Larissa L. S. Scholte

Bacteria and Genes Involved in Arsenic Speciation in Sediment Impacted by Long-Term Gold Mining

The bacterial community and genes involved in geobiocycling of arsenic (As) from sediment impacted by long-term gold mining were characterized through culture-based analysis of As-transforming bacteria and metagenomic studies of the arsC, arrA, and aioA genes. Sediment was collected from the historically gold mining impacted Mina stream, located in one of the world’s largest mining regions known as the “Iron Quadrangle”. A total of 123 As-resistant bacteria were recovered from the enrichment cultures, which were phenotypically and genotypically characterized for As-transformation. A diverse As-resistant bacteria community was found through phylogenetic analyses of the 16S rRNA gene. Bacterial isolates were affiliated with Proteobacteria, Firmicutes, and Actinobacteria and were represented by 20 genera. Most were AsV-reducing (72%), whereas AsIII-oxidizing accounted for 20%. Bacteria harboring the arsC gene predominated (85%), followed by aioA (20%) and arrA (7%). Additionally, we identified two novel As-transforming genera, Thermomonas and Pannonibacter. Metagenomic analysis of arsC, aioA, and arrA sequences confirmed the presence of these genes, with arrA sequences being more closely related to uncultured organisms. Evolutionary analyses revealed high genetic similarity between some arsC and aioA sequences obtained from isolates and clone libraries, suggesting that those isolates may represent environmentally important bacteria acting in As speciation. In addition, our findings show that the diversity of arrA genes is wider than earlier described, once none arrA-OTUs were affiliated with known reference strains. Therefore, the molecular diversity of arrA genes is far from being fully explored deserving further attention.

Evolutionary analysis of the cystatin family in three Schistosoma species

The cystatin family comprises cysteine protease inhibitors distributed in 3 subfamilies (I25A–C). Family members lacking cystatin activity are currently unclassified. Little is known about the evolution of Schistosoma cystatins, their physiological roles, and expression patterns in the parasite life cycle. The present study aimed to identify cystatin homologs in the predicted proteome of three Schistosoma species and other Platyhelminthes. We analyzed the amino acid sequence diversity focused in the identification of protein signatures and to establish evolutionary relationships among Schistosoma and experimentally validated human cystatins. Gene expression patterns were obtained from different developmental stages in Schistosoma mansoni using microarray data. In Schistosoma, only I25A and I25B proteins were identified, reflecting little functional diversification. I25C and unclassified subfamily members were not identified in platyhelminth species here analyzed. The resulting phylogeny placed cystatins in different clades, reflecting their molecular diversity. Our findings suggest that Schistosoma cystatins are very divergent from their human homologs, especially regarding the I25B subfamily. Schistosoma cystatins also differ significantly from other platyhelminth homologs. Finally, transcriptome data publicly available indicated that I25A and I25B genes are constitutively expressed thus could be essential for schistosome life cycle progression. In summary, this study provides insights into the evolution, classification, and functional diversification of cystatins in Schistosoma and other Platyhelminthes, improving our understanding of parasite biology and opening new frontiers in the identification of novel therapeutic targets against helminthiases.

LRR-RLK family from two Citrus species: genome-wide identification and evolutionary aspects

BackgroundLeucine-rich repeat receptor-like kinases (LRR-RLKs) represent the largest subfamily of plant RLKs. The functions of most LRR-RLKs have remained undiscovered, and a few that have been experimentally characterized have been shown to have important roles in growth and development as well as in defense responses. Although RLK subfamilies have been previously studied in many plants, no comprehensive study has been performed on this gene family in Citrus species, which have high economic importance and are frequent targets for emerging pathogens. In this study, we performed in silico analysis to identify and classify LRR-RLK homologues in the predicted proteomes of Citrus clementina (clementine) and Citrus sinensis (sweet orange). In addition, we used large-scale phylogenetic approaches to elucidate the evolutionary relationships of the LRR-RLKs and further narrowed the analysis to the LRR-XII group, which contains several previously described cell surface immune receptors.ResultsWe built integrative protein signature databases for Citrus clementina and Citrus sinensis using all predicted protein sequences obtained from whole genomes. A total of 300 and 297 proteins were identified as LRR-RLKs in C. clementina and C. sinensis, respectively. Maximum-likelihood phylogenetic trees were estimated using Arabidopsis LRR-RLK as a template and they allowed us to classify Citrus LRR-RLKs into 16 groups. The LRR-XII group showed a remarkable expansion, containing approximately 150 paralogs encoded in each Citrus genome. Phylogenetic analysis also demonstrated the existence of two distinct LRR-XII clades, each one constituted mainly by RD and non-RD kinases. We identified 68 orthologous pairs from the C. clementina and C. sinensis LRR-XII genes. In addition, among the paralogs, we identified a subset of 78 and 62 clustered genes probably derived from tandem duplication events in the genomes of C. clementina and C. sinensis, respectively.ConclusionsThis work provided the first comprehensive evolutionary analysis of the LRR-RLKs in Citrus. A large expansion of LRR-XII in Citrus genomes suggests that it might play a key role in adaptive responses in host-pathogen co-evolution, related to the perennial life cycle and domestication of the citrus crop species.

