Hereditary Amyloidosis: Insights Into a Fibrinogen A Variant Protein.

Amyloidosis are a group of diseases in which soluble proteins aggregate and deposit in fibrillar conformation extracellularly in tissues. The effectiveness of therapeutic strategies depends on the specific protein involved, being crucial to accurately determine its nature. Moreover, following the diagnosis, the search for the mutation within relatives allows the clinical advice. Here we report the precise diagnosis and explored the possible reasons of the structural pathogenicity for a renal amyloidosis related to a fibrinogen Aα-chain variant. Whole-exome sequencing and GATK calling pipeline were leveraged to characterize the protein variant present in a patient with kidney failure. Bioinformatics strategies were applied to suggest potential explanations of the variants aggregation. Our pipeline allowed the identification of a single-point variant of fibrinogen Aα-chain, which opened the possibility of curative transplantation. In silico structural analysis suggested that the pathogenicity of the variant may be attributed to a heightened susceptibility to yield a peptide prone to deposit as an oligomer with a ß-sheet structure. Exploiting the comprehensive coverage of whole-genome sequencing, we managed to fill a vacant stage in the diagnosis of hereditary amyloidosis and to stimulate the advancement in biomedicine.

Pathogenicity of microsclerotia from Metarhizium robertsii against Aedes aegypti larvae and antimicrobial peptides expression by mosquitoes during fungal-host interaction.

Aedes aegypti is a vector of various disease-causing arboviruses. Chemical insecticide-based methods for mosquito control have increased resistance in different parts of the world. Thus, alternative control agents such as the entomopathogenic fungi are excellent candidates to control mosquitoes as part of an ecofriendly strategy. There is evidence of the potential of entomopathogenic fungal conidia and blastospores for biological control of eggs, larval and adult stages, as well as the pathogenicity of fungal microsclerotia against adults and eggs. However, there are no studies on the pathogenicity of microsclerotia against either aquatic insects or insects that develop part of their life cycle in the water, such as the A. aegypti larvae. In this study, we assayed the production of microsclerotia and their pathogenicity against A. aegypti larvae of two isolates of Metarhizium robertsii, i.e., CEP 423 isolated in La Plata, Argentina, and the model ARSEF 2575. Both isolates significantly reduced the survival of A. aegypti exposed to their microsclerotia. The fungus-larva interaction resulted in a delayed response in the host. This was evidenced by the expression of some humoral immune system genes such as defensins and cecropin on the 9th day post-infection, when the fungal infection was consolidated as a successful process that culminates in larvae mortality. In conclusion, M. robertsii microsclerotia are promising propagules to be applied as biological control agents against mosquitoes since they produce pathogenic conidia against A. aegypti larvae.

Dark Pigments in Entomopathogenic Fungal Microsclerotia: Preliminary Evidence of a 1,8-Dihydroxynaphthalene-melanin-like Compound in Metarhizium robertsii.

Metarhizium robertsii microsclerotia are fungal aggregates composed of compacted, pigmented hyphae. As they are highly tolerant to desiccation and produce infective conidia, they are promising candidates to be formulated as bioinsecticides. Despite this potential, the nature of the pigments within these structures remains unclear. In this study, routine culture media used for the differentiation of M. robertsii microsclerotia were supplemented with four melanin inhibitors, and the resulting propagules were characterized. Inhibitors of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthetic pathway such as tricyclazole and guaiacol induced significant phenotypic and molecular modifications in the obtained M. robertsii propagules, which exhibited a more spherical shape, reduced size, and increased susceptibility to desiccation, heat, and oxidative stress than microsclerotia obtained without inhibitors. Additionally, genes encoding for a polyketide synthase (Mrpks2) and a putative 1,3,6,8-tetrahydroxynaphthalene reductase (Mrthnr), potentially involved in the DHN-melanin biosynthetic pathway, were upregulated in fungi grown in the inhibitor-added media. In conclusion, M. robertsii microsclerotia contain melanins of type DHN that might play a role in both microsclerotia differentiation and environmental stress tolerance.

