RESUMO
Aedes aegypti females are natural vectors of important arboviruses such as dengue, zika, and yellow fever. Mosquitoes activate innate immune response signaling pathways upon infection, as a resistance mechanism to fight pathogens and limit their propagation. Despite the beneficial effects of immune activation for insect vectors, phenotypic costs ultimately affect their fitness. However, the underlying mechanisms that mediate these fitness costs remain poorly understood. Given the high energy required to mount a proper immune response, we hypothesized that systemic activation of innate immunity would impair flight muscle mitochondrial function, compromising tissue energy demand and flight activity. Here, we investigated the dynamic effects of activation of innate immunity by intra-thoracic zymosan injection on A. aegypti flight muscle mitochondrial metabolism. Zymosan injection significantly increased defensin A expression in fat bodies in a time-dependent manner that compromised flight activity. Although oxidant levels in flight muscle were hardly altered, ATP-linked respiratory rates driven by mitochondrial pyruvate+proline oxidation were significantly reduced at 24 h upon zymosan injection. Oxidative phosphorylation coupling was preserved regardless of innate immune response activation along 24 h. Importantly, rotenone-sensitive respiration and complex I-III activity were specifically reduced 24 h upon zymosan injection. Also, loss of complex I activity compromised ATP-linked and maximal respiratory rates mediated by mitochondrial proline oxidation. Finally, the magnitude of innate immune response activation negatively correlated with respiratory rates, regardless of the metabolic states. Collectively, we demonstrate that activation of innate immunity is strongly associated with reduced flight muscle complex I activity with direct consequences to mitochondrial proline oxidation and flight activity. Remarkably, our results indicate a trade-off between dispersal and immunity exists in an insect vector, underscoring the potential consequences of disrupted flight muscle mitochondrial energy metabolism to arbovirus transmission.
RESUMO
Trypanosomatids are protozoan parasites that infect thousands of globally dispersed hosts, potentially affecting their physiology. Several species of trypanosomatids are commonly found in phytophagous insects. Leptomonas wallacei is a gut-restricted insect trypanosomatid only retrieved from Oncopeltus fasciatus. The insects get infected by coprophagy and transovum transmission of L. wallacei cysts. The main goal of the present study was to investigate the effects of a natural infection by L. wallacei on the hemipteran insect O. fasciatus, by comparing infected and uninfected individuals in a controlled environment. The L. wallacei-infected individuals showed reduced lifespan and morphological alterations. Also, we demonstrated a higher infection burden in females than in males. The infection caused by L. wallacei reduced host reproductive fitness by negatively impacting egg load, oviposition, and eclosion, and promoting an increase in egg reabsorption. Moreover, we associated the egg reabsorption observed in infected females, with a decrease in the intersex gene expression. Finally, we suggest alterations in population dynamics induced by L. wallacei infection using a mathematical model. Collectively, our findings demonstrated that L. wallacei infection negatively affected the physiology of O. fasciatus, which suggests that L. wallacei potentially has a vast ecological impact on host population growth.
Assuntos
Heterópteros/fisiologia , Trypanosomatina/patogenicidade , Animais , Estudos de Casos e Controles , Feminino , Heterópteros/parasitologia , Longevidade , Masculino , Modelos Teóricos , Oviposição , Dinâmica Populacional , Caracteres SexuaisRESUMO
Blood-feeding arthropods are vectors of infectious diseases such as dengue, Zika, Chagas disease, and malaria [1], and vector control is essential to limiting disease spread. Because these arthropods ingest very large amounts of blood, a protein-rich meal, huge amounts of amino acids are produced during digestion. Previous work on Rhodnius prolixus, a vector of Chagas disease, showed that, among all amino acids, only tyrosine degradation enzymes were overexpressed in the midgut compared to other tissues [2]. Here we demonstrate that tyrosine detoxification is an essential trait in the life history of blood-sucking arthropods. We found that silencing Rhodnius tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD), the first two enzymes of the phenylalanine/tyrosine degradation pathway, caused the death of insects after a blood meal. This was confirmed by using the HPPD inhibitor mesotrione, which selectively killed hematophagous arthropods but did not affect non-hematophagous insects. In addition, mosquitoes and kissing bugs died after feeding on mice that had previously received a therapeutic effective oral dose (1 mg/kg) of nitisinone, another HPPD inhibitor used in humans for the treatment of tyrosinemia type I [3]. These findings indicate that HPPD (and TAT) can be a target for the selective control of blood-sucking disease vector populations. Because HPPD inhibitors are extensively used as herbicides and in medicine, these compounds may provide an alternative less toxic to humans and more environmentally friendly than the conventional neurotoxic insecticides that are currently used, with the ability to affect only hematophagous arthropods.
