In dipteran insects, invading pathogens are selectively recognized by four major pathways, namely Toll, IMD, JNK, and JAK/STAT, and trigger the activation of several immune effectors. Although ...substantial advances have been made in understanding the immunity of model insects such as Drosophila melanogaster, knowledge on the activation of immune responses in other arthropods such as ticks remains limited. Herein, we have deepened our understanding of the intracellular signalling pathways likely to be involved in tick immunity by combining a large-scale in silico approach with high-throughput gene expression analysis. Data from in silico analysis revealed that although both the Toll and JAK/STAT signalling pathways are evolutionarily conserved across arthropods, ticks lack central components of the D. melanogaster IMD pathway. Moreover, we show that tick immune signalling-associated genes are constitutively transcribed in BME26 cells (a cell lineage derived from embryos of the cattle tick Rhipicephalus microplus) and exhibit different transcriptional patterns in response to microbial challenge. Interestingly, Anaplasma marginale, a pathogen that is naturally transmitted by R. microplus, causes downregulation of immune-related genes, suggesting that this pathogen may manipulate the tick immune system, favouring its survival and vector colonization.
•Toll and JAK/STAT signalling pathways are evolutionarily conserved across arthropods.•Ticks lack central components of the D. melanogaster IMD pathway.•Anaplasma marginale causes downregulation of immune-related genes.•A. marginale may manipulate the tick immune system favouring its vector colonization.
The maintenance of genomic integrity is the responsibility of a complex network, denominated the DNA damage response (DDR), which controls the lesion detection and DNA repair. The main repair ...pathways are base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination repair (HR) and non-homologous end joining repair (NHEJ). They correct double-strand breaks (DSB), single-strand breaks, mismatches and others, or when the damage is quite extensive and repair insufficient, apoptosis is activated.
In this study we used the BLAST reciprocal best-hit methodology to search for DDR orthologs proteins in Aedes aegypti. We also provided a comparison between Ae. aegypti, D. melanogaster and human DDR network.
Our analysis revealed the presence of ATR and ATM signaling, including the H2AX ortholog, in Ae. aegypti. Key DDR proteins (orthologs to RAD51, Ku and MRN complexes, XP-components, MutS and MutL) were also identified in this insect. Other proteins were not identified in both Ae. aegypti and D. melanogaster, including BRCA1 and its partners from BRCA1-A complex, TP53BP1, PALB2, POLk, CSA, CSB and POLβ. In humans, their absence affects DSB signaling, HR and sub-pathways of NER and BER. Seven orthologs not known in D. melanogaster were found in Ae. aegypti (RNF168, RIF1, WRN, RAD54B, RMI1, DNAPKcs, ARTEMIS).
The presence of key DDR proteins in Ae. aegypti suggests that the main DDR pathways are functional in this insect, and the identification of proteins not known in D. melanogaster can help fill gaps in the DDR network. The mapping of the DDR network in Ae. aegypti can support mosquito biology studies and inform genetic manipulation approaches applied to this vector.
2,3-Butanediol (BD) and acetoin (AC) are products of the non-oxidative metabolism of microorganisms, presenting industrial importance due to their wide range of applications and high market value. ...Their optical isomers have particular applications, justifying the efforts on the selective bioproduction. Each microorganism produces different isomer mixtures, as a consequence of having different butanediol dehydrogenase (BDH) enzymes. However, the whole scene of the isomer bioproduction, considering the several enzymes and conditions, has not been completely elucidated. Here we show the BDH classification as R, S or meso by bioinformatics analysis uncovering the details of the isomers production. The BDH was compared to diacetyl reductases (DAR) and the new enoyl reductases (ER). We observed that R-BDH is the most singular BDH, while meso and S-BDHs are similar and may be better distinguished through their stereo-selective triad. DAR and ER showed distinct stereo-triads from those described for BDHs, agreeing with kinetic data from the literature and our phylogenetic analysis. The ER family probably has meso-BDH like activity as already demonstrated for a single sequence from this group. These results are of great relevance, as they organize BD producing enzymes, to our known, never shown before in the literature. This review also brings attention to nontraditional enzymes/pathways that can be involved with BD/AC synthesis, as well as oxygen conditions that may lead to the differential production of their isomers. Together, this information can provide helpful orientation for future studies in the field of BD/AC biological production, thus contributing to achieve their production on an industrial scale.