Isolation of Beauveria bassiana from the Chagas Disease Vector Triatoma infestans in the Gran Chaco Region of Argentina: Assessment of Gene Expression during Host-Pathogen Interaction.

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 the median survival time or insect mortality. However, Bb-C001 showed a higher expression than GHA of the bassianolide synthetase gene (BbbslS) during infection, and similar levels of the beauvericin synthetase gene (BbbeaS). Immune-related genes of T. infestans nymphs (limpet-2 and defensin-1, -2, and -6) were later expressed and thus insects failed to stop the infection process. These results showed that B. bassiana Bb-C001 is a promised fungal strain to be incorporated in the current biological control programs of T. infestans in Salta province, Argentina.

The entomopathogenic fungus Beauveria bassiana produces microsclerotia-like pellets mediated by oxidative stress and peroxisome biogenesis.

Several filamentous fungi are known to produce macroscopic pigmented hyphal aggregates named sclerotia. In recent years, some entomopathogenic fungi were reported to produce small sclerotia termed 'microsclerotia', becoming new potential propagules for biocontrol strategies. In this study, we described the production of microsclerotia-like pellets by the entomopathogenic fungus Beauveria bassiana. The carbon: nitrogen ratio equal to or higher than 12.5:1 amended with Fe2+ induced the germination of conidia, producing hyphal aggregate that formed sclerotial structures in submerged liquid cultures. These aggregates were able to tolerate desiccation as they germinated and subsequently produced viable conidia. Conidia derived from microsclerotial aggregates formulated with diatomaceous earth effectively kill 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.

Alkane-grown Beauveria bassiana produce mycelial pellets displaying peroxisome proliferation, oxidative stress, and cell surface alterations.

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 with high peroxidase activity, exhibiting peroxisome proliferation and an apparent surface thickening. Alkane-grown conidia appeared to be more hydrophobic and cell surfaces displayed different topography than glucose-grown cells. We also found a significant induction in several genes encoding for peroxins, catalases, superoxide dismutases, and hydrophobins. These results show that both morphological and metabolic changes are triggered in mycelial pellets derived from alkane-grown B. bassiana.

Insights into Hydrocarbon Assimilation by Eurotialean and Hypocrealean Fungi: Roles for CYP52 and CYP53 Clans of Cytochrome P450 Genes.

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 comparative analysis of CYP genes belonging to clans CYP52 and CYP53 in Aspergillus niger, Beauveria bassiana, Metarhizium robertsii (formerly M. anisopliae var. anisopliae), and Penicillium chrysogenum. These species were able to assimilate n-hexadecane, n-octacosane, and phenanthrene, exhibiting a species-dependent modification in pH of the nutrient medium during this process. Modeling of the molecular docking of the hydrocarbons to the cytochrome P450 active site revealed that both phenanthrene and n-octacosane are energetically favored as substrates for the enzymes codified by genes belonging to both CYP52 and CYP53 clans, and thus appear to be involved in this oxidation step. Analyses of gene expression revealed that CYP53 members were significantly induced by phenanthrene in all species studied, but only CYP52X1 and CYP53A11 from B. bassiana were highly induced with n-alkanes. These findings suggest that the set of P450 enzymes involved in hydrocarbon assimilation by fungi is dependent on phylogeny and reveal distinct substrate and expression specificities.

Biochemical and structural characterization of a novel arginine kinase from the spider Polybetes pythagoricus.

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) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Finally, these results contribute to knowledge of mechanistic details of the function of arginine kinase.

Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis.

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 fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells.

Cystathionine γ-lyase, an enzyme related to the reverse transsulfuration pathway, is functional in Leishmania spp.

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 ß-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.

Functional characterization of enzymes involved in cysteine biosynthesis and H(2)S production in Trypanosoma cruzi.

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 ß 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 γ 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.

Biochemical characterization of serine acetyltransferase and cysteine desulfhydrase from Leishmania major.

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.