Papers by Marianela Santana
Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are ... more Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus (PpAK), from its complementary DNA to the crystal structure. The PpAK cDNA from muscle was cloned, and it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant (Km) was 1.7 mM with a kcat of 75 s-1. Two crystal structures are presented, the apoPvAK and PpAK bound to arginine, both in the open conformation with the active site lid (residues 310-320) completel...
The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons as ... more The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons as the sole carbon source, inducing several enzymes involved in alkane assimilation and concomitantly increasing virulence against insect hosts. In this study, we describe some physiological and molecular processes implicated in growth, nutritional stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology was investigated using light and transmission electron microscopy (TEM) while the surface topography was examined using atomic force microscopy (AFM). Fungal hydrophobicity was also measured on the cell surface. Additionally, the expression pattern of several genes associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to mycelial pellets described in other alkane-free fungi, which were able to germinate and produce viable conidia in media with...
Entomologia Experimentalis et Applicata, 2022
Journal of Fungi, 2020
A native strain of the entomopathogenic fungus Beauveria bassiana (Bb-C001) was isolated from a n... more A native strain of the entomopathogenic fungus Beauveria bassiana (Bb-C001) was isolated from a naturally infected Triatoma infestans, Klug (Hemiptera: Reduviidae) adult cadaver in the Gran Chaco region, Salta province, Argentina. The isolate was both phenotypic and molecularly characterized in a context of fungus-insect interaction, by measuring the expression pattern of toxin genes during infection and immune response of T. infestans. The commercial strain GHA of B. bassiana, which was previously used in field interventions to control these vectors, was used as reference in this study. The phylogenetic trees based on both ribosomal internal transcribed spacer (ITS) and elongation factor 1-alpha (EF1-α) indicated that Bb-C001 fits into a B. bassiana cluster, and the sequence-characterized amplified regions (SCAR) showed that Bb-C001 is different from the GHA strain. There were no differences between both strains regarding viability, radial growth, and conidia production, either in ...
Innovación y Desarrollo Tecnológico y Social, 2020
En diciembre de 2019, se produjo un nuevo brote de enfermedad por coronavirus (COVID-19) en Wuhan... more En diciembre de 2019, se produjo un nuevo brote de enfermedad por coronavirus (COVID-19) en Wuhan, China. El síndrome respiratorio agudo severo-coronavirus-2 (SARS-CoV-2), que es el séptimo coronavirus conocido que infecta a los humanos, es altamente infeccioso y se ha expandido rápidamente en todo el mundo desde su descubrimiento. El diagnóstico de la infección por SARS-CoV-2 se basa en la detección del genoma viral (ARN) a través de técnicas de biología molecular. Con este fin, se extrae el ARN total para su posterior detección mediante PCR cuantitativa en tiempo real (RT-qPCR). Las pruebas cuantitativas de ácidos nucleicos se han convertido en el “estándar de oro” para el diagnóstico y guía en la toma de decisiones clínicas. Sin embargo, los ensayos de RT-qPCR dirigidos al SARS-CoV-2 tienen varios desafíos, especialmente en términos de diseño de cebadores / sondas y de desarrollo de metodologías que permitan estimar la carga viral en pacientes con diagnóstico de COVID-19.
Environmental Microbiology Reports, 2019
Tribolium castaneum larvae. Optical and transmission microscopical imaging, qPCR and spectrophoto... more Tribolium castaneum larvae. Optical and transmission microscopical imaging, qPCR and spectrophotometric analysis revealed that an oxidative stress scenario is involved in conidial differentiation into microsclerotia-like pellets, inducing fungal antioxidant response with high peroxidase activity-mainly detected in peroxisomes and mitochondria-and progress with active peroxisome proliferation. The results provide clues about B. bassiana microsclerotial differentiation and indicate that these pigmented aggregates are promising propagules for production, formulation and potentially application in the control of soil-inhabiting arthropod pests.
Fungal biology, Jun 1, 2018
The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons, in... more The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons, inducing alkane assimilation pathways and concomitantly increasing virulence against insect hosts. In this study, we describe some physiological and molecular processes implicated in growth, nutritional stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology was investigated using light and transmission electron microscopy while the surface topography was examined using atomic force microscopy. Additionally, the expression pattern of several genes associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to mycelial pellets described in other alkane-free fungi, which were able to produce viable conidia and to be pathogenic against larvae of the beetles Tenebrio molitor and Tribolium castaneum. Mycelial pellets were formed by hyphae cumulates wit...
Applied Biochemistry and Biotechnology, 2017
Several filamentous fungi are able to concomitantly assimilate both aliphatic and polycyclic arom... more Several filamentous fungi are able to concomitantly assimilate both aliphatic and polycyclic aromatic hydrocarbons that are the biogenic by-products of some industrial processes. Cytochrome P450 monooxygenases catalyze the first oxidation reaction for both types of substrate. Among the cytochrome P450 (CYP) genes, the family CYP52 is implicated in the first hydroxylation step in alkane-assimilation processes, while genes belonging to the family CYP53 have been linked with oxidation of aromatic hydrocarbons. Here, we perform a
Biochemical Journal, 2017
Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de... more Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive...
