Trypanosoma cruzi iron superoxide dismutases: insights from phylogenetics to chemotherapeutic target assessment.

BACKGROUND: Components of the antioxidant defense system in Trypanosoma cruzi are potential targets for new drug development. Superoxide dismutases (SODs) constitute key components of antioxidant defense systems, removing excess superoxide anions by converting them into oxygen and hydrogen peroxide. The main goal of the present study was to investigate the genes coding for iron superoxide dismutase (FeSOD) in T. cruzi strains from an evolutionary perspective. METHODS: In this study, molecular biology methods and phylogenetic studies were combined with drug assays. The FeSOD-A and FeSOD-B genes of 35 T. cruzi strains, belonging to six discrete typing units (Tcl-TcVI), from different hosts and geographical regions were amplified by PCR and sequenced using the Sanger method. Evolutionary trees were reconstructed based on Bayesian inference and maximum likelihood methods. Drugs that potentially interacted with T. cruzi FeSODs were identified and tested against the parasites. RESULTS: Our results suggest that T. cruzi FeSOD types are members of distinct families. Gene copies of FeSOD-A (n = 2), FeSOD-B (n = 4) and FeSOD-C (n = 4) were identified in the genome of the T. cruzi reference clone CL Brener. Phylogenetic inference supported the presence of two functional variants of each FeSOD type across the T. cruzi strains. Phylogenetic trees revealed a monophyletic group of FeSOD genes of T. cruzi TcIV strains in both distinct genes. Altogether, our results support the hypothesis that gene duplication followed by divergence shaped the evolution of T. cruzi FeSODs. Two drugs, mangafodipir and polaprezinc, that potentially interact with T. cruzi FeSODs were identified and tested in vitro against amastigotes and trypomastigotes: mangafodipir had a low trypanocidal effect and polaprezinc was inactive. CONCLUSIONS: Our study contributes to a better understanding of the molecular biodiversity of T. cruzi FeSODs. Herein we provide a successful approach to the study of gene/protein families as potential drug targets.

Perspectives From Systems Biology to Improve Knowledge of Leishmania Drug Resistance.

Neglected Tropical Diseases include a broad range of pathogens, hosts, and vectors, which represent evolving complex systems. Leishmaniasis, caused by different Leishmania species and transmitted to humans by sandflies, are among such diseases. Leishmania and other Trypanosomatidae display some peculiar features, which make them a complex system to study. Leishmaniasis chemotherapy is limited due to high toxicity of available drugs, long-term treatment protocols, and occurrence of drug resistant parasite strains. Systems biology studies the interactions and behavior of complex biological processes and may improve knowledge of Leishmania drug resistance. System-level studies to understand Leishmania biology have been challenging mainly because of its unusual molecular features. Networks integrating the biochemical and biological pathways involved in drug resistance have been reported in literature. Antioxidant defense enzymes have been identified as potential drug targets against leishmaniasis. These and other biomarkers might be studied from the perspective of systems biology and systems parasitology opening new frontiers for drug development and treatment of leishmaniasis and other diseases. Our main goals include: 1) Summarize current advances in Leishmania research focused on chemotherapy and drug resistance. 2) Share our viewpoint on the application of systems biology to Leishmania studies. 3) Provide insights and directions for future investigation.

Temperature Dramatically Shapes Mosquito Gene Expression With Consequences for Mosquito-Zika Virus Interactions.

Vector-borne flaviviruses are emerging threats to human health. For successful transmission, the virus needs to efficiently enter mosquito cells and replicate within and escape several tissue barriers while mosquitoes elicit major transcriptional responses to flavivirus infection. This process will be affected not only by the specific mosquito-pathogen pairing but also by variation in key environmental variables such as temperature. Thus far, few studies have examined the molecular responses triggered by temperature and how these responses modify infection outcomes, despite substantial evidence showing strong relationships between temperature and transmission in a diversity of systems. To define the host transcriptional changes associated with temperature variation during the early infection process, we compared the transcriptome of mosquito midgut samples from mosquitoes exposed to Zika virus (ZIKV) and non-exposed mosquitoes housed at three different temperatures (20, 28, and 36°C). While the high-temperature samples did not show significant changes from those with standard rearing conditions (28°C) 48 h post-exposure, the transcriptome profile of mosquitoes housed at 20°C was dramatically different. The expression of genes most altered by the cooler temperature involved aspects of blood-meal digestion, ROS metabolism, and mosquito innate immunity. Further, we did not find significant differences in the viral RNA copy number between 24 and 48 h post-exposure at 20°C, suggesting that ZIKV replication is limited by cold-induced changes to the mosquito midgut environment. In ZIKV-exposed mosquitoes, vitellogenin, a lipid carrier protein, was most up-regulated at 20°C. Our results provide a deeper understanding of the temperature-triggered transcriptional changes in Aedes aegypti and can be used to further define the molecular mechanisms driven by environmental temperature variation.

