First Confirmed Diagnosis of Nocardioform Placentitis (Amycolatopsis lexingtonensis) in South America.

Nocardioform placentitis is a pathologically unique form of placental disease first diagnosed in central Kentucky in the mid-80s. Since then, the occurrence of nocardioform placentitis in the region has varied over the years, from sporadic cases to outbreaks. The disease has been sporadically detected in other countries and has not been confirmed in South America. A 13-year-old multiparous Mangalarga delivered a healthy filly at 340d gestation. The mare passed the fetal membranes 33 minute after foaling. Gross examination of the fetal membranes identified two focal lesions on the chorionic surface consistent with focal mucoid placentitis. Histopathologic evaluation revealed hyperplasia and degeneration of the allantoic mesoderm, intense mononuclear inflammatory infiltrates with marked lymphocytes and plasma, and occasional macrophages and neutrophils in the microvilli. Necrotic debris and exudate were identified in the chorionic epithelium, with macrophages, plasma cells, and neutrophils confirming the diagnosis of focal mucoid placentitis. The exudate culture revealed white, firm, punctiform colonies of ∼1 mm diameter. Gram staining revealed bacilli with rounded ends and branching aspect typical of actinomycetes. PCR using primers for the 16S rRNA identified the genera of bacteria as Amycolatopsis. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis identified the isolate as Amycolatopsis lexingtonensis. In conclusion, we described the first confirmed case of nocardioform placentitis in South America. The present case was associated with the birth of a full-term healthy live foal; this result is consistent with Amycolatopsis spp and, in this case, was caused by A. lexingtonensis.

Molecular Characterization of Trypanosoma evansi Mevalonate Kinase (TeMVK).

The mevalonate pathway is an essential part of isoprenoid biosynthesis leading to production of a diverse class of >30,000 biomolecules including cholesterol, heme, and all steroid hormones. In trypanosomatids, the mevalonate pathway also generates dolichols, which play an essential role in construction of glycosylphosphatidylinositol (GPI) molecules that anchor variable surface proteins (VSGs) to the plasma membrane. Isoprenoid biosynthesis involves one of the most highly regulated enzymes in nature, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which catalyzes the conversion of HMG-CoA to mevalonic acid. The enzyme mevalonate kinase (MVK) subsequently converts mevalonic acid to 5-phosphomevalonic acid. Trypanosoma evansi is a flagellate protozoan parasite that causes the disease "Surra" in domesticated large mammals, with great economic impact. T. evansi has only a trypomastigote bloodstream form and requires constant modification of the variant surface glycoprotein (VSG) coat for protection against the host immune system. We identified MVK of T. evansi (termed TeMVK) and performed a preliminary characterization at molecular, biochemical, and cellular levels. TeMVK from parasite extract displayed molecular weight ~36 kDa, colocalized with aldolase (a glycosomal marker enzyme) in glycosomes, and is structurally similar to Leishmania major MVK. Interestingly, the active form of TeMVK is the tetrameric oligomer form, in contrast to other MVKs in which the dimeric form is active. Despite lacking organized mitochondria, T. evansi synthesizes both HMGCR transcripts and protein. Both MVK and HMGCR are expressed in T. evansi during the course of infection in animals, and therefore are potential targets for therapeutic drug design.

The diversity and expansion of the trans-sialidase gene family is a common feature in Trypanosoma cruzi clade members.

Trans-sialidase (TS) is a polymorphic protein superfamily described in members of the protozoan genus Trypanosoma. Of the eight TS groups recently described, TS group I proteins (some of which have catalytic activity) are present in the distantly related Trypanosoma brucei and Trypanosoma cruzi phylogenetic clades, whereas other TS groups have only been described in some species belonging to the T. cruzi clade. In the present study we analyzed the repertoire, distribution and phylogenetic relationships of TS genes among species of the T. cruzi clade based on sequence similarity, multiple sequence alignment and tree-reconstruction approaches using TS sequences obtained with the aid of PCR-based strategies or retrieved from genome databases. We included the following representative isolates of the T. cruzi clade from South America: T. cruzi, T. cruzi Tcbat, Trypanosoma cruzi marinkellei, Trypanosoma dionisii, Trypanosoma rangeli and Trypanosoma conorhini. The cloned sequences encoded conserved TS protein motifs Asp-box and VTVxNVxLYNR but lacked the FRIP motif (conserved in TS group I). The T. conorhini sequences were the most divergent. The hybridization patterns of TS probes with chromosomal bands confirmed the abundance of these sequences in species in the T. cruzi clade. Divergence and relationship analysis placed most of the TS sequences in the groups defined in T. cruzi. Further examination of members of TS group II, which includes T. cruzi surface glycoproteins implicated in host cell attachment and invasion, showed that sequences of T. cruzi Tcbat grouped with those of T. cruzi genotype TcI. Our analysis indicates that different members of the T. cruzi clade, with different vertebrate hosts, vectors and pathogenicity, share the extensive expansion and sequence diversification of the TS gene family. Altogether, our results are congruent with the evolutionary history of the T. cruzi clade and represent a contribution to the understanding of the molecular evolution and role of TS proteins in trypanosomes.

