Repeat-enriched proteins are related to host cell invasion and immune evasion in parasitic protozoa.

Proteins containing repetitive amino acid domains are widespread in all life forms. In parasitic organisms, proteins containing repeats play important roles such as cell adhesion and invasion and immune evasion. Therefore, extracellular and intracellular parasites are expected to be under different selective pressures regarding the repetitive content in their genomes. Here, we investigated whether there is a bias in the repetitive content found in the predicted proteomes of 6 exclusively extracellular and 17 obligate intracellular protozoan parasites, as well as 4 free-living protists. We also attempted to correlate the results with the distinct ecological niches they occupy and with distinct protein functions. We found that intracellular parasites have higher repetitive content in their proteomes than do extracellular parasites and free-living protists. In intracellular parasites, these repetitive proteins are located mainly at the parasite surface or are secreted and are enriched in amino acids known to be part of N- and O-glycosylation sites. Furthermore, in intracellular parasites, the developmental stages that are able to invade host cells express a higher proportion of proteins with perfect repeats relative to other life cycle stages, and these proteins have molecular functions associated with cell invasion. In contrast, in extracellular parasites, degenerate repetitive motifs are enriched in proteins that are likely to play roles in evading host immune response. Altogether, our results support the hypothesis that both the ability to invade host cells and to escape the host immune response may have shaped the expansion and maintenance of perfect and degenerate repeats in the genomes of intra- and extracellular parasites.

Identification of genes encoding hypothetical proteins in open-reading frame expressed sequence tags from mammalian stages of Trypanosoma cruzi.

Approximately 50% of the predicted protein-coding genes of the Trypanosoma cruzi CL Brener strain are annotated as hypothetical or conserved hypothetical proteins. To further characterize these genes, we generated 1161 open-reading frame expressed sequence tags (ORESTES) from the mammalian stages of the VL10 human strain. Sequence clustering resulted in 435 clusters, consisting of 339 singletons and 96 contigs. Significant matches to the T. cruzi predicted gene database were found for ~94% contigs and ~69% singletons. These included genes encoding surface proteins, known to be intensely expressed in the parasite mammalian stages and implicated in host cell invasion and/or immune evasion mechanisms. Among 151 contigs and singletons with similarity to predicted hypothetical protein-coding genes and conserved hypothetical protein-coding genes, 83% showed no match with T. cruzi EST and/or proteome databases. These ORESTES are the first experimental evidence that the corresponding genes are in fact transcribed. Sequences with no significant match were searched against several T. cruzi and National Center for Biotechnology Information non-redundant sequence databases. The ORESTES analysis indicated that 124 predicted conserved hypothetical protein-coding genes and 27 predicted hypothetical protein-coding genes annotated in the CL Brener genome are transcribed in the VL10 mammalian stages. Six ORESTES annotated as hypothetical protein-coding genes showing no match to EST and/or proteome databases were confirmed by Northern blot in VL10. The generation of this set of ORESTES complements the T. cruzi genome annotation and suggests new stage-regulated genes encoding hypothetical proteins.

Epidemiologic aspects of an outbreak of Trypanosoma vivax in a dairy cattle herd in Minas Gerais state, Brazil.

The aim of this study was to assess the epidemiological situation of bovine trypanosomiasis caused by Trypanosoma vivax in a dairy cattle herd from Igarapé, Minas Gerais state, Brazil. The herd was monitored from September 2007 to February 2009 by sampling blood for determination of packed cell volume (PCV), microhaematocrit centrifugation test of parasitaemia (MHCT), serology (IFA), morphological identification of T. vivax and molecular diagnosis by polymerase chain reaction (PCR). During all the experimental period, 25 animals were MHCT and PCR positive, considering that in each sample collection a mean of 70 animals was evaluated. The morphometric characteristics of trypomastigote forms confirmed the infection by T. vivax. The seroprevalence ranged from 7.4% in September 2007 to 48% in February 2009, and the highest incidence observed could be correlated with an increased population of Stomoxys calcitrans flies in that region. Anaemia was the most important change found in infected animals, which showed lower averages of PCV than parasitologically negative animals (p<0.0001). Infected individuals showed lower averages of PCV than parasitologically negative animals (p<0.0001), indicating higher anaemia in the former compared with the latter group.

Molecular cloning and characterization of a gene encoding the 29-kDa proteasome subunit from Trypanosoma cruzi.

Antiserum raised against purified Trypanosoma cruzi proteasomes was used to isolate two cDNA clones, tcpr29 and tcpr29B, and the corresponding genomic sequence, termed tcpr29A. Both cDNAs and the gene contain a 798-bp ORF, coding for a 266-amino acid protein, with a predicted molecular mass of 29 kDa. Sequence comparisons show that the protein encoded by tcpr29 belongs to the alpha6 subfamily of proteasome subunits. Southern analysis indicated that tcpr29 subunit is encoded by a single-copy gene which maps to chromosome 20 of the CL Brener clone. Allelic variants were found in other T. cruzi isolates, suggesting heterozygosity for the gene in some and homozygosity in other strains. A spliced-leader addition site was identified 123 bp upstream from the start codon, generating a stable 1.5-kb transcript. Western analysis revealed that tcpr29A is constitutively expressed during the life cycle of the parasite.

Molecular cloning and characterization of the DNA mismatch repair gene class 2 from the Trypanosoma cruzi.

Genes with homology to the bacterial mutS gene, which encodes a protein involved in post-replication DNA mismatch repair, are known in several organisms. Using a degenerate PCR strategy, we cloned a Trypanosoma cruzi genomic DNA fragment homologous to the mutS gene class two (MSH2). This fragment was used as a probe to select the corresponding cDNAs from a T. cruzi cDNA library. The complete sequence of the gene (3304 bp), denominated TcMSH2, was obtained. The sequence contained an open reading frame of 2889 bp coding for a putative protein of 962 amino acids. Computational analyses of the amino acid sequence showed 36% identity with MSH2 proteins from other eukaryotes and revealed the presence of all functional domains of MutS proteins. Hybridization analyses indicated that the TcMSH2 gene is present as a single copy gene that is expressed in all forms of the T. cruzi life cycle. The role of the product of the TcMSH2 gene in mismatch repair was investigated by negative dominance phenotype analyses in Escherichia coli. When eukaryotic muts genes are expressed in a prokaryotic system, they increase the bacterial mutation rate. The same phenomenon was observed with the TcMSH2 cDNA, indicating that T. cruzi MSH2 interferes with the bacterial mismatch system. Phylogenetic analyses showed that the T. cruzi gene grouped with the MSH2 clade confirming the nature of the gene isolated in this work.