Deep serological profiling of the Trypanosoma cruzi TSSA antigen reveals different epitopes and modes of recognition by Chagas disease patients.

BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, displays a highly structured population, with multiple strains that can be grouped into 6-7 evolutionary lineages showing variable eco-epidemiological traits and likely also distinct disease-associated features. Previous works have shown that antibody responses to 'isoforms' of the polymorphic parasite antigen TSSA enable robust and sensitive identification of the infecting strain with near lineage-level resolution. To optimize the serotyping performance of this molecule, we herein used a combination of immunosignaturing approaches based on peptide microarrays and serum samples from Chagas disease patients to establish a deep linear B-cell epitope profiling of TSSA. METHODS/PRINCIPLE FINDINGS: Our assays revealed variations in the seroprevalence of TSSA isoforms among Chagas disease populations from different settings, hence strongly supporting the differential distribution of parasite lineages in domestic cycles across the Americas. Alanine scanning mutagenesis and the use of peptides of different lengths allowed us to identify key residues involved in antibody pairing and the presence of three discrete B-cell linear epitopes in TSSAII, the isoform with highest seroprevalence in human infections. Comprehensive screening of parasite genomic repositories led to the discovery of 9 novel T. cruzi TSSA variants and one TSSA sequence from the phylogenetically related bat parasite T. cruzi marinkellei. Further residue permutation analyses enabled the identification of diagnostically relevant or non-relevant substitutions among TSSA natural polymorphisms. Interestingly, T. cruzi marinkellei TSSA displayed specific serorecognition by one chronic Chagas disease patient from Colombia, which warrant further investigations on the diagnostic impact of such atypical TSSA. CONCLUSIONS/SIGNIFICANCE: Overall, our findings shed new light into TSSA evolution, epitope landscape and modes of recognition by Chagas disease patients; and have practical implications for the design and/or evaluation of T. cruzi serotyping strategies.

Evolutionary volatile Cysteines and protein disorder in the fast evolving tunicate Oikopleura dioica.

Cysteine (Cys) is regarded as the most conservative amino acid in nature, something that does not occur in the tunicate Oikopleura dioica, where this amino acid is one of the fastest evolving. In this work we analyze some of the causes of this intriguing absence of conservation. Considering the well-known stabilizing role of Cys, it was first investigated whether the lack of conservation was accompanied by an increase in intrinsic protein disorder. In contrast to expectations, it was found that O. dioica is the chordate that has the lowest levels of intrinsic disorder, while vertebrates (represented by Bos taurus) contain the most disordered proteins. Oikopleura proteins are shorter than their homologs in other Chordates (Ciona and B. taurus proteins are respectively 11% and 18% longer). This process of protein shortening was more intense in intrinsic disordered regions. As a result proteins became not only shorter but also more compact. It is also reported here that the conservation/divergence behavior of Cys depends on whether they are located in ordered or disordered regions. In the four species analyzed, disordered Cys are majorly (> 75%) not conserved at all. Ordered Cys instead, are much more free to diverge in Oikopleura than in the other chordates. We hypothesize that the preferential deletion of disordered regions resulted in a decreased protein disorder and a direct elimination (by deletion) of many ancestral Cys. Besides, the alterations (shortening or complete elimination) of some disordered regions (loops/random coils) probably promoted further Cys evolutionary volatility, because some ancestral Cys (and other amino acids which play a role in stability like Trp) located outside deleted regions became redundant due to the loss of their stabilizing partners.

Peculiar patterns of amino acid substitution and conservation in the fast evolving tunicate Oikopleura dioica.

We analyze the patterns and rates of amino acid evolution in tunicates with special interest on the extremely fast evolving Oikopleura dioica. We show that this species, on average, is twice as fast as the already fast evolving Ciona intestinalis. The acceleration in both species seems to be affected by similar evolutionary forces yet to different extent, since a substantial proportion of the most and less accelerated genes are orthologous between the two species. Among the possible causes that underlie the genome wide acceleration in Oikopleura, relaxation of functional constraints appears to be an important one, since all amino acids exhibit surprisingly homogenous levels of divergence. Such homogeneity, however, is not observed in Ciona. Apart from the genome wide acceleration, detailed analysis of functional groups of genes revealed that genes associated with regulatory functions (transcription regulators, chromatin remodeling proteins and metabolic regulators), have been subjected to an even more extreme process of acceleration, suggesting that adaptive evolution is the most probable cause of their unusual exacerbated rates. Another remarkable observation is that cysteine is among the less conserved amino acids, contrary to what is commonly observed in other species. The possible causes of this particular behavior are discussed.