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.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Beca Presidente de la Republica-ChileUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilUniv Antofagasta, Lab Bioquim, Dept Biomed, Antofagasta, ChileUniv Bandeirante São Paulo, São Paulo, BrazilUniv Brasilia, Dept Biol Celular, Inst Biol, Brasilia, DF, BrazilFiocruz MS, Ctr Pesquisa Rene Rachou CPqRR, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilWeb of Scienc

Metagenomics of Atacama lithobiontic extremophile life unveils highlights on fungal communities, biogeochemical cycles and carbohydrate-active enzymes

Halites, which are typically found in various Atacama locations, are evaporitic rocks that are considered as micro-scaled salterns. Both structural and functional metagenomic analyses of halite nodules were performed. Structural analyses indicated that the halite microbiota is mainly composed of NaCl-adapted microorganisms. In addition, halites appear to harbor a limited diversity of fungal families together with a biodiverse collection of protozoa. Functional analysis indicated that the halite microbiome possesses the capacity to make an extensive contribution to carbon, nitrogen, and sulfur cycles, but possess a limited capacity to fix nitrogen. The halite metagenome also contains a vast repertory of carbohydrate active enzymes (CAZY) with glycosyl transferases being the most abundant class present, followed by glycosyl hydrolases (GH). Amylases were also present in high abundance, with GH also being identified. Thus, the halite microbiota is a potential useful source of novel enzymes that could have biotechnological applicability. This is the first metagenomic report of fungi and protozoa as endolithobionts of halite nodules, as well as the first attempt to describe the repertoire of CAZY in this community. In addition, we present a comprehensive functional metagenomic analysis of the metabolic capacities of the halite microbiota, providing evidence for the first time on the sulfur cycle in Atacama halites

A Microethnographic and Ethnobotanical Approach to Llayta Consumption among Andes Feeding Practices

Llayta is a dietary supplement that has been used by rural communities in Perú and northern Chile since pre-Columbian days. Llayta is the biomass of colonies of a Nostoc cyanobacterium grown in wetlands of the Andean highlands, harvested, sun-dried and sold as an ingredient for human consumption. The biomass has a substantial content of essential amino acids (58% of total amino acids) and polyunsaturated fatty acids (33% total fatty acids). This ancestral practice is being lost and the causes were investigated by an ethnographic approach to register the social representations of Llayta, to document how this Andean feeding practice is perceived and how much the community knows about Llayta. Only 37% of the participants (mostly adults) have had a direct experience with Llayta; other participants (mostly children) did not have any knowledge about it. These social responses reflect anthropological and cultural tensions associated with a lack of knowledge on Andean algae, sites where to find Llayta, where it is commercialized, how it is cooked and on its nutritional benefits. The loss of this ancestral feeding practice, mostly in northern Chile, is probably associated with cultural changes, migration of the rural communities, and very limited access to the available information. We propose that Llayta consumption can be revitalized by developing appropriate educational strategies and investigating potential new food derivatives based on the biomass from the isolated Llayta cyanobacterium

UV-A Irradiation Increases Scytonemin Biosynthesis in Cyanobacteria Inhabiting Halites at Salar Grande, Atacama Desert

Microbial consortia inhabiting evaporitic salt nodules at the Atacama Desert are dominated by unculturable cyanobacteria from the genus Halothece. Halite nodules provide transparency to photosynthetically active radiation and diminish photochemically damaging UV light. Atacama cyanobacteria synthesize scytonemin, a heterocyclic dimer, lipid soluble, UV-filtering pigment (in vivo absorption maximum at 370 nm) that accumulates at the extracellular sheath. Our goal was to demonstrate if UV-A irradiations modulate scytonemin biosynthesis in ground halites containing uncultured Halothece sp. cyanobacteria. Pulverized halite nodules with endolithic colonization were incubated under continuous UV-A radiation (3.6 W/m2) for 96 h, at 67% relative humidity, mimicking their natural habitat. Scytonemin content and relative transcription levels of scyB gene (a key gene in the biosynthesis of scytonemin) were evaluated by spectrophotometry and quantitative RT-PCR, respectively. After 48 h under these experimental conditions, the ratio scytonemin/chlorophyll a and the transcription of scyB gene increased to a maximal 1.7-fold value. Therefore, endolithic Halothece cyanobacteria in halites are metabolically active and UV radiation is an environmental stressor with a positive influence on scyB gene transcription and scytonemin biosynthesis. Endolithobiontic cyanobacteria in Atacama show a resilient evolutive and adaptive strategy to survive in one of the most extreme environments on Earth

Absence of Cyanotoxins in Llayta, Edible Nostocaceae Colonies from the Andes Highlands

Edible Llayta are cyanobacterial colonies consumed in the Andes highlands. Llayta and four isolated cyanobacteria strains were tested for cyanotoxins (microcystin, nodularin, cylindrospermopsin, saxitoxin and β-N-methylamino-L-alanine—BMAA) using molecular and chemical methods. All isolates were free of target genes involved in toxin biosynthesis. Only DNA from Llayta amplified the mcyE gene. Presence of microcystin-LR and BMAA in Llayta extracts was discarded by LC/MS analyses. The analysed Llayta colonies have an incomplete microcystin biosynthetic pathway and are a safe food ingredient

Staphylococcus sciuri Strain LCHXa is a Free-Living Lithium-Tolerant Bacterium Isolated from Salar de Atacama, Chile

In addition to the industrial and biomedical applications of lithium, information on the tolerance of microorganisms to high Li concentrations in natural biological systems is limited. Strain LCHXa is a novel free-living Gram-positive, non-motile bacterium strain isolated from water samples taken at Laguna Chaxa, a non-industrial water body with the highest soluble Li content (33 mM LiCl) within the Salar de Atacama basin in northern Chile. Enrichment was conducted in Luria-Bertani (LB) medium supplemented with 1 M LiCl. Strain LCHXa was a Novobiocin-resistant and coagulase negative Staphylococcus. Phylogenetically, strain LCHXa belongs to the species Staphylococcus sciuri. Strain LCHXa grew optimally in LB medium at pH 6–8 and 37 °C, and it was able to sustain growth at molar Li concentrations at 2 M LiCl, with a decrease in the specific growth rate of 85%. Osmoregulation in strain LCHXa partially involves glycine betaine and glycerol as compatible solutes

Cellulose Synthase in Atacama Cyanobacteria and Bioethanol Production from Their Exopolysaccharides

Cyanobacteria produce exopolysaccharides (EPSs) as an adaptative mechanism against ultraviolet radiation and desiccation. Cellulose is present in the extracellular polymeric substance in some cyanobacteria genera and it has been proposed as a raw material for biofuel production. The goal of this work was to evaluate the cellulose presence in EPS of Atacama cyanobacteria strains and its use as an alternative and innovative biological source to produce bioethanol. The presence of cellulose was evaluated using techniques of molecular biology, bioinformatics, and electronic microscopy. The conserved motif D,D,D,35QXXRW, characteristic of processive β-glycosyltransferase in all cellulose-producing organisms, was identified in the genome of the LLA-10 strain. This is evidence that cellulose synthase in the LLA-10 strain is a functional enzyme. EPS from Atacama cyanobacteria was hydrolyzed by β-glucosidases (cellobiase and cellulase) and the released glucose was yeast-fermented to ethanol. Ethanol production reached 172.69 ± 0.02 mg ethanol/g EPS after 48 h of incubation. These results are the first step in the evaluation of EPS produced by native cyanobacteria isolated from northern Chile for future biotechnological applications such as the production of bioethanol