Novel arsenic-transforming bacteria and the diversity of their arsenic-related genes and enzymes arising from arsenic-polluted freshwater sediment

Bacteria are essential in arsenic cycling. However, few studies have addressed 16S rRNA and arsenic-related functional gene diversity in long-term arsenic-contaminated tropical sediment. Here, using culture-based, metagenomic and computational approaches, we describe the diversity of bacteria, genes and enzymes involved in AsIII and AsV transformation in freshwater sediment and in anaerobic AsIII- and AsV-enrichment cultures (ECs). The taxonomic profile reveals significant differences among the communities. Arcobacter, Dechloromonas, Sedimentibacter and Clostridium thermopalmarium were exclusively found in ECs, whereas Anaerobacillus was restricted to AsV-EC. Novel taxa that are both AsV-reducers and AsIII-oxidizers were identified: Dechloromonas, Acidovorax facilis, A. delafieldii, Aquabacterium, Shewanella, C. thermopalmarium and Macellibacteroides fermentans. Phylogenic discrepancies were revealed among the aioA, arsC and arrA genes and those of other species, indicating horizontal gene transfer. ArsC and AioA have sets of amino acids that can be used to assess their functional and structural integrity and familial subgroups. The positions required for AsV reduction are conserved, suggesting strong selective pressure for maintaining the functionality of ArsC. Altogether, these findings highlight the role of freshwater sediment bacteria in arsenic mobility, and the untapped diversity of dissimilatory arsenate-reducing and arsenate-resistant bacteria, which might contribute to arsenic toxicity in aquatic environments.

Evolutionary relationships among protein lysine deacetylases of parasites causing neglected diseases

The availability of the genomic data of diverse parasites provides an opportunity to identify new drug candidates against neglected tropical diseases affecting people worldwide. Histone modifying enzymes (HMEs) are potential candidates since they play key roles in the regulation of chromatin modifications, thus globally regulating gene expression. Furthermore, aberrant epigenetic states are often associated with human diseases, leading to great interest in HMEs as therapeutic targets. Our work focused on two families of protein lysine deacetylases (HDACs and sirtuins). First, we identified potential homologues in the predicted proteomes of selected taxa by using hidden Markov model profiles. Then, we reconstructed the evolutionary relationships of protein sequences by Bayesian inference and maximum likelihood method. In addition, we constructed homology models for five parasite HDACs to provide information for experimental validation and structure-based optimization of inhibitors. Our results showed that parasite genomes code for diverse HDACs and sirtuins. The evolutionary pattern of protein deacetylases with additional experimental data points to these enzymes as common drug targets among parasites. This work has improved the functional annotation of approximately 63% HDACs and 51% sirtuins in the selected taxa providing insights for experimental design. Homology models pointed out structural conservation and differences among parasite and human homologues and highlight potential candidates for further inhibitor development. Some of these parasite proteins are undergoing RNA interference or knockout analyses to validate the function of their corresponding genes. In the future, we will investigate the main functions performed by these proteins, related phenotypes, and their potential as therapeutic targets.

