The repetitive cytoskeletal protein H49 of Trypanosoma cruzi is a calpain-like protein located at the flagellum attachment zone

Paracoccidioides brasiliensis causes paracoccidioidomycosis, a systemic mycosis in Latin America. Formamidases hydrolyze formamide, putatively plays a role infungal nitrogen metabolism. An abundant 45-kDa protein was identified as the P. brasiliensis formamidase. In this study, recombinant formamidase was over-expressed in bacteria and a polyclonal antibody to this protein was produced. Weidentified a 180-kDa protein species reactive to the antibody produced in miceagainst the P. brasiliensis recombinant purified formamidase of 45 kDa. The180-kDa purified protein yielded a heat-denatured species of 45 kDa. Both protein species of 180 and 45 kDa were identified as formamidase by peptide massfinger printing using MS. The identical mass spectra generated by the 180 and the45-kDa protein species indicated that the fungal formamidase is most likely homotetrameric in its native conformation. Furthermore, the purified formami-dase migrated as a protein of 191 kDa in native polyacrylamide gel electrophoresis, thus revealing that the enzyme forms a homotetrameric structure in its native state. This enzyme is present in the fungus cytoplasm and the cell wall. Use of a yeast two-hybrid system revealed cell wall membrane proteins, in addition to cytosolic proteins interacting with formamidase. These data provide new insights intoformamidase structure as well as potential roles for formamidase and its interaction partners in nitrogen metabolism

Genome of the Avirulent Human-Infective Trypanosome—Trypanosoma rangeli

Background: Trypanosoma rangeli is a hemoflagellate protozoan parasite infecting humans and other wild and domestic mammals across Central and South America. It does not cause human disease, but it can be mistaken for the etiologic agent of Chagas disease, Trypanosoma cruzi. We have sequenced the T. rangeli genome to provide new tools for elucidating the distinct and intriguing biology of this species and the key pathways related to interaction with its arthropod and mammalian hosts.  Methodology/Principal Findings: The T. rangeli haploid genome is ,24 Mb in length, and is the smallest and least repetitive trypanosomatid genome sequenced thus far. This parasite genome has shorter subtelomeric sequences compared to those of T. cruzi and T. brucei; displays intraspecific karyotype variability and lacks minichromosomes. Of the predicted 7,613 protein coding sequences, functional annotations could be determined for 2,415, while 5,043 are hypothetical proteins, some with evidence of protein expression. 7,101 genes (93%) are shared with other trypanosomatids that infect humans. An ortholog of the dcl2 gene involved in the T. brucei RNAi pathway was found in T. rangeli, but the RNAi machinery is non-functional since the other genes in this pathway are pseudogenized. T. rangeli is highly susceptible to oxidative stress, a phenotype that may be explained by a smaller number of anti-oxidant defense enzymes and heatshock proteins.  Conclusions/Significance: Phylogenetic comparison of nuclear and mitochondrial genes indicates that T. rangeli and T. cruzi are equidistant from T. brucei. In addition to revealing new aspects of trypanosome co-evolution within the vertebrate and invertebrate hosts, comparative genomic analysis with pathogenic trypanosomatids provides valuable new information that can be further explored with the aim of developing better diagnostic tools and/or therapeutic targets

Interclonal Variations in the Molecular Karyotype of <i>Trypanosoma cruzi</i>: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

<div><p><i>Trypanosoma cruzi</i> comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the <i>T. cruzi</i> G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for <i>Leishmania infantum,</i> have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the <i>T. cruzi</i> arsenal for responding to environmental pressure.</p></div

Identification of homologous chromosomal bands of similar molecular sizes in the G strain and clone D11.

<p>Hybridization profile of specific chromosomal markers hybridized to one or more bands of similar molecular size in both isolates after chromosome separation by PFGE and Southern-blot hybridization. The markers used are TEUF0099, rDNA18S, TEUF0242 and ADC. Gene identification and GenBank accession number of each marker are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063738#pone-0063738-t001" target="_blank">Table 1</a>.</p

Location of molecular markers on G strain and clone D11 chromosomal bands.

#<p>Chromosomal bands separated by PFGE and stained with ethidium bromide.</p

Identification of a rearrangement involving a large fragment containing the α- tubulin gene in clone D11.

<p><b>Panel A</b>) Mapping of the α-tubulin gene on chromosomal bands of the G strain and clone D11 showing a translocation event involving large chromosomes. β-tubulin, hypothetical protein XM_804243 and endomembrane protein (XM_ 812238) were also mapped and showed the same hybridization profile. The positions of markers used as probes are indicated in the diagrammatic representation of in silico chromosomes TcChr14. <b>Panel B</b>) Restriction fragment analysis of α-tubulin gene loci was carried out by digesting genomic DNA with <i>Pst</i>I (P) or double-digesting it with <i>Bgl</i>II and <i>Pst</i>I (B/P). Phage lambda DNA digested with <i>Hae</i>III, used as a molecular weight marker, is shown on the left. <b>Panel C</b>) Restriction analysis of whole chromosomes in agarose blocks was performed using the rare-cutting enzymes <i>Pac</i>I and <i>Sfi</i>I. The molecular weights of fragments recognized by the probe are shown on the left.</p

Allele sizes (bp) for each microsatellite locus amplified for the G strain and clone D11.

a<p>Microsatellite loci with the same alleles in the G strain and D11 clone.</p>b<p>Microsatellite loci with a common allele in the G strain and D11 clone.</p>c<p>Microsatellite loci with different alleles in the G strain and D11 clone.</p

Telomere length polymorphism of the G strain and clone D11.

<p><b>Panel A</b>) Southern-blot hybridization of restriction fragments generated by <i>Hae</i>III and <i>Msp</i>I probed with the telomeric repeat (TTAGGG). <i>Hae</i>III-digested phage lambda DNA (used as a molecular weight marker) is shown on the left. <b>Panel B</b>) Analysis of the subtelomeric length of the G strain and clone D11 chromosomes was performed by Southern-blot hybridization of <i>Sfi</i>I restriction fragments with the telomeric repeat. The size of the larger subtelomeric fragment of clone D11 is shown on the left.</p

Karyotype polymorphism between the G strain and clone D11.

<p><b>Panel A)</b> Chromosomal bands were separated by Pulsed-Field Gel Electrophoresis (PFGE) and stained with SYBR Green I. The bands from the G strain were numbered using Arabic numerals (1–19) as in a previous study (Souza et al., 2011) while capital letters (A – U) were used for clone D11, starting from the smallest band. <b>Panel B)</b> Diagrammatic representation of the molecular karyotypes of the G strain and clone D11. The rectangles represent a unique distinguishable band visualized after SYBR Green I staining. The thickness of the rectangles represents the proportional staining of each chromosomal band. The number and letter of chromosomal bands as well as their molecular weight are indicated to the left and right of each strip, respectively.</p