Chromosomal polymorphism in the Sporothrix schenckii complex.

Sporotrichosis is a polymorphic disease caused by a complex of thermodimorphic fungi including S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa and S. luriei. Humans and animals can acquire the disease through traumatic inoculation of propagules into the subcutaneous tissue. Despite the importance of sporotrichosis as a disease that can take epidemic proportions there are just a few studies dealing with genetic polymorphisms and genomic architecture of these pathogens. The main objective of this study was to investigate chromosomal polymorphisms and genomic organization among different isolates in the S. schenckii complex. We used pulsed field gel electrophoresis (PFGE) to separate chromosomal fragments of isolated DNA, followed by probe hybridization. Nine loci (ß-tubulin, calmodulin, catalase, chitin synthase 1, Internal Transcribed Spacer, Pho85 cyclin-dependent kinase, protein kinase C Ss-2, G protein α subunit and topoisomerase II) were mapped onto chromosomal bands of Brazilian isolates of S. schenckii s. str. and S. brasiliensis. Our results revealed the presence of intra and interspecies polymorphisms in chromosome number and size. The gene hybridization analysis showed that closely related species in phylogenetic analysis had similar genetic organizations, mostly due to identification of synteny groups in chromosomal bands of similar sizes. Our results bring new insights into the genetic diversity and genome organization among pathogenic species in the Sporothrix schenckii complex.

Anatomy and evolution of telomeric and subtelomeric regions in the human protozoan parasite Trypanosoma cruzi.

BACKGROUND: The subtelomeres of many protozoa are highly enriched in genes with roles in niche adaptation. T. cruzi trypomastigotes express surface proteins from Trans-Sialidase (TS) and Dispersed Gene Family-1 (DGF-1) superfamilies which are implicated in host cell invasion. Single populations of T. cruzi may express different antigenic forms of TSs. Analysis of TS genes located at the telomeres suggests that chromosome ends could have been the sites where new TS variants were generated. The aim of this study is to characterize telomeric and subtelomeric regions of T. cruzi available in TriTrypDB and connect the sequences of telomeres to T. cruzi working draft sequence. RESULTS: We first identified contigs carrying the telomeric repeat (TTAGGG). Of 49 contigs identified, 45 have telomeric repeats at one end, whereas in four contigs the repeats are located internally. All contigs display a conserved telomeric junction sequence adjacent to the hexamer repeats which represents a signature of T. cruzi chromosome ends. We found that 40 telomeric contigs are located on T. cruzi chromosome-sized scaffolds. In addition, we were able to map several telomeric ends to the chromosomal bands separated by pulsed-field gel electrophoresis.The subtelomeric sequence structure varies widely, mainly as a result of large differences in the relative abundance and organization of genes encoding surface proteins (TS and DGF-1), retrotransposon hot spot genes (RHS), retrotransposon elements, RNA-helicase and N-acetyltransferase genes. While the subtelomeric regions are enriched in pseudogenes, they also contain complete gene sequences matching both known and unknown expressed genes, indicating that these regions do not consist of nonfunctional DNA but are instead functional parts of the expressed genome. The size of the subtelomeric regions varies from 5 to 182 kb; the smaller of these regions could have been generated by a recent chromosome breakage and telomere healing event. CONCLUSIONS: The lack of synteny in the subtelomeric regions suggests that genes located in these regions are subject to recombination, which increases their variability, even among homologous chromosomes. The presence of typical subtelomeric genes can increase the chance of homologous recombination mechanisms or microhomology-mediated end joining, which may use these regions for the pairing and recombination of free ends.