Assuntos
4-Hidroxifenilpiruvato Dioxigenase/genética , Inativação Gênica , Proteínas de Insetos/genética , Rhodnius/genética , Tirosina Transaminase/genética , Tirosina/metabolismo , 4-Hidroxifenilpiruvato Dioxigenase/metabolismo , Animais , Feminino , Inativação Metabólica , Proteínas de Insetos/metabolismo , Masculino , Ninfa/genética , Ninfa/crescimento & desenvolvimento , Ninfa/metabolismo , Rhodnius/crescimento & desenvolvimento , Rhodnius/metabolismo , Tirosina Transaminase/metabolismoRESUMO
Rhodnius prolixus not only has served as a model organism for the study of insect physiology, but also is a major vector of Chagas disease, an illness that affects approximately seven million people worldwide. We sequenced the genome of R. prolixus, generated assembled sequences covering 95% of the genome (â¼ 702 Mb), including 15,456 putative protein-coding genes, and completed comprehensive genomic analyses of this obligate blood-feeding insect. Although immune-deficiency (IMD)-mediated immune responses were observed, R. prolixus putatively lacks key components of the IMD pathway, suggesting a reorganization of the canonical immune signaling network. Although both Toll and IMD effectors controlled intestinal microbiota, neither affected Trypanosoma cruzi, the causal agent of Chagas disease, implying the existence of evasion or tolerance mechanisms. R. prolixus has experienced an extensive loss of selenoprotein genes, with its repertoire reduced to only two proteins, one of which is a selenocysteine-based glutathione peroxidase, the first found in insects. The genome contained actively transcribed, horizontally transferred genes from Wolbachia sp., which showed evidence of codon use evolution toward the insect use pattern. Comparative protein analyses revealed many lineage-specific expansions and putative gene absences in R. prolixus, including tandem expansions of genes related to chemoreception, feeding, and digestion that possibly contributed to the evolution of a blood-feeding lifestyle. The genome assembly and these associated analyses provide critical information on the physiology and evolution of this important vector species and should be instrumental for the development of innovative disease control methods.
Assuntos
Adaptação Fisiológica/genética , Doença de Chagas , Interações Hospedeiro-Parasita/genética , Insetos Vetores , Rhodnius , Trypanosoma cruzi/fisiologia , Animais , Sequência de Bases , Transferência Genética Horizontal , Humanos , Insetos Vetores/genética , Insetos Vetores/parasitologia , Dados de Sequência Molecular , Rhodnius/genética , Rhodnius/parasitologia , Wolbachia/genéticaRESUMO
In ticks, the digestion of blood occurs intracellularly and proteolytic digestion of hemoglobin takes place in a dedicated type of lysosome, the digest vesicle, followed by transfer of the heme moiety of hemoglobin to a specialized organelle that accumulates large heme aggregates, called hemosomes. In the present work, we studied the uptake of fluorescent metalloporphyrins, used as heme analogs, and amitraz, one of the most regularly used acaricides to control cattle tick infestations, by Rhipicephalus (Boophilus) microplus midgut cells. Both compounds were taken up by midgut cells in vitro and accumulated inside the hemosomes. Transport of both molecules was sensitive to cyclosporine A (CsA), a well-known inhibitor of ATP binding cassette (ABC) transporters. Rhodamine 123, a fluorescent probe that is also a recognized ABC substrate, was similarly directed to the hemosome in a CsA-sensitive manner. Using an antibody against conserved domain of PgP-1-type ABC transporter, we were able to immunolocalize PgP-1 in the digest vesicle membranes. Comparison between two R. microplus strains that were resistant and susceptible to amitraz revealed that the resistant strain detoxified both amitraz and Sn-Pp IX more efficiently than the susceptible strain, a process that was also sensitive to CsA. A transcript containing an ABC transporter signature exhibited 2.5-fold increased expression in the amitraz-resistant strain when compared with the susceptible strain. RNAi-induced down-regulation of this ABC transporter led to the accumulation of metalloporphyrin in the digestive vacuole, interrupting heme traffic to the hemosome. This evidence further confirms that this transcript codes for a heme transporter. This is the first report of heme transport in a blood-feeding organism. While the primary physiological function of the hemosome is to detoxify heme and attenuate its toxicity, we suggest that the use of this acaricide detoxification pathway by ticks may represent a new molecular mechanism of resistance to pesticides.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Proteínas de Artrópodes/química , Heme/química , Intestinos/fisiologia , Rhipicephalus/fisiologia , Acaricidas/química , Trifosfato de Adenosina/química , Animais , Anticorpos/química , Bovinos , Cromatografia Líquida de Alta Pressão , Ciclosporina/química , Feminino , Metaloporfirinas/química , Metaloporfirinas/farmacocinética , Ligação Proteica , Estrutura Terciária de Proteína , Protoporfirinas/química , Interferência de RNA , Rodamina 123/química , Rodamina 123/farmacocinética , Infestações por Carrapato/tratamento farmacológico , Toluidinas/química , Toluidinas/farmacocinéticaRESUMO
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.
