Schwann cell reprogramming into repair cells increases miRNA-21 expression in exosomes promoting axonal growth.

Functional recovery after peripheral nerve damage is dependent on the reprogramming of differentiated Schwann cells (dSCs) into repair Schwann cells (rSCs), which promotes axonal regeneration and tissue homeostasis. Transition into a repair phenotype requires expression of c-Jun and Sox2, which transcriptionally mediates inhibition of the dSC program of myelination and activates a non-cell-autonomous repair program, characterized by the secretion of neuronal survival and regenerative molecules, formation of a cellular scaffold to guide regenerating axons and activation of an innate immune response. Moreover, rSCs release exosomes that are internalized by peripheral neurons, promoting axonal regeneration. Here, we demonstrate that reprogramming of Schwann cells (SCs) is accompanied by a shift in the capacity of their secreted exosomes to promote neurite growth, which is dependent on the expression of c-Jun (also known as Jun) and Sox2 by rSCs. Furthermore, increased expression of miRNA-21 is responsible for the pro-regenerative capacity of rSC exosomes, which is associated with PTEN downregulation and PI3-kinase activation in neurons. We propose that modification of exosomal cargo constitutes another important feature of the repair program of SCs, contributing to axonal regeneration and functional recovery after nerve injury.

Campylobacter ornithocola sp. nov., a novel member of the Campylobacter lari group isolated from wild bird faecal samples.

During a study on the prevalence and diversity of campylobacteria in wild birds faecal samples from the city of Valdivia (southern Chile) 17 Gram-stain-negative, curved-rod-shaped isolates, were initially identified as Campylobacter lari by PCR-RFLP. Further identification by 16S rRNA sequence analysis revealed that they formed a distinct group in the genus Campylobacter. This unique position was confirmed by the results of analysis of rpoB, atpA and cpn60 gene sequences. The average nucleotide identity between the representative strain WBE38T and the type strain of the most closely related taxon C. larisubsp.concheus (LMG 11760) was 90.8 %. The oxidase and urease activity of the novel isolates enabled them to be phenotypically differentiated from species of the genus Campylobacter with validly published names. Therefore, on the basis of phenotypic, genetic and genomic characterizations, the results of this study clearly indicate that these strains represent a novel species within the genus Campylobacter, for which the name Campylobacter ornithocola sp. nov. is proposed, with the type strain WBE38T (=CECT 9147T=LMG 29815T).

Paracrine Signaling Mediated by the Cytosolic Tryparedoxin Peroxidase of Trypanosoma cruzi.

Peroxiredoxins are abundant and ubiquitous proteins that participate in different cellular functions, such as oxidant detoxification, protein folding, and intracellular signaling. Under different cellular conditions, peroxiredoxins can be secreted by different parasites, promoting the induction of immune responses in hosts. In this work, we demonstrated that the cytosolic tryparedoxin peroxidase of Trypanosoma cruzi (cTXNPx) is secreted by epimastigotes and trypomastigotes associated with extracellular vesicles and also as a vesicle-free protein. By confocal microscopy, we show that cTXNPx can enter host cells by an active mechanism both through vesicles and as a recombinant protein. Transcriptomic analysis revealed that cTXNPx induces endoplasmic reticulum stress and interleukin-8 expression in epithelial cells. This analysis also suggested alterations in cholesterol metabolism in cTXNPx-treated cells, which was confirmed by immunofluorescence showing the accumulation of LDL and the induction of LDL receptors in both epithelial cells and macrophages. BrdU incorporation assays and qPCR showed that cTXNPx has a mitogenic, proliferative, and proinflammatory effect on these cells in a dose-dependent manner. Importantly, we also demonstrated that cTXNPx acts as a paracrine virulence factor, increasing the susceptibility to infection in cTXNPx-pretreated epithelial cells by approximately 40%. Although the results presented in this work are from in vitro studies and likely underestimate the complexity of parasite-host interactions, our work suggests a relevant role for this protein in establishing infection.

Genome-wide chromatin interaction map for Trypanosoma cruzi.

Trypanosomes are eukaryotic, unicellular parasites, such as Trypanosoma brucei, which causes sleeping sickness, and Trypanosoma cruzi, which causes Chagas disease. Genomes of these parasites comprise core regions and species-specific disruptive regions that encode multigene families of surface glycoproteins. Few transcriptional regulators have been identified in these parasites, and the role of spatial organization of the genome in gene expression is unclear. Here we mapped genome-wide chromatin interactions in T. cruzi using chromosome conformation capture (Hi-C), and we show that the core and disruptive regions form three-dimensional chromatin compartments named C and D. These chromatin compartments differ in levels of DNA methylation, nucleosome positioning and chromatin interactions, affecting genome expression dynamics. Our data reveal that the trypanosome genome is organized into chromatin-folding domains and transcription is affected by the local chromatin structure. We propose a model in which epigenetic mechanisms affect gene expression in trypanosomes.

New Insights into the Role of the Trypanosoma cruzi Aldo-Keto Reductase TcAKR.