Helminth secretomes reflect different lifestyles and parasitized hosts.

Helminths cause a number of medical and agricultural problems and are a major cause of parasitic infections in humans, animals and plants. Comparative analysis of helminth genes and genomes are important to understand the genomic biodiversity and evolution of parasites and their hosts in terms of different selective pressures in their habitats. The interactions between the infective organisms and their hosts are mediated in large part by secreted proteins, known collectively as the "secretome". Proteins secreted by parasites are able to modify a host's environment and modulate their immune system. The composition and function of this set of proteins varies depending on the ecology, lifestyle and environment of an organism. The present study aimed to predict, in silico, the secretome in 44 helminth species including Nematoda (31 species) and Platyhelminthes (13 species) and, understand the diversity and evolution of secretomes. Secretomes from plant helminths range from 7.6% (943 proteins) to 13.9% (2,077 proteins) of the filtered proteome with an average of 10.2% (1,412 proteins) and from free-living helminths range from 4.4% (870 proteins) to 13% (3,121 proteins) with an average of 9.8% (2,126 proteins), respectively, and thus are considerably larger secretomes in relation to animal helminth secretomes which range from 4.2% (431 proteins) to 11.8% (2,419 proteins) of the proteomes, with an average of 7.1% (804 proteins). Across 44 secretomes in different helminth species, we found five conserved domains: (i) PF00014 (Kunitz/Bovine pancreatic trypsin inhibitor domain), (ii) PF00046 (Homeobox domain), (iii) PF00188 (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), (iv) PF00085 (Thioredoxin) and (v) PF07679 (Immunoglobulin I-set domain). Our results detected secreted proteins associated with invasion, infection, adhesion and immunoregulation processes as protease inhibitors and cytokines, among other functions. In summary, this study will contribute towards the understanding of host-parasite interactions and possibly identify new molecular targets for the treatment or diagnosis of helminthiases.

Effectiveness of ITS and sub-regions as DNA barcode markers for the identification of Basidiomycota (Fungi).

BACKGROUND: Fungi are among the most abundant and diverse organisms on Earth. However, a substantial amount of the species diversity, relationships, habitats, and life strategies of these microorganisms remain to be discovered and characterized. One important factor hindering progress is the difficulty in correctly identifying fungi. Morphological and molecular characteristics have been applied in such tasks. Later, DNA barcoding has emerged as a new method for the rapid and reliable identification of species. The nrITS region is considered the universal barcode of Fungi, and the ITS1 and ITS2 sub-regions have been applied as metabarcoding markers. In this study, we performed a large-scale analysis of all the available Basidiomycota sequences from GenBank. We carried out a rigorous trimming of the initial dataset based in methodological principals of DNA Barcoding. Two different approaches (PCI and barcode gap) were used to determine the performance of the complete ITS region and sub-regions. RESULTS: For most of the Basidiomycota genera, the three genomic markers performed similarly, i.e., when one was considered a good marker for the identification of a genus, the others were also; the same results were observed when the performance was insufficient. However, based on barcode gap analyses, we identified genomic markers that had a superior identification performance than the others and genomic markers that were not indicated for the identification of some genera. Notably, neither the complete ITS nor the sub-regions were useful in identifying 11 of the 113 Basidiomycota genera. The complex phylogenetic relationships and the presence of cryptic species in some genera are possible explanations of this limitation and are discussed. CONCLUSIONS: Knowledge regarding the efficiency and limitations of the barcode markers that are currently used for the identification of organisms is crucial because it benefits research in many areas. Our study provides information that may guide researchers in choosing the most suitable genomic markers for identifying Basidiomycota species.

The Schistosoma mansoni phylome: using evolutionary genomics to gain insight into a parasite's biology.