Assuntos
Proteínas de Insetos/genética , Rhodnius/genética , Transcriptoma , Animais , Feminino , Trato Gastrointestinal , Proteínas de Insetos/biossíntese , América Latina , Masculino , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
In insects, eggshell hardening involves cross-linking of chorion proteins via their tyrosine residues. This process is catalyzed by peroxidases at the expense of H2O2 and confers physical and biological protection to the developing embryo. Here, working with Rhodnius prolixus, the insect vector of Chagas disease, we show that an ovary dual oxidase (Duox), a NADPH oxidase, is the source of the H2O2 that supports dityrosine-mediated protein cross-linking and eggshell hardening. RNAi silencing of Duox activity decreased H2O2 generation followed by a failure in embryo development caused by a reduced resistance to water loss, which, in turn, caused embryos to dry out following oviposition. Phenotypes of Duox-silenced eggs were reversed by incubation in a water-saturated atmosphere, simultaneous silencing of the Duox and catalase genes, or H2O2 injection into the female hemocoel. Taken together, our results show that Duox-generated H2O2 fuels egg chorion hardening and that this process plays an essential role during eggshell waterproofing.
Assuntos
NADPH Oxidases/metabolismo , Rhodnius/enzimologia , Sequência de Aminoácidos , Animais , Córion/fisiologia , Feminino , Genes de Insetos , Peróxido de Hidrogênio/metabolismo , Dados de Sequência Molecular , NADPH Oxidases/química , NADPH Oxidases/genética , Oogênese/genética , Oogênese/fisiologia , Ovário/enzimologia , Filogenia , Estrutura Terciária de Proteína , Interferência de RNA , Rhodnius/genética , Rhodnius/fisiologia , Homologia de Sequência de AminoácidosRESUMO
In this opinion paper, we discuss the potential and challenges of using the symbiont Wolbachia to block mosquito transmitted diseases such as dengue, malaria and chikungunya in Latin America.
Assuntos
Culicidae/microbiologia , Insetos Vetores/microbiologia , Controle Biológico de Vetores/métodos , Wolbachia/fisiologia , Infecções por Alphavirus/prevenção & controle , Animais , Febre de Chikungunya , Dengue/prevenção & controle , Humanos , América Latina , Malária/prevenção & controleRESUMO
Insect oocytes grow in close association with the ovarian follicular epithelium (OFE), which escorts the oocyte during oogenesis and is responsible for synthesis and secretion of the eggshell. We describe a transcriptome of OFE of the triatomine bug Rhodnius prolixus, a vector of Chagas disease, to increase our knowledge of the role of FE in egg development. Random clones were sequenced from a cDNA library of different stages of follicle development. The transcriptome showed high commitment to transcription, protein synthesis, and secretion. The most abundant cDNA was a secreted (S) small, proline-rich protein with maximal expression in the vitellogenic follicle, suggesting a role in oocyte maturation. We also found Rp45, a chorion protein already described, and a putative chitin-associated cuticle protein that was an eggshell component candidate. Six transcripts coding for proteins related to the unfolded-protein response (UPR) by were chosen and their expression analyzed. Surprisingly, transcripts related to UPR showed higher expression during early stages of development and downregulation during late stages, when transcripts coding for S proteins participating in chorion formation were highly expressed. Several transcripts with potential roles in oogenesis and embryo development are also discussed. We propose that intense protein synthesis at the FE results in reticulum stress (RS) and that lowering expression of a set of genes related to cell survival should lead to degeneration of follicular cells at oocyte maturation. This paradoxical suppression of UPR suggests that ovarian follicles may represent an interesting model for studying control of RS and cell survival in professional S cell types.
