RESUMO
This study aimed to evaluate the genomic profile of the Antarctic marine Curtobacterium sp. CBMAI 2942, as well as to optimize the conditions for chitinase production and antifungal potential for biological control. Assembly and annotation of the genome confirmed the genomic potential for chitinase synthesis, revealing two ChBDs of chitin binding (Chi C). The optimization enzyme production using an experimental design resulted in a 3.7-fold increase in chitinase production. The chitinase enzyme was identified by SDS-PAGE and confirmed through mass spectrometry analysis. The enzymatic extract obtained using acetone showed antifungal activity against the phytopathogenic fungus Aspergillus sp. series Nigri CBMAI 1846. The genetic capability of Curtobacterium sp. CBMAI 2942 for chitin degradation was confirmed through genomic analysis. The basal culture medium was adjusted, and the chitinase produced by this isolate from Antarctica showed significant inhibition against Aspergillus sp. Nigri series CBMAI 1846, which is a tomato phytopathogenic fungus. This suggests that this marine bacterium could potentially be used as a biological control of agricultural pests.
Assuntos
Antifúngicos , Quitinases , Proteômica , Quitinases/metabolismo , Quitinases/genética , Quitinases/farmacologia , Antifúngicos/farmacologia , Regiões Antárticas , Proteômica/métodos , Genômica/métodos , Aspergillus/enzimologia , Aspergillus/genética , Genoma Bacteriano , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Organismos Aquáticos , Quitina/farmacologia , Quitina/metabolismo , Quitina/químicaRESUMO
With the emergence of multidrug-resistant microorganisms, microbial agents have become a serious global threat, affecting human health and various plants. Therefore, new therapeutic alternatives, such as chitin-binding proteins, are necessary. Chitin is an essential component of the fungal cell wall, and chitin-binding proteins exhibit antifungal activity. In the present study, chitin-binding peptides isolated from Capsicum chinense seeds were characterized and evaluated for their in vitro antimicrobial effect against the growth of Candida and Fusarium fungi. Proteins were extracted from the seeds and subsequently the chitin-binding proteins were separated by chitin affinity chromatography. After chromatography, two fractions, Cc-F1 (not retained on the column) and Cc-F2 (retained on the column), were obtained. Electrophoresis revealed major protein bands between 6.5 and 26.6 kDa for Cc-F1 and only a ~ 6.5 kDa protein band for Cc-F2, which was subsequently subjected to mass spectrometry. The protein showed similarity with hevein-like and endochitinase and was then named Cc-Hev. Data are available via ProteomeXchange with identifier PXD054607. Next, we predicted the three-dimensional structure of the peptides and performed a peptide docking with (NAG)3. Subsequently, growth inhibition assays were performed to evaluate the ability of the peptides to inhibit microorganism growth. Cc-Hev inhibited the growth of C. albicans (up to 75% inhibition) and C. tropicalis (100% inhibition) and induced a 65% decrease in cell viability for C. albicans and 100% for C. tropicalis. Based on these results, new techniques to combat fungal diseases could be developed through biotechnological applications; therefore, further studies are needed.
Assuntos
Antifúngicos , Candida , Capsicum , Quitina , Quitinases , Fusarium , Sementes , Sementes/química , Antifúngicos/farmacologia , Antifúngicos/isolamento & purificação , Antifúngicos/química , Antifúngicos/metabolismo , Quitina/metabolismo , Quitina/farmacologia , Fusarium/efeitos dos fármacos , Quitinases/farmacologia , Quitinases/metabolismo , Quitinases/química , Quitinases/isolamento & purificação , Candida/efeitos dos fármacos , Candida/enzimologia , Lectinas de Plantas/farmacologia , Lectinas de Plantas/química , Lectinas de Plantas/isolamento & purificação , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Simulação de Acoplamento Molecular , Proteínas de Plantas/farmacologia , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Peptídeos Catiônicos AntimicrobianosRESUMO
Chitinases are promising enzymes for a multitude of applications, including chitooligosaccharide (COS) synthesis for food and pharmaceutical uses and marine waste management. Owing to fungal diversity, fungal chitinases may offer alternatives for chitin degradation and industrial applications. The rapid reproduction cycle, inexpensive growth media, and ease of handling of fungi may also contribute to reducing enzyme production costs. Thus, this study aimed to identify fungal species with chitinolytic potential and optimize chitinase production by submerged culture and enzyme characterization using shrimp chitin. Three fungal species, Coriolopsis byrsina, Trichoderma reesei, and Trichoderma harzianum, were selected for chitinase production. The highest endochitinase production was achieved in C. byrsina after 168 h cultivation (0.3 U mL- 1). The optimal temperature for enzyme activity was similar for the three fungal species (up to 45 and 55 ºC for endochitinases and exochitinases, respectively). The effect of pH on activity indicated maximum hydrolysis in acidic pH (4-7). In addition, the crude T. reesei extract showed promising properties for removing Candida albicans biofilms. This study showed the possibility of using shrimp chitin to induce chitinase production and enzymes that can be applied in different industrial sectors.
