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  • br Material and methods br Results br Discussion Cysticerci

    2019-07-11


    Material and methods
    Results
    Discussion Cysticerci located in host tissues are exposed to several effector mechanisms of the immune system (Flisser et al., 1986; Sciutto et al., 2000; Amit et al., 2011; Singh et al., 2013). In addition, the extensive uptake of host proteins by cysticerci has led to the proposal that these proteins could play a role in the worm physiology. A striking example is porcine serum haptoglobin, which seems to modulate the host-parasite relation in T. solium cysticercosis (Navarrete-Perea et al., 2014, Navarrete-Perea et al., 2016). The survival of T. solium metacestode in host tissues depends on several mechanisms to evade or modulate the host immune response (Baig et al., 2005; Sciutto et al., 2007; Terrazas, 2008; Mendlovic et al., 2015). Little is known about the mechanisms used by T. solium to migrate in early stages of infection and establish in different host tissues. The establishment of T. solium cysticerci in the central nervous system may represent an advantage for the survival of the parasite in this tissue with a privileged immune status (Garcia et al., 2004; Sciutto et al., 2007). Understanding the role that several cysticercus proteins play in parasite migration and establishment will provide us with a more comprehensive view of the survival mechanisms applied by cysticerci lodged in host tissues. Enolase, a glycolytic enzyme, has been recently described as a potential vaccine candidate (Yang et al., 2010; Chen et al., 2012), since it is highly expressed in cysticercus tegument, and has been proposed to play a role in the maintenance of host-parasite relation through its Plg-activating ability, already described for T. pisiformis (Zhang et al., 2015). Four enolase genes have been described for vertebrates: Eno1, Eno2, Eno3, and Eno4 (Pearce et al., 1976; Schmechel et al., 1978; Ueta et al., 2004). However, invertebrate enolase gene D-Pantothenic acid has not been characterized. Four tapeworm enolase genes were identified in public genome databases. Coincidently, four genes have also been described in vertebrate organisms. However, few studies on enolase evolution are available (Tracy and Hedges, 2000; Harper and Keeling, 2004; Piast et al., 2005), and these studies included few examples of invertebrate enolases. Thus, to the best of our knowledge, no evidence on the relation between vertebrate and invertebrate enolase isoforms was available. This fact led us to conduct a new phylogenetic analysis that included 75 enolase amino acid sequences from organisms belonging to six kingdoms. ML phylogenetic analysis showed that T. solium and other tapeworms have four enolase isoforms (Fig. 2). The topology of our tree showed that the origin of enolase isoforms in vertebrates, except for Eno4, is independent of the enolase isoforms in invertebrates (Fig. 2). Herein we propose a new designation (EnoA, EnoB, EnoC, and Eno4) to emphasize that invertebrate enolase isoforms are not orthologues of their vertebrate counterparts. Three of the T. solium enolase isoforms (TsEnoA, TsEnoB and TsEnoC) were 433–450 amino acid residues long, whereas TsEno4 was about 40% smaller: 250 residues long. It is noteworthy that this enolase lacks the Plg-binding motif. Studies to determine whether this isoform, clearly expressed in cysticerci, retains its Plg-binding activity are currently underway. With respect to the gene expression level of the enolase isoforms in T. solium cysticerci, RT-PCR assays using primer pairs specific for each isoform showed that TsEnoA, TsEnoC and TsEno4 were clearly expressed, while no TsEnoB was detected, possibly because it is expressed in another stage of the parasite life cycle, like the oncosphere or the adult worm. A line of evidence supporting a high expression of TsEnoA is given by a random sequencing of 75 000 ESTs from T. solium cysticerci and adults (Tsai et al., 2013, http://www.genedb.org/Homepage/Tsolium); 674 hits for TsEnoA were recorded in cysticercus ESTs, whereas only one hit was reported for Eno4 (data not shown). The high expression of TsEnoA could be due to its involvement in glycolysis, a key metabolic process for T. solium, since this parasite is well known for its high glucose consumption (Willms et al., 2005).