Assuntos
Perfilação da Expressão Gênica , Insetos Vetores/metabolismo , Rhodnius/metabolismo , Animais , Morte Celular , DNA Complementar/química , DNA Complementar/isolamento & purificação , Bases de Dados Genéticas , Feminino , Proteínas de Insetos/biossíntese , Proteínas de Insetos/metabolismo , Folículo Ovariano/crescimento & desenvolvimento , Folículo Ovariano/metabolismo , Análise de Sequência de DNA , Resposta a Proteínas não DobradasRESUMO
In mammalian cells, heme can be degraded by heme-oxygenases (HO). Heme-oxygenase 1 (HO-1) is known to be the heme inducible isoform, whereas heme-oxygenase 2 (HO-2) is the constitutive enzyme. Here we investigated the presence of HO during erythroid differentiation in human bone marrow erythroid precursors and K562 cells. HO-1 mRNA and protein expression levels were below limits of detection in K562 cells. Moreover, heme was unable to induce HO-1, at the protein and mRNA profiles. Surprisingly, HO-2 expression was inhibited upon incubation with heme. To evaluate the physiological relevance of these findings, we analyzed HO expression during normal erythropoiesis in human bone marrow. Erythroid precursors were characterized by lack of significant expression of HO-1 and by progressive reduction of HO-2 during differentiation. FLVCR expression, a recently described heme exporter found in erythroid precursors, was also analyzed. Interestingly, the disruption in the HO detoxification system was accompanied by a transient induction of FLVCR. It will be interesting to verify if the inhibition of HO expression, that we found, is preventing a futile cycle of concomitant heme synthesis and catabolism. We believe that a significant feature of erythropoiesis could be the replacement of heme breakdown by heme exportation, as a mechanism to prevent heme toxicity.
Assuntos
Células da Medula Óssea/enzimologia , Eritropoese , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1/metabolismo , Adulto , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células Eritroides/citologia , Células Eritroides/efeitos dos fármacos , Células Eritroides/enzimologia , Eritropoese/efeitos dos fármacos , Glicoforinas/metabolismo , Heme/farmacologia , Heme Oxigenase (Desciclizante)/antagonistas & inibidores , Hemoglobinas/biossíntese , Humanos , Células K562 , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , Receptores Virais/metabolismoRESUMO
BACKGROUND: Aedes aegypti is the main vector of the virus causing Dengue fever, a disease that has increased dramatically in importance in recent decades, affecting many tropical and sub-tropical areas of the globe. It is known that viruses and other parasites can potentially alter vector behavior. We investigated whether infection with Dengue virus modifies the behavior of Aedes aegypti females with respect to their activity level. METHODS/PRINCIPAL FINDINGS: We carried out intrathoracic Dengue 2 virus (DENV-2) infections in Aedes aegypti females and recorded their locomotor activity behavior. We observed an increase of up to â¼50% in the activity of infected mosquitoes compared to the uninfected controls. CONCLUSIONS: Dengue infection alters mosquito locomotor activity behavior. We speculate that the higher levels of activity observed in infected Aedes aegypti females might involve the circadian clock. Further studies are needed to assess whether this behavioral change could have implications for the dynamics of Dengue virus transmission.
Assuntos
Aedes/virologia , Vírus da Dengue/fisiologia , Dengue/virologia , Atividade Motora/fisiologia , Animais , FemininoRESUMO
The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme.
Assuntos
Aedes/microbiologia , Heme/metabolismo , Hemoglobinas/metabolismo , Proteínas de Insetos/metabolismo , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Animais , Heme/farmacologia , Hemoglobinas/farmacologia , Humanos , CoelhosRESUMO
This study reports the cloning, expression analysis and localization of calreticulin (CRT) in the endoplasmic reticulum (ER) during late oogenesis and early embryogenesis of the insect Rhodnius prolixus. CRT was cloned and sequenced from cDNA extracted from unfertilized eggs. Real-time PCR showed that CRT expression remains at lower levels during late oogenesis when compared to vitellogenic oocytes or day 0 laid fertilized eggs. Immunofluorescence microscopy showed that this protein is located in the periphery of the egg, in a differential peripheral ooplasm surrounding the yolk-rich internal ooplasm, only identified by transmission electron microscopy (TEM) of thin sections. Using immunogold electron microscopy, the ER ultrastructure (CRT labeled) was identified in the peripheral ooplasm as dispersed lamellae, randomly distributed in the peripheral ooplasm. No massive alterations of ER ultrastructure were found before or right after (30 min) fertilization, but an increase in CRT expression levels and assembly of typical rough ER (parallel cisternae with associated ribosomes) were observed 18-24 h after oviposition. The lack of ER assembly at fertilization and the later formation of rough ER together with the increase in CRT expression levels, suggest that the major functions of ER might be of great importance during the early events of development. The possible involvement of ER in the early steps of embryogenesis will be discussed.