Celotno besedilo
Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Eukaryotic cells have evolved an intricate system to resolve DNA damage to prevent its transmission to daughter cells. This system, collectively known as the DNA damage response (DDR) network, ...includes many proteins that detect DNA damage, promote repair, and coordinate progression through the cell cycle. Because defects in this network can lead to cancer, this network constitutes a barrier against tumorigenesis. The modular BRCA1 carboxyl-terminal (BRCT) domain is frequently present in proteins involved in the DDR, can exist either as an individual domain or as tandem domains (tBRCT), and can bind phosphorylated peptides. We performed a systematic analysis of protein-protein interactions involving tBRCT in the DDR by combining literature curation, yeast two-hybrid screens, and tandem affinity purification coupled to mass spectrometry. We identified 23 proteins containing conserved BRCT domains and generated a human protein-protein interaction network for seven proteins with tBRCT. This study also revealed previously unknown components in DNA damage signaling, such as COMMD1 and the target of rapamycin complex mTORC2. Additionally, integration of tBRCT domain interactions with DDR phosphoprotein studies and analysis of kinase-substrate interactions revealed signaling subnetworks that may aid in understanding the involvement of tBRCT in disease and DNA repair.
Tsetse flies are responsible for the transmission of Trypanossoma sp. to vertebrate animals in Africa causing huge health issues and economic loss. The availability of the genome sequence of Glossina ...morsitans enabled the discovery of several genes related to medically important phenotypes and novel physiological features. However, a genome-wide scan for coding regions that underwent positive selection is still missing, which is surprising given the evolution of traits associated with the hematophagy in this lineage. In this study, we employed an experimental design that controlled for the rate of false positives and we performed a scan of 3,318 G. morsitans genes. We found 145 genes with significant historical signal of positive selection. These genes were categorized into 18 functional classes after careful manual annotation. Based on their attributed functions, we identified candidate genes related with feeding habits and embryonic development. When our results were contrasted with gene expression data, we confirmed that most genes that underwent adaptive molecular evolution were frequently expressed in organs associated with key physiological evolutionary innovations in the G. morsitans lineage, namely, the salivary gland, the midgut, fat body tissue, and in the spermatophore.
The adult females of Aedes aegypti mosquitoes are facultative hematophagous insects but they are unable to feed on blood right after pupae emergence. The maturation process that takes place during ...the first post-emergence days, hereafter named hematophagic and gonotrophic capacitation, comprises a set of molecular and physiological changes that prepare the females for the first gonotrophic cycle. Notwithstanding, the molecular bases underlying mosquito hematophagic and gonotrophic capacitation remain obscure. Here, we investigated the molecular and biochemical changes in adult Ae. aegypti along the first four days post-emergence, prior to a blood meal. We performed a RNA-Seq analysis of the head and body, comparing male and female gene expression time courses. A total of 811 and 203 genes were differentially expressed, respectively in the body and head, and both body parts showed early, mid, and late female-specific expression profiles. Female-specific up-regulation of genes involved in muscle development and the oxidative phosphorylation pathway were remarkable features observed in the head. Functional assessment of mitochondrial oxygen consumption in heads showed a gradual increase in respiratory capacity and ATP-linked respiration as a consequence of induced mitochondrial biogenesis and content over time. This pattern strongly suggests that boosting oxidative phosphorylation in heads is a required step towards blood sucking habit. Several salivary gland genes, proteases, and genes involved in DNA replication and repair, ribosome biogenesis, and juvenile hormone signaling were up-regulated specifically in the female body, which may reflect the gonotrophic capacitation. This comprehensive description of molecular and biochemical mechanisms of the hematophagic and gonotrophic capacitation in mosquitoes unravels potentially new targets for vector control.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut ...is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Chagas disease or American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi. Once known as an endemic health problem of poor rural ...populations in Latin American countries, it has now spread worldwide. The parasite is transmitted by triatomine bugs, of which Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae) is one of the vectors and a model organism. This species occurs mainly in Central and South American countries where the disease is endemic. Disease prevention focuses on vector control programs that, in general, rely intensely on insecticide use. However, the massive use of chemical insecticides can lead to resistance. One of the major mechanisms is known as metabolic resistance that is associated with an increase in the expression or activity of detoxification genes. Three of the enzyme families that are involved in this process – carboxylesterases (CCE), glutathione s-transferases (GST) and cytochrome P450s (CYP) – are analyzed in the R. prolixus genome. A similar set of detoxification genes to those of the Hemipteran Acyrthosiphon pisum but smaller than in most dipteran species was found in R. prolixus genome. All major CCE classes (43 genes found) are present but the pheromone/hormone processing class had fewer genes than usual. One main expansion was detected on the detoxification/dietary class. The phosphotriesterase family, recently associated with insecticide resistance, was also represented with one gene. One microsomal GST gene was found and the cytosolic GST gene count (14 genes) is extremely low when compared to the other hemipteran species with sequenced genomes. However, this is similar to Apis mellifera, a species known for its deficit in detoxification genes. In R. prolixus 88 CYP genes were found, with representatives in the four clans (CYP2, CYP3, CYP4 and mitochondrial) usually found in insects. R. prolixus seems to have smaller species-specific expansions of CYP genes than mosquitoes and beetles, among others. The number of R. prolixus CYP genes is similar to the hemipteran Ac. pisum, although with a bigger expansion in CYP3 and CYP4 clans, along with several gene fragments, mostly in CYP4 clan. Eleven founding members of new families were detected, consisting of ten genes in the CYP3 clan and 1 gene in the CYP4 clan. Members of these clans were proposed to have important detoxification roles in insects. The identification of CCE, GST and CYP genes is of utmost importance for directing detoxification studies on triatomines that can help insecticide management strategies in control programs.