Chagas disease is a zoonotic infectious disease caused by the protozoan parasite Trypanosoma cruzi. It is distributed worldwide, affecting around 7 million people; there is no effective treatment, and it constitutes a leading cause of disability and premature death in the Americas. Only two drugs are currently approved for the treatment, Benznidazole and Nifurtimox, and both have to be activated by reducing the nitro-group. The T. cruzi aldo-keto reductase (TcAKR) has been related to the metabolism of benznidazole. TcAKR has been extensively studied, being most efforts focused on characterizing its implication in trypanocidal drug metabolism; however, little is known regarding its biological role. Here, we found that TcAKR is confined, throughout the entire life cycle, into the parasite mitochondria providing new insights into its biological function. In particular, in epimastigotes, TcAKR is associated with the kinetoplast, which suggests additional roles of the protein. The upregulation of TcAKR, which does not affect TcOYE expression, was correlated with an increase in PGF2α, suggesting that this enzyme is related to PGF2α synthesis in T. cruzi. Structural analysis showed that TcAKR contains a catalytic tetrad conserved in the AKR superfamily. Finally, we found that TcAKR is also involved in Nfx metabolization.

Nanopore Long Read DNA Sequencing of Protozoan Parasites: Hybrid Genome Assembly of Trypanosoma cruzi.

Due to highly repetitive genome sequences, short-read-based Trypanosoma cruzi genomes are extremely fragmented. Contiguous trypanosomatid genomes assemblies have resulted in the advent of third-generation sequencing technologies. Long reads span several to hundreds of kbps allowing to correct assemblies of repeated and low complexity DNA regions. However, these techniques present higher error rates. Hybrid assembly strategies that combine error-prone long reads with much more accurate Illumina short reads represent a very convenient approach for enhancing genome completeness. Here, we describe how to perform a hybrid assembly for genomic analysis of protozoan pathogens using Illumina and Oxford Nanopore sequencing.

Maxicircle architecture and evolutionary insights into Trypanosoma cruzi complex.

We sequenced maxicircles from T. cruzi strains representative of the species evolutionary diversity by using long-read sequencing, which allowed us to uncollapse their repetitive regions, finding that their real lengths range from 35 to 50 kb. T. cruzi maxicircles have a common architecture composed of four regions: coding region (CR), AT-rich region, short (SR) and long repeats (LR). Distribution of genes, both in order and in strand orientation are conserved, being the main differences the presence of deletions affecting genes coding for NADH dehydrogenase subunits, reinforcing biochemical findings that indicate that complex I is not functional in T. cruzi. Moreover, the presence of complete minicircles into maxicircles of some strains lead us to think about the origin of minicircles. Finally, a careful phylogenetic analysis was conducted using coding regions of maxicircles from up to 29 strains, and 1108 single copy nuclear genes from all of the DTUs, clearly establishing that taxonomically T. cruzi is a complex of species composed by group 1 that contains clades A (TcI), B (TcIII) and D (TcIV), and group 2 (1 and 2 do not coincide with groups I and II described decades ago) containing clade C (TcII), being all hybrid strains of the BC type. Three variants of maxicircles exist in T. cruzi: a, b and c, in correspondence with clades A, B, and C from mitochondrial phylogenies. While A and C carry maxicircles a and c respectively, both clades B and D carry b maxicircle variant; hybrid strains also carry the b- variant. We then propose a new nomenclature that is self-descriptive and makes use of both the phylogenetic relationships and the maxicircle variants present in T. cruzi.

Host-pathogen transcriptomics: Trypanosoma cruzi as a model for studying RNA contamination.

Cellular infection assays constitute essential tools to understand host-pathogen interactions, particularly for intracellular microorganisms that are produced in cell lines are needed to propagate the microorganism. In this work, we demonstrate that RNA derived from Vero cells is an important contaminant to consider in order to avoid false positive results in transcriptomic experiments. We study the cross-contamination on a Trypanosoma cruzi cell infection model, the etiological agent of Chagas disease. We implemented the most frequently used trypanosome-purification protocols and, for all of them, we detected RNAs derived from Vero cells in trypomastigote extracts. For some of the protocols we also detected Vero RNAs in infected human cells. We also found this type of contamination in microarray experiments of human samples infected with T. cruzi. Concerning Illumina RNA-Seq data, we found that the contamination with Vero cells is probably introducing spurious results. Finally, we recommend a protocol to purify trypomastigotes, which showed a high percentage of trypomastigote recovery and the absence of Vero contamination in infected human samples. Avoiding this type of contamination should be an important factor to consider during experimental design, in order to minimize false positive results in transcriptomic studies as well as RNA contamination in vaccine production. SIGNIFICANCE: Transcriptomic studies are widely used to understand host-pathogen interactions. When the pathogen is an intracellular microorganism, an additional mammalian cell system can be needed to propagate it. In this work we demonstrate that pathogens purified from infected monolayers can carry RNAs from these mammalian cells, and that this ambient RNA contamination is probably producing false positive results in subsequent transcriptomic studies performed with qRT-PCR, microarrays or Next Generation Sequencing.

Transcriptome Studies in Trypanosoma cruzi Using RNA-seq.