BACKGROUND: Schistosoma mansoni is one of the causative agents of schistosomiasis, a neglected tropical disease that affects about 237 million people worldwide. Despite recent efforts, we still lack a general understanding of the relevant host-parasite interactions, and the possible treatments are limited by the emergence of resistant strains and the absence of a vaccine. The S. mansoni genome was completely sequenced and still under continuous annotation. Nevertheless, more than 45% of the encoded proteins remain without experimental characterization or even functional prediction. To improve our knowledge regarding the biology of this parasite, we conducted a proteome-wide evolutionary analysis to provide a broad view of the S. mansoni's proteome evolution and to improve its functional annotation. RESULTS: Using a phylogenomic approach, we reconstructed the S. mansoni phylome, which comprises the evolutionary histories of all parasite proteins and their homologs across 12 other organisms. The analysis of a total of 7,964 phylogenies allowed a deeper understanding of genomic complexity and evolutionary adaptations to a parasitic lifestyle. In particular, the identification of lineage-specific gene duplications pointed to the diversification of several protein families that are relevant for host-parasite interaction, including proteases, tetraspanins, fucosyltransferases, venom allergen-like proteins, and tegumental-allergen-like proteins. In addition to the evolutionary knowledge, the phylome data enabled us to automatically re-annotate 3,451 proteins through a phylogenetic-based approach rather than solely sequence similarity searches. To allow further exploitation of this valuable data, all information has been made available at PhylomeDB (http://www.phylomedb.org). CONCLUSIONS: In this study, we used an evolutionary approach to assess S. mansoni parasite biology, improve genome/proteome functional annotation, and provide insights into host-parasite interactions. Taking advantage of a proteome-wide perspective rather than focusing on individual proteins, we identified that this parasite has experienced specific gene duplication events, particularly affecting genes that are potentially related to the parasitic lifestyle. These innovations may be related to the mechanisms that protect S. mansoni against host immune responses being important adaptations for the parasite survival in a potentially hostile environment. Continuing this work, a comparative analysis involving genomic, transcriptomic, and proteomic data from other helminth parasites, other parasites, and vectors will supply more information regarding parasite's biology as well as host-parasite interactions.

Evolutionary histories of expanded peptidase families in Schistosoma mansoni.

Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family), cercarial elastases (serine peptidase S1 family) and cathepsin D proteins (aspartic peptidase A1 family). Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.

Evolutionary histories of expanded peptidase families in Schistosoma mansoni

Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family), cercarial elastases (serine peptidase S1 family) and cathepsin D proteins (aspartic peptidase A1 family). Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.

Adapting to a changing world: RAG genomics and evolution.

The origin of the recombination-activating genes (RAGs) is considered to be a foundation hallmark for adaptive immunity, characterised by the presence of antigen receptor genes that provide the ability to recognise and respond to specific peptide antigens. In vertebrates, a diverse repertoire of antigen-specific receptors, T cell receptors and immunoglobulins is generated by V(D)J recombination performed by the RAG-1 and RAG-2 protein complex. RAG homologues were identified in many jawed vertebrates. Despite their crucial importance, no homologues have been found in jawless vertebrates and invertebrates. This paper focuses on the RAG homologues in humans and other vertebrates for which the genome is completely sequenced, and also discusses the main contribution of the use of RAG homologues in phylogenetics and vertebrate evolution. Since mutations in both genes cause a spectrum of severe combined immunodeficiencies, including the Omenn syndrome (OS), these topics are discussed in detail. Finally, the relevance to genomic diversity and implications to immunomics are addressed. The search for homologues could enlighten us about the evolutionary processes that shaped the adaptive immune system. Understanding the diversity of the adaptive immune system is crucially important for the design and development of new therapies to modulate the immune responses in humans and/or animal models.

Diversification of Ramphastinae (Aves, Ramphastidae) prior to the Cretaceous/Tertiary boundary as shown by molecular clock of mtDNA sequences

Partial cytochrome b and 12S rDNA mitochondrial DNA sequences of eight representatives of the Ramphastidae family were analyzed. We applied the linearized tree method to identify sequences evolving at similar rates and estimated the divergence times among some of the taxa analyzed. After excluding Ramphastos tucanus and Capito dayi from our data set, the remaining taxa presented a constant rate of DNA substitution, and branch lengths could be re-estimated with a clock constraint using the maximum likelihood method. Branch lengths were calibrated assuming that Galliformes and Piciformes split around 100 million years ago (mya). Our results indicate that Ramphastinae, and probably Capitoninae, diverged from other Piciformes in the Late Cretaceous (82 mya), suggesting that Piciformes is another avian order that survived the mass extinction event occurred 65 mya at the Cretaceous/Tertiary (K/T) boundary. The divergence times estimated within the Ramphastinae genera cover the period from the Middle Eocene (around 47 mya) through the Late Miocene (9.5 mya). Our estimate of divergence time is coincidental with the split of the African and the South American continents and other intense geologic activities and modifications of the areas which correspond to the current Neotropics. These events might have influenced the diversification of Ramphastinae in South America.