Assuntos
Calreticulina/genética , Calreticulina/metabolismo , Desenvolvimento Embrionário/genética , Retículo Endoplasmático/metabolismo , Oogênese/genética , Rhodnius/embriologia , Rhodnius/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Calreticulina/química , Calreticulina/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Fertilização , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Dados de Sequência Molecular , Óvulo/citologia , Óvulo/metabolismo , Óvulo/ultraestrutura , Rhodnius/citologia , Rhodnius/ultraestrutura , Alinhamento de SequênciaRESUMO
Infectious diseases are a major cause of mortality in the world and, among them, dengue is considered the main human arbovirus. No effective vaccines or antiviral drugs are available for this illness, and it is estimated that 2.5 billion people live at risk, leading to millions of dengue cases annually. The most common method for dengue virus (DENV) quantitation is the plaque assay, but there are DENV strains that do not form plaques. For this reason, a PCR protocol able to detect and quantify DENV in the different kinds of samples employed for DENV study is of great value. In this study, a real-time PCR method suitable not only for clinical objectives but also for laboratory routine is described. Sequences from several strains of DENV comprising the four serotypes were aligned. A fragment located at the 5'UTR region of the virus genome was used to generate the primers and the probe for real-time PCR. This method was successfully used to identify and quantify distinct dengue virus strains and serotypes in clinical samples, in sera from patients infected with dengue virus, and in the mosquito Aedes aegypti, as well as to study virus replication in different cell lines.
Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Aedes/virologia , Animais , Sequência de Bases , Dengue/sangue , Dengue/virologia , Vírus da Dengue/classificação , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , Sorotipagem , Ensaio de Placa ViralRESUMO
BACKGROUND: Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion can exert cellular deleterious effects. Mitochondria perform multiple roles in cell biology acting as the site of aerobic energy-transducing pathways, and also an important source of reactive oxygen species (ROS), modulating redox metabolism. Therefore, regulation of mitochondrial function should be relevant for hematophagous arthropods. Here, we investigated the effects of blood-feeding on flight muscle (FM) mitochondria from the mosquito Aedes aegypti, a vector of dengue and yellow fever. METHODOLOGY/PRINCIPAL FINDINGS: Blood-feeding caused a reversible reduction in mitochondrial oxygen consumption, an event that was parallel to blood digestion. These changes were most intense at 24 h after blood meal (ABM), the peak of blood digestion, when oxygen consumption was inhibited by 68%. Cytochromes c and a+a(3) levels and cytochrome c oxidase activity of the electron transport chain were all reduced at 24 h ABM. Ultrastructural and molecular analyses of FM revealed that mitochondria fuse upon blood meal, a condition related to reduced ROS generation. Consistently, BF induced a reversible decrease in mitochondrial H(2)O(2) formation during blood digestion, reaching their lowest values at 24 h ABM where a reduction of 51% was observed. CONCLUSION: Blood-feeding triggers functional and structural changes in hematophagous insect mitochondria, which may represent an important adaptation to blood feeding.
Assuntos
Aedes/fisiologia , Sangue/metabolismo , Voo Animal , Mitocôndrias Musculares/metabolismo , Aedes/metabolismo , Ração Animal , Ciências da Nutrição Animal , Animais , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Peróxido de Hidrogênio/química , Microscopia Eletrônica de Transmissão/métodos , Modelos Biológicos , Oxirredução , Consumo de Oxigênio , RNA/metabolismo , Coelhos , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A blood-sucking habit appeared independently several times in the course of arthropod evolution. However, from more than a million species of insects and arachnids presently living on earth, only about 14,000 species developed the capacity to feed on vertebrate blood. This figure suggests the existence of severe physiological constraints for the evolution of hematophagy, implying the selective advantage of special adaptations related to the use of blood as a food source. Digestion of vertebrate hemoglobin in the midgut of blood-feeding arthropods results in the production of large amounts of heme, a potentially cytotoxic molecule. Here we will review mechanisms by which heme can exert biological damage, together with a wide spectrum of adaptations developed by blood-feeding insects and ticks to counteract its deleterious effects. In spite of the existence of a great molecular diversity of protective mechanisms, different hematophagous organisms developed convergent solutions that may be physiologically equivalent.