Helminths and Cancers From the Evolutionary Perspective

Helminths include free-living and parasitic Platyhelminthes and Nematoda which infect millions of people worldwide. Some Platyhelminthes species of blood flukes (Schistosoma haematobium, Schistosoma japonicum, and Schistosoma mansoni) and liver flukes (Clonorchis sinensis and Opisthorchis viverrini) are known to be involved in human cancers. Other helminths are likely to be carcinogenic. Our main goals are to summarize the current knowledge of human cancers caused by Platyhelminthes, point out some helminth and human biomarkers identified so far, and highlight the potential contributions of phylogenetics and molecular evolution to cancer research. Human cancers caused by helminth infection include cholangiocarcinoma, colorectal hepatocellular carcinoma, squamous cell carcinoma, and urinary bladder cancer. Chronic inflammation is proposed as a common pathway for cancer initiation and development. Furthermore, different bacteria present in gastric, colorectal, and urogenital microbiomes might be responsible for enlarging inflammatory and fibrotic responses in cancers. Studies have suggested that different biomarkers are involved in helminth infection and human cancer development; although, the detailed mechanisms remain under debate. Different helminth proteins have been studied by different approaches. However, their evolutionary relationships remain unsolved. Here, we illustrate the strengths of homology identification and function prediction of uncharacterized proteins from genome sequencing projects based on an evolutionary framework. Together, these approaches may help identifying new biomarkers for disease diagnostics and intervention measures. This work has potential applications in the field of phylomedicine (evolutionary medicine) and may contribute to parasite and cancer research.

Insights into the Genome Sequence of Chromobacterium amazonense Isolated from a Tropical Freshwater Lake

Members of the genus Chromobacterium have been isolated from geographically diverse ecosystems and exhibit considerable metabolic flexibility, as well as biotechnological and pathogenic properties in some species. This study reports the draft assembly and detailed sequence analysis of Chromobacterium amazonense strain 56AF. The de novo-assembled genome is 4,556,707 bp in size and contains 4294 protein-coding and 95 RNA genes, including 88 tRNA, six rRNA, and one tmRNA operon. A repertoire of genes implicated in virulence, for example, hemolysin, hemolytic enterotoxins, colicin V, lytic proteins, and Nudix hydrolases, is present. The genome also contains a collection of genes of biotechnological interest, including esterases, lipase, auxins, chitinases, phytoene synthase and phytoene desaturase, polyhydroxyalkanoates, violacein, plastocyanin/azurin, and detoxifying compounds. Importantly, unlike other Chromobacterium species, the 56AF genome contains genes for pore-forming toxin alpha-hemolysin, a type IV secretion system, among others. The analysis of the C. amazonense strain 56AF genome reveals the versatility, adaptability, and biotechnological potential of this bacterium. This study provides molecular information that may pave the way for further comparative genomics and functional studies involving Chromobacterium-related isolates and improves our understanding of the global genomic diversity of Chromobacterium species.

Heterologous expression in Caenorhabditis elegans as an alternative approach to functional studies in Schistosoma mansoni.

The lack of an accurate diagnosis has been a serious obstacle to the advancement of the anti-Trypanosoma cruzi chemotherapy and long-term infection can result in different health risks to human. PCRs are alternative methods, more sensitive than conventional parasitological techniques, which due to their low sensitivities are considered unsuitable for these purposes. The aim of this study was to investigate a sensitive diagnostic strategy to quantify blood and cardiac tissues parasites based on real-time PCR tools during acute and chronic phases of murine Chagas disease, as well as to monitor the evolution of infection in those mice under specific treatment. In parallel, fresh blood examination, immunological analysis and quantification of cardiac inflammation were also performed to confront and improve real-time PCR data. Similar profiles of parasitemia curves were observed in both quantification techniques during the acute phase of the infection. In contrast, parasites could be quantified only by real-time PCR at 60 and 120 days of infection. In cardiac tissue, real-time PCR detected T. cruzi DNA in 100% of infected mice, and using this tool a significant Pearson correlation between parasite load in peripheral blood and in cardiac tissue during acute and chronic phases was observed. Levels of serum CCL2, CCL5 and nitric oxide were coincident with parasite load but focal and diffuse mononuclear infiltrates was observed, even with significant (p < 0.05) reduction of parasitism after 60 days of infection. Later, this methodology was used to monitor the evolution of infection in animals treated with itraconazole (Itz). Itz-treatment induced a reduction of parasite load in both blood and cardiac muscle at the treatment period, but after the end of chemotherapy an increase of parasitism was detected. Interestingly, inflammatory mediators levels and heart inflammation intensity had similar evolution to the parasite load, in the group of animals treated. Taken together, our data show that real-time PCR strategy used was suitable for studies of murine T. cruzi infection and may prove useful in investigations involving experimental chemotherapy of the disease and the benefits of treatment in relation to parasitism and inflammatory respons.