigeria) (Leeflang et al., 1976) and was kindly provided by the International Livestock Research Institute, Nairobi, Kenya. Cultures were carried out as described previously (D'Archivio et al., 2011).

Expression of sialidases in E. coli and protein purification

TvTS1–5 were amplified by PCR on genomic T. vivax Y486 DNA using specific primers. Fragments were cloned into the pET-24a vector (Novagen). E. coli BL21 bacteria were transfected and expression conducted according to the manufacturer's instructions. Recombinant proteins were always expressed as inclusion bodies, regardless of temperature and induction. Following purification of the inclusion bodies, recombinant proteins were electroeluted after separation on SDS-PAGE.

Expression of sialidases in P. pastoris and protein purification

TvTS2 was expressed in the EasySelect Pichia system (Invitrogen) according to the manufacturer's instructions. Gene was amplified by PCR on genomic T. vivax Y486 DNA using specific primers. Fragments were cloned into the pPICZ A vector (Invitrogen). Expression in the P. pastoris strain X-33 and subsequent purification using ion-exchange chromatography were performed as described (Coustou et al., 2012).

Enzyme activity assays and kinetic studies

Sialidase (SA) and trans-sialidase (TS) activities were measured as previously described (Coustou et al., 2012). Details are provided in the supplemental experimental procedures.

Experimental infection of mice

Eight-week-old female were purchased from Charles River Laboratories (L'Arbresle Cedex France). All animal studies adhered to protocols approved by the University of Bordeaux 2 animal care and the ethics committee. CD1 mice were inoculated by intraperitoneal injection with 2·106 BSF of T. vivax Y486. Parasitaemia was monitored daily by microscopic observation. Haematocrit was determined in two ways. Blood samples were collected by tail bleed in 100 μl capillary tubes coated with Na-heparin, sealed, centrifuged (12 000 g, 12 min), and analysed with the haematocrit reader supplied by the manufacturer (Heraeus) to determine the PCV; or a 5 μl sample of tail blood was placed in 100 μl of distilled water and the OD405 of a 40-fold dilution in distilled water was measured. The second method used much less blood and avoided bleeding-induced anaemia. Our model revealed a perfect correlation between PCV and OD405, so data were always expressed as PCV.

Reticulocyte numbers were determined by mixing 5 μl of blood and 5 μl of brilliant cresyl blue (150 mg l−1 in 0.58% NaCl) for 30 min followed by a thin smear prepared on a glass slide. The reticulocyte number among at least 1000 RBC was determined by microscopy and expressed as percentage.

Ex vivo erythrophagocytosis assay

Mouse peritoneal macrophages were obtained by washing the peritoneal cavity with 10 ml of phosphate-buffered-saline solution (PBS: 150 mM NaCl, 12 mM Na2HPO4 and 3 mM KH2PO4). The cell suspension was centrifuged (350 g, 4°C, 5 min), and the pellet was resuspended in DMEM medium containing 20% of fetal calf serum (FCS, Gibco). Of this suspension, 500 μl containing 1–5·105 macrophages was transferred into 24-well plates and incubated in a 5% CO2 humidified atmosphere at 37°C for 3 h. Non-adherent cells were removed by repeated washing with DMEM medium. In parallel, 40 μl of murine blood was collected on heparin, mixed with 1 ml of PBS, centrifuged (3300 g, 4°C, 2 min) and washed three times. After counting, 108 erythrocytes were labelled with 500 μl of calcein-AM 5 mM (Sigma) for 30 min at 37°C. Erythrocytes were washed eight times with 1 ml of PBS (3300 g, 4°C, 2 min) and resuspended in PBS (106 cells per 10 μl). In addition, serum was prepared from 1 ml of blood from the same mouse by allowing blood to clot for 30 min at 37°C followed by 10 min on ice. After centrifugation (3000 g, 10 min), ∼ 500 μl of serum was obtained.

Following this, 230 μl of DMEM, 10% of mouse serum and 106 labelled erythrocytes were mixed and incubated for 4 h or 24 h at 37°C in a 5% CO2 humidified atmosphere. After incubation, non-internalized RBC were removed by washing three times with PBS. Macrophages were released by trypsin treatment (0.5 g l−1, Sigma) for 5 min at 37°C. To inactivate trypsin, 350 μl of RPMI medium without phenol red containing 10% FCS were added. Finally macrophages were labelled using rat APC-conjugated anti-CD16/32 (0.2 μg ml−1, Beckman Coulter) for 10 min and the cell suspension was analysed by flow cytometry. Data were collected on a FACS Canto II (Becton Dickinson) flow cytometer and analysed using the FACSDiva software. The light-scatter channels were set on linear gain and the fluorescence channels on a logarithmic scale. Cells were gated for forward and side-angle scatters and 5000 fluorescent particles of each gated population were analysed. The analytical method was described in the results.

