Served in aTS and iTS proteins from all DTUs. In support, a residual enzyme activity has been recently found for iTS protein [35] emphasizing that it has similar properties to aTS in sequence and folding. Furthermore, in vitro assays have demonstrated the costimulatory properties of iTS proteins on the immune system [36]. The strong sequence conservation 25033180 in all iTS genes supports thatFigure 2. UPGMA tree based on TS genes sequence alignment (with ambiguous states). Each circle grouped all 38 T. cruzi strains in their respective previous assigned DTU, except CAN III 
and 3.1 that were previously assigned to TcIV. Significant bootstrap values for TcI, TcIII and TcIV are reported, bootstrap values for other DTUs were ,50. doi:10.1371/journal.pone.INCB-039110 site 0058967.gTrans-Sialidase Genes in T. cruzi PopulationsiTS plays an evolutionary selectable role, instead of representing just a collection of pseudogenes. Therefore, 
an involvement in parasite attachment/invasion to host cells can be postulated because iTS acts as a lectin, able to bind not only small oligosaccharides but also sialylated glycoproteins [32,34], a relevant feature in the physiological scenario of parasite infection. Interestingly, our findings also reveal the existence of parasites with highly reduced TS genes content that provide models to develop genomic KO, a largely expected tool to extend the study of the biological relevance of TS whose generation has been hampered by the high gene copy numbers always reported for TS. Moreover, the ongoing transfection assays with the iTS gene might provide with a nice opportunity to test the actual relevance of iTS in parasite biology and pathogenesis. In 2009, an expert committee revised the information available about T. cruzi evolution and clustering. They remember that the partition of T. cruzi in six principal DTUs could be explained by two alternative models for their origin: the `Two Hybridization’ model giving rise to TcIII and then to TcV and TcVI through hybridization of two ancestors (TcI and TcII) [51] and the `Three Ancestor’ where the ancestors TcI, TcII and TcIII gave rise to the hybrids TcV and TcVI [52]. The current distribution of aTS/iTS suggests a closer relationship of TcI with TcIII-TcIV than with the other DTUs as well as a related evolution of TcII, TcV and TcVI. Indeed, the sequence analysis that reflect the variability of a set of genes coding for the same virulence factor (TS) fits with the six DTUs clustering, although TcII, TcV and TcVI DTU were not supported by significant bootstrap values because the hybrid nature of TcV and TcVI, reduces the bootstrap values, and if these strains are removed from the analysis (see Figure S2), TcI and TcII DTUs are everyone very well supported by high bootstrap value (93.8 and 98.3 GNF-7 biological activity respectively), and TcIII and TcIV are grouped together with a lower bootstrap value (60.9). However, this group is further divided into two clusters, one including CanIII, M5631, X109/2 and 3.1 strains (bootstrap value of 95.8) and the other comprising STC16Rcl1, STC10R and 92122102R strains (boot-strap value of 71.4). Although several scenarios can explain the current variability of the TS genes within DTUs, considering that TcI and TcII are ancestors [51,52] and that iTS may have originated from aTS genes through a single mutation event, the common ancestor of TcI and TcII should not have had iTS. After iTS consolidation in TcII, its delivery during subsequent hybridization events could explain its p.Served in aTS and iTS proteins from all DTUs. In support, a residual enzyme activity has been recently found for iTS protein [35] emphasizing that it has similar properties to aTS in sequence and folding. Furthermore, in vitro assays have demonstrated the costimulatory properties of iTS proteins on the immune system [36]. The strong sequence conservation 25033180 in all iTS genes supports thatFigure 2. UPGMA tree based on TS genes sequence alignment (with ambiguous states). Each circle grouped all 38 T. cruzi strains in their respective previous assigned DTU, except CAN III and 3.1 that were previously assigned to TcIV. Significant bootstrap values for TcI, TcIII and TcIV are reported, bootstrap values for other DTUs were ,50. doi:10.1371/journal.pone.0058967.gTrans-Sialidase Genes in T. cruzi PopulationsiTS plays an evolutionary selectable role, instead of representing just a collection of pseudogenes. Therefore, an involvement in parasite attachment/invasion to host cells can be postulated because iTS acts as a lectin, able to bind not only small oligosaccharides but also sialylated glycoproteins [32,34], a relevant feature in the physiological scenario of parasite infection. Interestingly, our findings also reveal the existence of parasites with highly reduced TS genes content that provide models to develop genomic KO, a largely expected tool to extend the study of the biological relevance of TS whose generation has been hampered by the high gene copy numbers always reported for TS. Moreover, the ongoing transfection assays with the iTS gene might provide with a nice opportunity to test the actual relevance of iTS in parasite biology and pathogenesis. In 2009, an expert committee revised the information available about T. cruzi evolution and clustering. They remember that the partition of T. cruzi in six principal DTUs could be explained by two alternative models for their origin: the `Two Hybridization’ model giving rise to TcIII and then to TcV and TcVI through hybridization of two ancestors (TcI and TcII) [51] and the `Three Ancestor’ where the ancestors TcI, TcII and TcIII gave rise to the hybrids TcV and TcVI [52]. The current distribution of aTS/iTS suggests a closer relationship of TcI with TcIII-TcIV than with the other DTUs as well as a related evolution of TcII, TcV and TcVI. Indeed, the sequence analysis that reflect the variability of a set of genes coding for the same virulence factor (TS) fits with the six DTUs clustering, although TcII, TcV and TcVI DTU were not supported by significant bootstrap values because the hybrid nature of TcV and TcVI, reduces the bootstrap values, and if these strains are removed from the analysis (see Figure S2), TcI and TcII DTUs are everyone very well supported by high bootstrap value (93.8 and 98.3 respectively), and TcIII and TcIV are grouped together with a lower bootstrap value (60.9). However, this group is further divided into two clusters, one including CanIII, M5631, X109/2 and 3.1 strains (bootstrap value of 95.8) and the other comprising STC16Rcl1, STC10R and 92122102R strains (boot-strap value of 71.4). Although several scenarios can explain the current variability of the TS genes within DTUs, considering that TcI and TcII are ancestors [51,52] and that iTS may have originated from aTS genes through a single mutation event, the common ancestor of TcI and TcII should not have had iTS. After iTS consolidation in TcII, its delivery during subsequent hybridization events could explain its p.
