Identification of glycoproteins targeted by Trypanosoma cruzi trans-sialidase, a virulence factor that disturbs lymphocyte glycosylation.

Trypanosoma cruzi, the agent of the American trypanosomiasis or Chagas disease, bypasses its lack of de novo synthesis of sialic acids by expressing a surface-anchored trans-sialidase. This enzyme transfers sialic acid residues from the host's sialylglycoconjugates to the parasite's galactosylglycoconjugates. In addition to carrying out a pivotal role in parasite persistence/replication within the infected mammal, the trans-sialidase is shed into the bloodstream and induces alterations in the host immune system by modifying the sialylation of the immune cells. A major obstacle to understand these events is the difficulty to identify the transferred sialic acid among all those naturally occurring on the cell surface. Here, we report the use of azido-modified unnatural sialic acid to identify those molecules that act as cell surface acceptors of the sialyl residue in the trans-sialidase-catalyzed reaction, which might then be involved in the immune alterations induced. In living parasites, we readily observed the transfer of azido-sialic acid to surface mucins. When evaluating mouse thymocytes and splenocytes as acceptors of the azido-sugar, a complex pattern of efficiently tagged glycoproteins was revealed. In both leukocyte populations, the main proteins labeled were identified as different CD45 isoforms. Disruption of the cell architecture increased the number and the molecular weight distribution of azido-sialic acid tagged proteins. Nevertheless, CD45 remained to be the main acceptor. Mass spectrometry assays allowed us to identify other acceptors, mainly integrins. The findings reported here provide a molecular basis to understand the abnormalities induced in the immune system by the trans-sialidase during T. cruzi infection.

Stage-specific expression of Trypanosoma cruzi trans-sialidase involves highly conserved 3' untranslated regions.

trans-Sialidases (TSs) are virulence factors that allow some trypanosomatids to incorporate sialic acid from host molecules. Trypanosoma cruzi bears a complex gene family coding for TS members, which can be broadly divided into two groups: one translated in stages present in the mammalian host (trypomastigote TS, tTS) and one translated in the insect vector stages (epimastigote TS, eTS). The molecular basis underlying the expression of different, nonoverlapping sets of TS proteins in either host is poorly understood, particularly because of the lack of transcription initiation control in this organism. Here we show that 3' untranslated regions (3'UTRs) of tTS and eTS are highly conserved within each gene group but completely different between both groups. Importantly, tTS-3'UTR but not eTS-3'UTR promoted high expression of the green fluorescent protein reporter gene in the mammalian-dwelling stages. In epimastigotes, both 3'UTRs lead to a comparatively low expression of the reporter gene, although eTS-3'UTR was more efficient than tTS-3'UTR. These results stress the importance of posttranscriptional events, mainly driven by specific 3'UTRs, in gene expression regulation in T. cruzi.