The TIR domain consists of approximately 200 amino acids that form five β sheets surrounded by five α helixes. In animals and plants, as well as in free-living amebae, the TIR domain participates in diverse biological activities such as pathogen resistance, immune recognition and feeding[13, 24–27]. In this study, we identified a thioredoxin in the E. histolytica proteome which contains a putative TIR domain in a thioredoxin protein. Primary structure of this TIR domain differ from other TIR domains found in plants and animals, but secondary structures show more similar regions, mainly in Box1 and Box2 that are essential regions to fold the protein, and both are conformed by two helixes. On the other hand, the specific one-on-one 3D structure alignment showed that the thioredoxin has structural analogues of TIR domains in mammalian protein IL-1R and TLR2. This supports the idea that three-dimensional structure is much more closely associated with function than its lineal sequence, tertiary structure is more evolutionarily conserved than primary structure (http://www.ncbi.nlm.nih.gov/books/NBK22362). Both TIR domains, of mammalian IL-1R and TLR2, mediate homotypic protein–protein interactions in the signal transduction that facilitate the activation and expression of mechanisms which increase several biological process, including phagocytic activity[14, 28, 29].
The participation of this domain on phagocytosis during infection resistance and feeding observed in the evolutionary related ameba Dictyostelium discoideum[13, 15, 30], led us to think that the thioredoxin found, may participate in the phagocytosis of natural sources of nutrients through human commensal bacteria (such as Escherichia coli) and erythrocytes to uptake iron. In E. histolytica, phagocytosis process is Gal/GalNAc lectin and PATMK dependent.
In this report, we observed reduction in phagocytosis of human erythrocytes and E. coli after incubation with small interfering double stranded-RNAs for thioredoxin, suggesting a role for the TIR domain-containing thioredoxin in trophozoites phagocytosis. These results could support the proteomic approach explored by McCoy et al., and Okada et al.[32, 33], which suggest that the phagocytosis process Gal/GalNAc lectin-mediated involves the recruitment of thiol-specific antioxidants (such as thioredoxin) for phagosome biogenesis and cytoskeletal rearrangement[8–10]. If this is right, PATMK and Gal/GalNAc lectin, both of which are involved in the interaction with the membrane of erythrocytes, could be involved in the formation of microdomains necessaries to signaling in phagocytosis, in which molecules like thioredoxin could participate. However this must be investigated further.
The above mentioned, is similar to reported to the majority of these TIR domain-containing proteins that have been described in plants, insects, and vertebrates and participate in phagocytic and cell activation[13, 25]. However, despite finding similarities among the reduction of phagocytosis when the expression of these transcripts is reduced, according to other reports, we cannot reject the possibility that the thioredoxin identified here participates in the phagocytic process via a mechanism that is not related to its TIR domain, for example the redox state thioredoxin-dependent processes which regulate cellular functions, such as endocytosis and cell adhesion[34–36].
A diverse number of specific functions and cofactor activities have been described for thioredoxins. These functions include growth factor activity, cell protection from toxic compounds (especially oxidants and electrophiles), activation of inflammatory pathways, regulation of transcription factors and protein binding, and catalysis of protein folding; each of these activities has an effect on cellular responses to toxic insults[11, 12]. However, no other thioredoxin is involved in the regulation of phagocytic processes. E. histolytica expresses other proteins with thioredoxin activity that participate in nicotinamide adenine dinucleotide phosphate (NADPH)-dependent hydrogen peroxide reduction; these detoxifying thioredoxins do not share the TIR domain identified here.
In this report we present evidence that protein sequence corresponding to 344–442 position in E. histolytica thioredoxin (GenPept:XP_649779) has a TIR-domain. Considering that several members of TIR domain family have been involved in signal transduction in many organisms, and silencing its expression decrease erythrocytes and bacteria phagocytosis, the thioredoxin could play a central role in signal transduction during this event, hence influencing the E. histolytica survival mechanisms.