Assuntos
Adaptação Fisiológica , Artrópodes/fisiologia , Heme/metabolismo , Animais , Antioxidantes/fisiologia , Artrópodes/parasitologia , Comportamento Alimentar , Hemeproteínas/fisiologia , Peroxidação de Lipídeos , Estresse OxidativoRESUMO
The tick Boophilus microplus is a bovine ectoparasite present in tropical and subtropical areas of the world and the use of vaccines is a promising method for tick control. BYC is an aspartic proteinase found in eggs that is involved in the embryogenesis of B. microplus and was proposed as an important antigen in the development of an anti-tick vaccine. The cDNA of BYC was amplified by PCR and cloned for expression in two forms with and without thioredoxin fusion protein (Trx), coding recombinant proteins named rBYC-Trx and rBYC, respectively. Expression, solubility, and yields of the two forms were analyzed. The recombinant proteins were expressed in inclusion bodies (IBs) and three denaturant agents (N-lauroyl sarcosine, guanidine hydrochloride, and urea) were tested for IBs solubilization. The N-lauroyl sarcosine solubilized 90.4 and 92.4% of rBYC-Trx and rBYC IBs, respectively, and was the most efficient denaturant. Two recombinant forms were affinity-purified by Ni2+-Sepharose under denaturing conditions. After dialysis, the yield of soluble protein was 84.1% for r-BYC-Trx and 5.9% for rBYC. These proteins were immune-reactive against sera from rabbit, mouse, and bovine previously immunized with native BYC, which confirms the antigenicity of the recombinant BYCs expressed in the Escherichia coli system.
Assuntos
Ácido Aspártico Endopeptidases/genética , Ácido Aspártico Endopeptidases/isolamento & purificação , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Corpos de Inclusão/genética , Carrapatos/genética , Animais , Ácido Aspártico Endopeptidases/metabolismo , Western Blotting , Clonagem Molecular , DNA Complementar/genética , Precursores Enzimáticos/metabolismo , Epitopos , Feminino , Regulação da Expressão Gênica , Guanidina/farmacologia , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Sarcosina/farmacologia , Solubilidade , Ureia/farmacologiaRESUMO
The biological activity of granulocyte-macrophage colony-stimulating factor (GM-CSF) is modulated by the sulfated glycosaminoglycans (GAGs) heparan sulfate and heparin. However, the molecular mechanisms involved in such interactions are still not completely understood. We have proposed previously that helix C, one of the four alpha-helices of human GM-CSF (hGM-CSF), contains a GAG-binding site in which positively charged residues are spatially positioned for interaction with the sulfate moieties of the GAGs (Wettreich, A., Sebollela, A., Carvalho, M. A., Azevedo, S. P., Borojevic, R., Ferreira, S. T., and Coelho-Sampaio, T. (1999) J. Biol. Chem. 274, 31468-31475). Protonation of two histidine residues (His83 and His87) in helix C of hGM-CSF appears to act as a pH-dependent molecular switch to control the interaction with GAGs. Based on these findings, we have now generated a triple mutant form of murine GM-CSF (mGM-CSF) in which three noncharged residues in helix C of the murine factor (Tyr83, Gln85, and Tyr87) were replaced by the corresponding basic residues present in hGM-CSF (His83, Lys85, and His87). Binding assays on heparin-Sepharose showed that, at acidic pH, the triple mutant mGM-CSF binds to immobilized heparin with significantly higher affinity than wild type (WT) mGM-CSF and that neither protein binds to the column at neutral pH. The fact that even WT mGM-CSF binds to heparin at acidic pH indicates the existence of a distinct, lower affinity heparin-binding site in the protein. Chemical modification of the single histidine residue (His15) located in helix A of WT mGM-CSF with diethyl pyrocarbonate totally abolished binding to immobilized heparin. Moreover, replacement of His15 for an alanine residue significantly reduced the affinity of mGM-CSF for heparin at pH 5.0 and completely blocked heparin binding to a synthetic peptide corresponding to helix A of GM-CSF. These results indicate a major role of histidine residues in the regulation of the binding of GM-CSF to GAGs, supporting the notion that an acidic microenvironment is required for GM-CSF-dependent regulation of target cells. In addition, our results provide insight into the molecular basis of the strict species specificity of the biological activity of GM-CSF.