RNA-seq is a powerful method to study in detail transcriptome changes in defined conditions, providing enormous amount of information on RNA stability and gene regulation. In this chapter, we describe a directional and a nondirectional library preparation protocol for RNA-seq in Trypanosoma cruzi, as well as a pipeline for bioinformatic analysis, which includes read trimming, alignment to a reference genome, and differential expression.

The Tritryps Comparative Repeatome: Insights on Repetitive Element Evolution in Trypanosomatid Pathogens.

The major human pathogens Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major are collectively known as the Tritryps. The initial comparative analysis of their genomes has uncovered that Tritryps share a great number of genes, but repetitive DNA seems to be extremely variable between them. However, the in-depth characterization of repetitive DNA in these pathogens has been in part neglected, mainly due to the well-known technical challenges of studying repetitive sequences from de novo assemblies using short reads. Here, we compared the repetitive DNA repertories between the Tritryps genomes using genome-wide, low-coverage Illumina sequencing coupled to RepeatExplorer analysis. Our work demonstrates that this extensively implemented approach for studying higher eukaryote repeatomes is also useful for protozoan parasites like trypanosomatids, as we recovered previously observed differences in the presence and amount of repetitive DNA families. Additionally, our estimations of repetitive DNA abundance were comparable to those obtained from enhanced-quality assemblies using longer reads. Importantly, our methodology allowed us to describe a previously undescribed transposable element in Leishmania major (TATE element), highlighting its potential to accurately recover distinctive features from poorly characterized repeatomes. Together, our results support the application of this low-cost, low-coverage sequencing approach for the extensive characterization of repetitive DNA evolutionary dynamics in trypanosomatid and other protozoan genomes.

Draft Genome Sequences of 40 Pathogenic Leptospira Strains Isolated from Cattle in Uruguay.

Pathogenic Leptospira species represent a major concern for livestock but also for human health, as they cause zoonotic infections. Forty strains representing L. interrogans, L. borgpetersenii, and L. noguchii were isolated from naturally infected cattle in Uruguay. Here, we report the whole-genome sequences for these strains.

Nanopore Sequencing Significantly Improves Genome Assembly of the Protozoan Parasite Trypanosoma cruzi.

Chagas disease was described by Carlos Chagas, who first identified the parasite Trypanosoma cruzi from a 2-year-old girl called Berenice. Many T. cruzi sequencing projects based on short reads have demonstrated that genome assembly and downstream comparative analyses are extremely challenging in this species, given that half of its genome is composed of repetitive sequences. Here, we report de novo assemblies, annotation, and comparative analyses of the Berenice strain using a combination of Illumina short reads and MinION long reads. Our work demonstrates that Nanopore sequencing improves T. cruzi assembly contiguity and increases the assembly size in ∼16 Mb. Specifically, we found that assembly improvement also refines the completeness of coding regions for both single-copy genes and repetitive transposable elements. Beyond its historical and epidemiological importance, Berenice constitutes a fundamental resource because it now constitutes a high-quality assembly available for TcII (clade C), a prevalent lineage causing human infections in South America. The availability of Berenice genome expands the known genetic diversity of these parasites and reinforces the idea that T. cruzi is intraspecifically divided in three main clades. Finally, this work represents the introduction of Nanopore technology to resolve complex protozoan genomes, supporting its subsequent application for improving trypanosomatid and other highly repetitive genomes.

Old Yellow Enzyme from Trypanosoma cruzi Exhibits In Vivo Prostaglandin F2α Synthase Activity and Has a Key Role in Parasite Infection and Drug Susceptibility.

The discovery that trypanosomatids, unicellular organisms of the order Kinetoplastida, are capable of synthesizing prostaglandins raised questions about the role of these molecules during parasitic infections. Multiple studies indicate that prostaglandins could be related to the infection processes and pathogenesis in trypanosomatids. This work aimed to unveil the role of the prostaglandin F2α synthase TcOYE in the establishment of Trypanosoma cruzi infection, the causative agent of Chagas disease. This chronic disease affects several million people in Latin America causing high morbidity and mortality. Here, we propose a prokaryotic evolutionary origin for TcOYE, and then we used in vitro and in vivo experiments to show that T. cruzi prostaglandin F2α synthase plays an important role in modulating the infection process. TcOYE overexpressing parasites were less able to complete the infective cycle in cell culture infections and increased cardiac tissue parasitic load in infected mice. Additionally, parasites overexpressing the enzyme increased PGF2α synthesis from arachidonic acid. Finally, an increase in benznidazole and nifurtimox susceptibility in TcOYE overexpressing parasites showed its participation in activating the currently anti-chagasic drugs, which added to its observed ability to confer resistance to hydrogen peroxide, highlights the relevance of this enzyme in multiple events including host-parasite interaction.

Whole-Genome Sequences of Mycobacterium bovis Strain MbURU-001, Isolated from Fresh Bovine Infected Samples.

Bovine tuberculosis in cattle has a high incidence in Uruguay, where it is considered a disease of national importance. We present the genome sequence of Mycobacterium bovis strain MbURU-001, isolated from pectoral lymph nodes of a bovine host from a cattle farm.