In vitro erythrocyte desialylation

Calcein-labelled mouse erythrocytes were desialylated by mixing 3·106 erythrocytes with bacterial neuraminidase NA (from Arthrobacter ureafaciens, Sigma), or trypanosomal SA [TvTS2, described above; TcoTS-A1 previously described (Coustou et al., 2012)] in 200 μl of PBS glucose (0.8 g l−1), containing a protease inhibitor cocktail (Complete mini EDTA-free; Roche Diagnostics GmbH). The mixture was incubated 3 h at 37°C, washed in 1 ml of PBS (3300 g, 2 min) and resuspended in PBS (106 cells per 10 μl) for the erythrophagocytosis assay or blotting.

Erythrocyte ghost analysis by SDS-PAGE and silver staining/periodic acid oxidation method

One hundred microlitres of murine blood was collected with heparin, mixed with 1 ml of PBS, centrifuged (3300 g, 4°C, 2 min) and washed three times. Erythrocytes were lysed in 1 ml of hypotonic buffer (5 mM Na2HPO4, 0.3 mM KH2PO4, protease inhibitor cocktail) at 4°C and centrifuged (16 000 g, 10 min, 4°C) three times. Ghost pellets obtained were resuspended in SDS 2% (1–5·106 μl−1) and incubated at 100°C for 5 min. Samples were submitted to SDS-PAGE 10 or 15%. Gels were silver stained with Proteosilver silver Stain kit (Sigma) either directly or after periodic acid oxidation treatment. For this, the gel was fixed (10 min, 30°C, 10% w/w trichloroacetic acid), washed two times (5% v/v acetic acid, 2 min), oxidized (1% w/v periodic acid, natrium m-periodate (Sigma), 20 min, 30°C), washed two times with 5% v/v acetic acid, two times with water, then reduced [0.5% w/v potassium disulfide (Prolabo), 12 min, 30°C].

Immunoblotting and lectin blotting

Erythrocyte ghost membranes were probed with monoclonal anti-human glycophorinA (ab14484, abcam) or anti-human glycophorin C (ab6398, abcam) antibodies (1:1000 in PBS 0.05% tween20, 5% BSA), or with anti-mouse integrin β1 (102203, Biolegend 0.5 μg ml−1) followed by HRP-conjugated anti-mouse immunoglobulin G (IgG) (Sigma; 1:10 000).

To reveal the presence of sialic acids on glycoconjugates, membranes were incubated for 1 h with biotin-conjugated SNA (GALAB Technologies; 10 μg ml−1), washed, and incubated with HRP-conjugated strepavidin (Sigma; 1/2000).

Antigen-antibody and streptavidin-biotin interactions were developed using Immobilon Western chemiluminescent HRP substrate (Millipore).

Immunofluorescence analyses

Cells were fixed with formaldehyde as described (Bringaud et al., 1998). Slides were then incubated with rabbit anti-TvTS2 and three antibodies, diluted 1:100 in PBS 0.1% (v/v) Triton X-100 0.1% (w/v), BSA for 30 min, washed three times with PBS and incubated for 30 min with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (diluted 1:100) (Molecular probes). For colocalization, slides were also incubated with monoclonal mouse anti-hsp60 (undiluted) (Bringaud et al., 1995) and with goat anti-rabbit IgG secondary antibody (diluted 1:100) (Molecular probes). Finally, cells were incubated for 5 min with 1 mg ml−1 4′,6′-diamidino-2-phenylindle (DAPI) and mounted in Vectashield (Vector Laboratories). Cells were observed with a Zeiss UV microscope and images were captured using a MicroMax-1300Y/HS camera (Princeton Instruments) and Metaview software (Universal Imaging Corporation).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We are grateful to S. Claverol, A.M. Lomenech and J.W. Dupuy from the Genomic Platform at Bordeaux 2 University for their technical support in mass spectrometry analyses. We are grateful to B. Rousseau from the Bordeaux 2 animal care and to J. Izotte (UMR5234, Bordeaux2) for technical help on experimental infection of mice and to D. Pillay and L. Rivière for their critical reading of the manuscript.

