Transition of an estuarine benthic meiofauna assemblage 1.7 and 2.8 years after a mining disaster.

Background: Estuaries are transitional coastal ecosystems that are threatened by multiple sources of human pollution. In 2015, mining tailings from an upstream dam failure caused massive metal contamination that impacted benthic assemblages on the Brazilian Rio Doce estuary. Methods: In this study, we investigate and compare meiofaunal assemblages with eDNA metabarcoding 1.7 years (2017) and 2.8 years (2018) after the initial contamination by mine tailings in order to evaluate the continued impact of sediment mine tailing contaminants on the structure of benthic assemblages after the disaster. Results: The community was dominated by Arthropoda and Nematoda 1.7 yr after the impacts (42 and 29% of meiofaunal sequence reads, respectively) but after 2.8 years Arthropoda (64.8% of meiofaunal sequence reads) and Rotifera (11.8%) were the most common taxa. This continued impact on meiofaunal assemblage revealed a lower phylogenetic diversity (7.8-fold) in 2018, despite overall decrease in metal concentration (Al, Ba, Cr, As, Fe, Zn, Mn, Pb, Cd, Co) in sediments. Our data suggests that differences in benthic assemblages and loss of diversity may be influenced by contaminants in sediments of this estuary, and indicate that broad eDNA assessments are greatly useful to understand the full range of biodiversity changes in dynamic estuarine ecosystems.

A Trypanosoma cruzi zinc finger protein that is implicated in the control of epimastigote-specific gene expression and metacyclogenesis.

Trypanosoma cruzi has three biochemically and morphologically distinct developmental stages that are programmed to rapidly respond to environmental changes the parasite faces during its life cycle. Unlike other eukaryotes, Trypanosomatid genomes contain protein coding genes that are transcribed into polycistronic pre-mRNAs and have their expression controlled by post-transcriptional mechanisms. Transcriptome analyses comparing three stages of the T. cruzi life cycle revealed changes in gene expression that reflect the parasite adaptation to distinct environments. Several genes encoding RNA binding proteins (RBPs), known to act as key post-transcriptional regulatory factors, were also differentially expressed. We characterized one T. cruzi RBP, named TcZH3H12, which contains a zinc finger domain and is up-regulated in epimastigotes compared to trypomastigotes and amastigotes. TcZC3H12 knockout (KO) epimastigotes showed decreased growth rates and increased capacity to differentiate into metacyclic trypomastigotes. Transcriptome analyses comparing wild type and TcZC3H12 KOs revealed a TcZC3H12-dependent expression of epimastigote-specific genes such as genes encoding amino acid transporters and proteins associated with differentiation (PADs). RNA immunoprecipitation assays showed that transcripts from the PAD family interact with TcZC3H12. Taken together, these findings suggest that TcZC3H12 positively regulates the expression of genes involved in epimastigote proliferation and also acts as a negative regulator of metacyclogenesis.

Chronic trace metals effects of mine tailings on estuarine assemblages revealed by environmental DNA.

Mine tailing disasters have occurred worldwide and contemporary release of tailings of large proportions raise concerns of the chronic impacts that trace metals may have on the aquatic biodiversity. Environmental metabarcoding (eDNA) offers an as yet poorly explored opportunity for biological monitoring of impacted aquatic ecosystems from mine tailings and contaminated sediments. eDNA has been increasingly recognized to be an effective method to detect previously unrecognized small-sized Metazoan taxa, but their ecological responses to environmental pollution has not been assessed by metabarcoding. Here, we evaluated chronic effects of trace metal contamination from sediment eDNA of the Rio Doce estuary, 1.7 years after the Samarco mine tailing disaster, which released over 40 million m3 of iron tailings in the Rio Doce river basin. We identified 123 new sequence variants environmental taxonomic units (eOTUs) of benthic taxa and an assemblage composition dominated by Nematoda, Crustacea and Platyhelminthes; typical of other estuarine ecosystems. We detected environmental filtering on the meiofaunal assemblages and multivariate analysis revealed strong influence of Fe contamination, supporting chronic impacts from mine tailing deposition in the estuary. This was in contrast to environmental filtering of meiofaunal assemblages of non-polluted estuaries. Here, we suggest that the eDNA metabarcoding technique provides an opportunity to fill up biodiversity gaps in coastal marine ecology and may become a valid method for long term monitoring studies in mine tailing disasters and estuarine ecosystems with high trace metals content.

Revisiting molecular serotyping of Streptococcus pneumoniae.