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

BACKGROUND: Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49) which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein. In the current study, the genomic complexity of H49 and its relationships to the T. cruzi calpain-like cysteine peptidase family, comprising active calpains and calpain-like proteins, is addressed. Immunofluorescence analysis and biochemical fractionation were used to demonstrate the cellular location of H49 proteins. METHODS AND FINDINGS: All of H49 repeats are associated with calpain-like sequences. Sequence analysis demonstrated that this protein, now termed H49/calpain, consists of an amino-terminal catalytic cysteine protease domain II, followed by a large region of 68-amino acid repeats tandemly arranged and a carboxy-terminal segment carrying the protease domains II and III. The H49/calpains can be classified as calpain-like proteins as the cysteine protease catalytic triad has been partially conserved in these proteins. The H49/calpains repeats share less than 60% identity with other calpain-like proteins in Leishmania and T. brucei, and there is no immunological cross reaction among them. It is suggested that the expansion of H49/calpain repeats only occurred in T. cruzi after separation of a T. cruzi ancestor from other trypanosomatid lineages. Immunofluorescence and immunoblotting experiments demonstrated that H49/calpain is located along the flagellum attachment zone adjacent to the cell body. CONCLUSIONS: H49/calpain contains large central region composed of 68-amino acid repeats tandemly arranged. They can be classified as calpain-like proteins as the cysteine protease catalytic triad is partially conserved in these proteins. H49/calpains could have a structural role, namely that of ensuring that the cell body remains attached to the flagellum by connecting the subpellicular microtubule array to it.

Genome size, karyotype polymorphism and chromosomal evolution in Trypanosoma cruzi.

BACKGROUND: The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites. METHODOLOGY/PRINCIPAL FINDINGS: The genome sizes, repetitive DNA contents, and the numbers and sizes of chromosomes of nine strains of T. cruzi from four lineages (TcI, TcII, TcV and TcVI) were determined. The genome of the TcI group was statistically smaller than other lineages, with the exception of the TcI isolate Tc1161 (José-IMT). Satellite DNA content was correlated with genome size for all isolates, but this was not accompanied by simultaneous amplification of retrotransposons. Regardless of chromosomal polymorphism, large syntenic groups are conserved among T. cruzi lineages. Duplicated chromosome-sized regions were identified and could be retained as paralogous loci, increasing the dosage of several genes. By comparing T. cruzi and T. brucei chromosomes, homologous chromosomal regions in T. brucei were identified. Chromosomes Tb9 and Tb11 of T. brucei share regions of syntenic homology with three and six T. cruzi chromosomal bands, respectively. CONCLUSIONS: Despite genome size variation and karyotype polymorphism, T. cruzi lineages exhibit conservation of chromosome structure. Several syntenic groups are conserved among all isolates analyzed in this study. The syntenic regions are larger than expected if rearrangements occur randomly, suggesting that they are conserved owing to positive selection. Mapping of the syntenic regions on T. cruzi chromosomal bands provides evidence for the occurrence of fusion and split events involving T. brucei and T. cruzi chromosomes.

New Trypanosoma cruzi repeated element that shows site specificity for insertion.

A new family of site-specific repeated elements identified in Trypanosoma cruzi, which we named TcTREZO, is described here. TcTREZO appears to be a composite repeated element, since three subregions may be defined within it on the basis of sequence similarities with other T. cruzi sequences. Analysis of the distribution of TcTREZO in the genome clearly indicates that it displays site specificity for insertion. Most TcTREZO elements are flanked by conserved sequences. There is a highly conserved 68-bp sequence at the 5' end of the element and a sequence domain of approximately 500 bp without a well-defined borderline at the 3' end. Northern blot hybridization and reverse transcriptase PCR analyses showed that TcTREZO transcripts are expressed as oligo(A)-terminated transcripts whose length corresponds to the unit size of the element (1.6 kb). Transcripts of approximately 0.2 kb derived from a small part of TcTREZO are also detected in steady-state RNA. TcTREZO transcripts are unspliced and not translated. The copy number of TcTREZO sequences was estimated to be approximately 173 copies per haploid genome. TcTREZO appears to have been assembled by insertions of sequences into a progenitor element. Once associated with each other, these subunits were amplified as a new transposable element. TcTREZO shows site specificity for insertion, suggesting that a sequence-specific endonuclease could be responsible for its insertion at a unique site.