Assuntos
Biofilmes , Quitina , Quitinases , Biofilmes/crescimento & desenvolvimento , Quitinases/metabolismo , Quitinases/biossíntese , Quitina/metabolismo , Concentração de Íons de Hidrogênio , Temperatura , Hypocreales/enzimologia , Hypocreales/metabolismo , Candida albicans/enzimologia , Hidrólise , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genéticaRESUMO
Faced with the emergence of multiresistant microorganisms that affect human health, microbial agents have become a serious global threat, affecting human health and plant crops. Antimicrobial peptides have attracted significant attention in research for the development of new microbial control agents. This work's goal was the structural characterization and analysis of antifungal activity of chitin-binding peptides from Capsicum baccatum and Capsicum frutescens seeds on the growth of Candida and Fusarium species. Proteins were initially submitted to extraction in phosphate buffer pH 5.4 and subjected to chitin column chromatography. Posteriorly, two fractions were obtained for each species, Cb-F1 and Cf-F1 and Cb-F2 and Cf-F2, respectively. The Cb-F1 (C. baccatum) and Cf-F1 (C. frutescens) fractions did not bind to the chitin column. The electrophoresis results obtained after chromatography showed two major protein bands between 3.4 and 14.2 kDa for Cb-F2. For Cf-F2, three major bands were identified between 6.5 and 14.2 kDa. One band from each species was subjected to mass spectrometry, and both bands showed similarity to nonspecific lipid transfer protein. Candida albicans and Candida tropicalis had their growth inhibited by Cb-F2. Cf-F2 inhibited the development of C. albicans but did not inhibit the growth of C. tropicalis. Both fractions were unable to inhibit the growth of Fusarium species. The toxicity of the fractions was tested in vivo on Galleria mellonella larvae, and both showed a low toxicity rate at high concentrations. As a result, the fractions have enormous promise for the creation of novel antifungal compounds.
Assuntos
Antifúngicos , Candida , Quitina , Fusarium , Simulação de Acoplamento Molecular , Antifúngicos/farmacologia , Antifúngicos/química , Antifúngicos/metabolismo , Quitina/química , Quitina/metabolismo , Fusarium/efeitos dos fármacos , Candida/efeitos dos fármacos , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Animais , Capsicum/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Testes de Sensibilidade Microbiana , Ligação Proteica , Conformação ProteicaRESUMO
Chitin is a biopolymer profusely present in nature and of pivotal importance as a structural component in cells. It is degraded by chitinases, enzymes naturally produced by different organisms. Chitinases are proteins enrolled in many cellular mechanisms, including the remodeling process of the fungal cell wall, the cell growth process, the autolysis of filamentous fungi, and cell separation of yeasts, among others. These enzymes also have properties with different biotechnological applications. They are used to produce polymers, for biological control, biofilm formation, and as antitumor and anti-inflammatory target molecules. Chitinases are classified into different glycoside hydrolase (GH) families and are widespread in microorganisms, including viruses. Among them, the GH18 family is highly predominant in the viral genomes, being present and active enzymes in baculoviruses and nucleocytoplasmic large DNA viruses (NCLDV), especially chloroviruses from the Phycodnaviridae family. These viral enzymes contain one or more GH domains and seem to be involved during the viral replication cycle. Curiously, only a few DNA viruses have these enzymes, and studying their properties could be a key feature for biological and biotechnological novelties. Here, we provide an overview of viral chitinases and their probable function in viral infection, showing evidence of at least two distinct origins for these enzymes. Finally, we discuss how these enzymes can be applied as biotechnological tools and what one can expect for the coming years on these GHs.