This work was supported by the CNRS, the Ministere de l'Education Nationale de la Recherche et de la Technologie, the Conseil Régional d'Aquitaine and the LabEx ParaFrap (French Parasitology Alliance for Health Care). This research was also supported by the Global Alliance for Livestock Veterinary Medicine (GALVmed) with funding from the UK Government's Department for International Development (DFID) as part of GALVmed's Animal African Trypanosomosis Programme (DFID Programme: Controlling African Animal TRYPANOSOMOSIS (AAT) (Aries code 202040-101), and by CEVA sante animale (Libourne, France).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
cmi12123-sup-0001-si.doc131K

Fig. S1. Related to Fig. 1. Phylogenetic analysis of the sialidase-encoding genes of T. vivax, T. congolense and T. brucei.

A. Analysis of the TvTS family.

B. Analysis of T. vivax, T. congolense and T. brucei sialidases. T. congolense sialidases (TcoTS) are indicated by a blue circle, T. brucei sialidases (TbSA and TbTS) are indicated by a green triangle, and T. vivax sialidases are indicated by a red square.

Phylogenetic trees were constructed using the neighbour-joining method on MEGA 5 software. Branch length is indicated.

cmi12123-sup-0002-si.doc357K

Fig. S2. Related to Fig. 2. Biochemical characterization of TvTS2.

A. Study of pH effect on SA activity. Enzyme activity was assayed at 37°C in AMT buffer at various pH.

B. Lineweaver-Burk plots of SA activity. The activity of the enzymes was measured at 37°C, pH 7.0, at varying concentrations of MUNeu5Ac.

C. Lineweaver-Burk plots of TS activity. Enzyme activity was measured at 37°C, pH 7.0, using fixed MU-Gal concentration and varying concentrations of sialyl lactose.

D. Kinetic parameters of T. vivax TvTS2 The apparent Vmax and Km were determined using the Lineweaver-Burk method.

Data are expressed as the mean of three independent experiments.

cmi12123-sup-0003-si.doc163K

Fig. S3. Related to Fig. 2. Western blot analysis of TvTS expression in T. vivax Y486 BSF. Blots were probed with anti-TvTS5 sera. Molecular weights (kDa) are indicated on the left.

cmi12123-sup-0004-si.tif768K

Fig. S4. Related to Fig. 6. Modification profiles of the mouse erythrocyte surface glycoproteins throughout T. vivax infection and in a bleeding-induced anaemic mouse model.

A. Lectin blot analysis of erythrocyte membrane glycoproteins throughout T. vivax infection with biotin-conjugated Peanut Agglutinin (PNA) specific for terminal galactose attached to galactosamide, visible after removal of terminal sialic acids.

B. Monitoring of haematocrit and reticulocytes in the bleeding-induced anaemic CD1 mouse used to analyse erythrocyte membrane profiles (G–H).

C. SNA lectin blot analysis of erythrocyte membranes throughout the bleeding-induced anaemia.

D. Western blot analysis of glycophorin A (GlyA) and integrin β1 (β1) expression throughout the bleeding-induced anaemia.

cmi12123-sup-0005-si.tif457K

Fig. S5. Effects of myricetin on SA activity in infected mice and on recombinant TvTS2 action on haematocrit.

A. Parasitaemia, haematocrit and SA activity were monitored during T. vivax Y486 infection in myricetin treated mice CD1 mice. Myricetin (1 mg kg−1) was injected intraperitoneally 1 day post infection (infection with 3–4·106 BSF of T. vivax Y486) for 4 consecutive days in five CD1 mice (treated mice). Haematocrit was expressed in PCV.

B. In vivo effect of injection of recombinant TvTS2 previously incubated with myricetin on mouse haematocrit. 650 μU of T. vivax TvTS2 were incubated with myricetin (30 μg) for 30 min then injected in three CD1 mice for 3 consecutive days (0, 1, 2). Haematocrit was monitored during 11 days. Each bar represents the mean of haematocrit values ± SD.

cmi12123-sup-0006-si.doc219K

Table S1. Related to Fig. 1. Identity percentage between TvTS family members and between TvTS1–5 and other trypanosomal sialidases.

A. Comparison of the five complete TvTS enzymes on the full-length protein (1), on the catalytic domain (2) and on the lectin-like domain (3).

B. Comparison of TvTS1–5 with one representative T. cruzi TcTS (Q26964) (1), with T. congolense TS family (TcoTS) (2) and with T. brucei SA and TS (TbTS and TbSA) (3).

cmi12123-sup-0007-si.doc62K

Table S2. Related to Fig. 2. Sialidase peptides identified by mass spectrometry in membrane preparations in T. vivax Y486 BSF. Y486 BSF membranes were prepared as described below and analysed by SDS-PAGE and silver staining. Protein bands from 60 to 200 kDa were analysed by mass spectrometry and the peptides identified were listed in the tables. The presence of the peptides in the TvTS family members is indicated by +.

Supplemental experimental procedures.

cmi12123-sup-0008-si.zip23KSupporting Information

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