BACKGROUND: Ninety-two Streptococcus pneumoniae serotypes have been described so far, but the pneumococcal conjugate vaccine introduced in the Brazilian basic vaccination schedule in 2010 covers only the ten most prevalent in the country. Pneumococcal serotype-shifting after massive immunization is a major concern and monitoring this phenomenon requires efficient and accessible serotyping methods. Pneumococcal serotyping based on antisera produced in animals is laborious and restricted to a few reference laboratories. Alternatively, molecular serotyping methods assess polymorphisms in the cps gene cluster, which encodes key enzymes for capsular polysaccharides synthesis in pneumococci. In one such approach, cps-RFLP, the PCR amplified cps loci are digested with an endonuclease, generating serotype-specific fingerprints on agarose gel electrophoresis. METHODS: In this work, in silico and in vitro approaches were combined to demonstrate that XhoII is the most discriminating endonuclease for cps-RFLP, and to build a database of serotype-specific fingerprints that accommodates the genetic diversity within the cps locus of 92 known pneumococci serotypes. RESULTS: The expected specificity of cps-RFLP using XhoII was 76% for serotyping and 100% for serogrouping. The database of cps-RFLP fingerprints was integrated to Molecular Serotyping Tool (MST), a previously published web-based software for molecular serotyping. In addition, 43 isolates representing 29 serotypes prevalent in the state of Minas Gerais, Brazil, from 2007 to 2013, were examined in vitro; 11 serotypes (nine serogroups) matched the respective in silico patterns calculated for reference strains. The remaining experimental patterns, despite their resemblance to their expected in silico patterns, did not reach the threshold of similarity score to be considered a match and were then added to the database. CONCLUSION: The cps-RFLP method with XhoII outperformed the antisera-based and other molecular serotyping methods in regard of the expected specificity. In order to accommodate the genetic variability of the pneumococci cps loci, the database of cps-RFLP patterns will be progressively expanded to include new variant in vitro patterns. The cps-RFLP method with endonuclease XhoII coupled with MST for computer-assisted interpretation of results may represent a relevant contribution to the real time detection of changes in regional pneumococci population diversity in response to mass immunization programs.

Genomic organization and expression profile of the mucin-associated surface protein (masp) family of the human pathogen Trypanosoma cruzi.

A novel large multigene family was recently identified in the human pathogen Trypanosoma cruzi, causative agent of Chagas disease, and corresponds to approximately 6% of the parasite diploid genome. The predicted gene products, mucin-associated surface proteins (MASPs), are characterized by highly conserved N- and C-terminal domains and a strikingly variable and repetitive central region. We report here an analysis of the genomic organization and expression profile of masp genes. Masps are not randomly distributed throughout the genome but instead are clustered with genes encoding mucin and other surface protein families. Masp transcripts vary in size, are preferentially expressed during the trypomastigote stage and contain highly conserved 5' and 3' untranslated regions. A sequence analysis of a trypomastigote cDNA library reveals the expression of multiple masp variants with a bias towards a particular masp subgroup. Immunofluorescence assays using antibodies generated against a MASP peptide reveals that the expression of particular MASPs at the cell membrane is limited to subsets of the parasite population. Western blots of phosphatidylinositol-specific phospholipase C (PI-PLC)-treated parasites suggest that MASP may be GPI-anchored and shed into the medium culture, thus contributing to the large repertoire of parasite polypeptides that are exposed to the host immune system.

Molecular characterization of ribonucleoproteic antigens containing repeated amino acid sequences from Trypanosoma cruzi.

Trypanosoma cruzi expresses several proteins containing antigenic amino acid repeats. Here we characterized TcRpL7a and TcRBP28, which carry similar repeat motifs and share homology to the eukaryotic L7a ribosomal protein and to a Trypanosoma brucei RNA binding protein, respectively. Analyses of the full length and truncated recombinant TcRpL7a showed that the humoral response of patients with Chagas disease is directed towards its repetitive domain. Sequence analyses of distinct copies of TcRpL7a genes present in the genome of six T. cruzi strains indicate that the number of repeats is higher in proteins from T. cruzi II than T. cruzi I strains. A serum panel of 59 T. cruzi infected patients showed that 73% reacted with TcRpL7a, 71% reacted with TcRBP28 and 80% reacted with 1:1 mixture of both antigens. Synthetic peptides harboring the TcRpL7a repeat motif reacted with 46% of the serum samples. Antibodies raised against both antigens identified equivalent amounts of the native proteins in all three stages of the parasite life cycle. Analyses of subcellular fractions indicated that TcRBP28 is present in the cytoplasm whereas TcRpL7a co-fractionates with polysomes. Confirming their predicted cellular localization, GFP fusions showed that, whereas GFP::TcRBP28 localizes in the cytoplasm, GFP::TcRpL7a accumulates in the nucleus, where ribosome biogenesis occurs.