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•We analyzed detoxification related enzymes families in Rhodnius prolixus' genome.•We mined the number and annotation of genes of esterases, GSTs and CYPs.•The number of genes found was similar to other hemipteran species.•Most gene families had smaller species specific expansions than dipteran species.•Genes belonging to all major groups within each enzymatic family have been found.
The inability of the yeast
Saccharomyces cerevisiae
to produce ethanol from xylose has hampered the biofuel production from lignocellulosic biomass. However, prior studies reveal that functional ...expression of xylose isomerase (XI) from
Burkholderia cenocepacia
(XylA
Bc
) in
S. cerevisiae
has remarkably improved xylose consumption and ethanol productivity. Yet, little is known about kinetic and structural properties of this enzyme. Hereby, a purified recombinant XylA was assayed in vitro, showing optimal enzyme activity at 37 °C and pH 7.2. The K
m
of XylA for
d
-xylose was at least threefold lower than the K
m
results for any XI published to date (e.g. XylA from
Piromyces
sp.). In addition, oligomerization behavior as a tetramer was observed for XylA in solution. Functional and structural comparative analyses amongst three microbial XIs were further performed as theoretical models, showing that xylose orientation at the active site was highly conserved among the XIs. Mg
2+
ions anchor the sugar and guide its pyranoside oxygen towards a histidine residue present at the active site, allowing an acid–base reaction, linearizing xylose. Electrostatic surface analyses showed that small variations in the net charge distribution and dipole moment could directly affect the way the substrate interacts with the protein, thus altering its kinetic properties. Accordingly, in silico modeling suggested the tetramer may be the major functional form. These analyses and the resulting model promote a better understanding of this protein family and pave the way to further protein engineering and application of XylA in the ethanol industry.
Iron is an essential element for most organisms However, free iron and heme, its complex with protoporphyrin IX, can be extremely cytotoxic, due to the production of reactive oxygen species, ...eventually leading to oxidative stress. Thus, eukaryotic cells control iron availability by regulating its transport, storage and excretion as well as the biosynthesis and degradation of heme. In the genome of
, the vector of Chagas disease, we identified 36 genes related to iron and heme metabolism We performed a comprehensive analysis of these genes, including identification of homologous genes described in other insect genomes. We observed that blood-meal modulates the expression of ferritin, Iron Responsive protein (IRP), Heme Oxygenase (HO) and the heme exporter Feline Leukemia Virus C Receptor (FLVCR), components of major pathways involved in the regulation of iron and heme metabolism, particularly in the posterior midgut (PM), where an intense release of free heme occurs during the course of digestion. Knockdown of these genes impacted the survival of nymphs and adults, as well as molting, oogenesis and embryogenesis at different rates and time-courses. The silencing of FLVCR caused the highest levels of mortality in nymphs and adults and reduced nymph molting. The oogenesis was mildly affected by the diminished expression of all of the genes whereas embryogenesis was dramatically impaired by the knockdown of ferritin expression. Furthermore, an intense production of ROS in the midgut of blood-fed insects occurs when the expression of ferritin, but not HO, was inhibited. In this manner, the degradation of dietary heme inside the enterocytes may represent an oxidative challenge that is counteracted by ferritins, conferring to this protein a major antioxidant role. Taken together these results demonstrate that the regulation of iron and heme metabolism is of paramount importance for
physiology and imbalances in the levels of these key proteins after a blood- meal can be extremely deleterious to the insects in their various stages of development.
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