Journal of Eukaryotic Microbiology, 2014
Leishmania parasites seem capable of producing cysteine by de novo biosynthesis, similarly to bac... more Leishmania parasites seem capable of producing cysteine by de novo biosynthesis, similarly to bacteria, some pathogenic protists, and plants. In Leishmania spp., cysteine synthase (CS) and cystathionine β-synthase (CBS) are expected to participate in this metabolic process. Moreover, the reverse transsulfuration pathway (RTP) is also predicted to be operative in this trypanosomatid because CBS also catalyzes the condensation of serine with homocysteine, and a gene encoding a putative cystathionine γ-lyase (CGL) is present in all the sequenced genomes. Our results show that indeed, Leishmania major CGL is able to rescue the wild-type phenotype of a Saccharomyces cerevisiae CGL-null mutant and is susceptible to inhibition by an irreversible CGL inhibitor, DL-propargylglycine (PAG). In Leishmania promastigotes, CGL and CS are cytosolic enzymes. The coexistence of de novo synthesis with the RTP is extremely rare in most living organisms; however, despite this potentially high redundancy in cysteine production, PAG arrests the proliferation of L. major promastigotes with an IC50 of approximately 65 μM. These findings raise new questions regarding the biological role of CGL in these pathogens and indicate the need for understanding the molecular mechanism of PAG action in vivo to identify the potential targets affected by this drug.
Plant Physiology, 1998
Although it is well established that the plant host encodes and synthesizes the apoprotein for le... more Although it is well established that the plant host encodes and synthesizes the apoprotein for leghemoglobin in root nodules, the source of the heme moiety has been uncertain. We recently found that the transcript for coproporphyrinogen III oxidase, one of the later enzymes of heme synthesis, is highly elevated in soybean (Glycine maxL.) nodules compared with roots. In this study we measured enzyme activity and carried out western-blot analysis and in situ hybridization of mRNA to investigate the levels during nodulation of the plant-specific coproporphyrinogen oxidase and four other enzymes of the pathway in both soybean and pea (Pisum sativum L.). We compared them with the activity found in leaves and uninfected roots. Our results demonstrate that all of these enzymes are elevated in the infected cells of nodules. Because these are the same cells that express apoleghemoglobin, the data strongly support a role for the plant in the synthesis of the heme moiety of leghemoglobin.
Molecular and Biochemical Parasitology, 2010
Cysteine metabolism exhibits atypical features in Leishmania parasites. The nucleotide sequence a... more Cysteine metabolism exhibits atypical features in Leishmania parasites. The nucleotide sequence annotated as LmjF32.2640 encodes a cysteine desulfhydrase, which specifically catalyzes the breakdown of cysteine into pyruvate, NH 3 and H 2 S. Like in other pathogens, this capacity might be associated with regulatory mechanisms to control the intracellular level of cysteine, a highly toxic albeit essential amino acid, in addition to generate pyruvate for energy production. Besides, our results provide the first insight into the biochemical properties of Leishmania major serine acetyltransferase (SAT), which is likely involved in the two routes for de novo synthesis of cysteine in this pathogen. When compared with other members of SAT family, the N-terminal region of L. major homologue is uniquely extended, and seems to be essential for proper protein folding. Furthermore, unlike plant and bacterial enzymes, the carboxy-terminal-C 10 sequence stretch of L. major SAT appears not to be implicated in forming a tight bi-enzyme complex with cysteine synthase.
Molecular and Biochemical Parasitology, 2012
Trypanosoma cruzi is expected to synthetize de novo cysteine by different routes, among which the... more Trypanosoma cruzi is expected to synthetize de novo cysteine by different routes, among which the two-step pathway involving serine acetyltransferase and cysteine synthase (CS) is comprised. Also, cystathionine  synthase (CBS) might contribute to the de novo generation of cysteine in addition to catalyze the first step of the reverse transsulfuration route producing cystathionine. However, neither the functionality of CS nor that of cystathionine ␥ lyase (CGL) has been assessed. Our results show that T. cruzi CS could participate notably more actively than CBS in the de novo synthesis of cysteine. Interestingly, at the protein level T. cruzi CS is more abundant in amastigotes than in epimastigotes. Unlike the mammalian homologues, T. cruzi CGL specifically cleaves cystathionine into cysteine and is unable to produce H 2 S. The expression pattern of T. cruzi CGL parallels that of CBS, which unexpectedly suggests that in addition to the de novo synthesis of cysteine, the reverse transsulfuration pathway could be operative in the mammalian and insect stages. Besides, T. cruzi CBS produces H 2 S by decomposing cysteine or via condensation of cysteine with homocysteine. The latter reaction leads to cystathionine production, and is catalyzed remarkably more efficiently than the breakdown of cysteine. In T. cruzi like in other organisms, H 2 S could exert regulatory effects on varied metabolic processes. Notably, T. cruzi seems to count on stage-specific routes involved in cysteine production, the multiple cysteine-processing alternatives could presumably reflect this parasite's high needs of reducing power for detoxification of reactive oxygen species.
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Papers by Marianela Santana