Assuntos
Quitinases , Humanos , Quitinases/química , Quitinases/genética , Quitinases/metabolismo , Proteínas , Quitina/química , Quitina/metabolismo , Biotecnologia , FungosRESUMO
The midgut of Zabrotes subfasciatus (Coleoptera) and other insects may have regions lacking a peritrophic membrane (matrix, PM) and covered with a jelly-like material known as peritrophic gel. This work was undertaken to test the hypothesis that the peritrophic gel is a vertebrate-like mucus. By histochemistry we identified mucins along the whole midgut, which contrasts with the known occurrence of PM only at the posterior midgut. We also analyzed the expression of the genes coding for mucus-forming mucins (Mf-mucins), peritrophins, chitin synthases and chitin deacetylases along the midgut and carcass (insect without midgut) by RNA-seq. Mf-mucins were identified as proteins with high O-glycosylation and multiple tandem repeats of Pro/Thr/Ser residues. Peritrophins were separated into PM proteins, cuticular proteins analogous to peritrophins (CPAPs) and ubiquitous-chitin-binding domain-(CBD)-containing proteins (UCBPs). PM proteins have at least 3, CPAP one or 3, and UCBPs have a varied number of CBDs. PM proteins are more expressed at midgut, CPAP at the carcass, and UCBP at both. The results showed that most PM proteins are mainly expressed at the posterior midgut, together with midgut chitin synthase and chitin deacetylase, and in agreement with the presence of PM only at the posterior midgut by visual inspection. The excretion of most midgut chitinase is avoided, suggesting that the shortened PM is functional. Mf-mucins are expressed along the whole midgut, probably forming the extracellular mucus layer observed by histochemistry. Thus, the lack of PM at anterior and middle midgut causes the exposure of a mucus, which may correspond to the previously described peritrophic gel. The putative functional interplay of mucus and PM is discussed. The major role of mucus is proposed to be tissue protection and of PM to enhancing digestive efficiency by allowing enzyme recycling.
Assuntos
Besouros , Animais , Besouros/metabolismo , Proteínas de Membrana/metabolismo , Mucinas/genética , Transcriptoma , Insetos/metabolismo , Quitina/metabolismo , Proteínas de Insetos/metabolismo , Larva/genéticaRESUMO
It is difficult to produce chitin oligosaccharides by hydrolyzing untreated natural chitinous waste directly. In this study, two fungi Talaromyces allahabadensis Hi-4 and Talaromyces funiculosus Hi-5 from rotten black soldier fly were isolated and identified through multigene phylogenetic and morphological analyses. The chitinolytic enzymes were produced by solid state fermentation, and the growth conditions were optimized by combining single-factor and central composite design. The best carbon sources were powder of molting of mealworms (MMP) and there was no need for additional nitrogen sources in two fungi, then the maximum chitinolytic enzyme production of 46.80 ± 3.30 (Hi-4) and 55.07 ± 2.48 (Hi-5) U/gds were achieved after analyzing the 3D response surface plots. Pure chitin (colloidal chitin) and natural chitinous substrates (represented by MMP) were used to optimize degradation abilities by crude enzymes obtained from the two fungi. The optimum temperature for hydrolyzing MMP (40 °C both in two fungi) were lower and closer to room temperature than colloidal chitin (55 °C for Hi-4 and 45 °C for Hi-5). Then colloidal chitin, MMP and the powder of shrimp shells (SSP) were used for analyzing the products after 5-day degradation. The amounts of chitin oligosaccharides from SSP and MMP were about 1/6 (Hi-4), 1/17 (Hi-5) and 1/8 (Hi-4), 1/10 (Hi-5), respectively, in comparison to colloidal chitin. The main components of the products were GlcNAc for colloidal chitin, (GlcNAc)2 for MMP, and oligosaccharides with higher degree of polymerization (4-6) were obtained when hydrolyzing SSP, which is significant for applications in medicine and health products.
Assuntos
Quitinases , Dípteros , Talaromyces , Animais , Quitina/metabolismo , Fermentação , Filogenia , Pós , Talaromyces/metabolismo , Oligossacarídeos , Quitinases/genética , Insetos , Dípteros/metabolismoRESUMO
Vibrio parahaemolyticus can degrade insoluble chitin with the help of chitinase enzymes that generate soluble N-acetyl glucosamine oligosaccharides (GlcNAcn) to induce a state of natural competence for the uptake of extracellular DNA. In this study, we had evaluated the role of various regulatory factors such as TfoX, CytR, OpaR, and RpoS during natural transformation of V. parahaemolyticus. The results suggest that TfoX regulates natural competence via CytR in a chitin-dependent manner. CytR controls the release of GlcNAc6 from insoluble chitin and conversion of GlcNAc6 into smaller GlcNAc residues inside the periplasm by modulating the expression of endochitinase and periplasmic chitinases. In addition, CytR was also responsible for GlcNAc6-mediated upregulation of competence-related genes such as pilA, pilB, comEA, and qstR. Next, we found that the quorum sensing regulator OpaR affects the natural transformation through its regulation of extracellular nuclease Dns. The ΔopaR mutant showed increased expression of Dns, which might degrade the eDNA. As a consequence, the transformation efficiency was decreased and eDNA-dependent growth was hugely enhanced. However, when Dns-containing DASW was substituted with fresh DASW, the transformation was detectable in ΔopaR mutant and eDNA-dependent growth was less. These results suggest that the occurrence of natural transformation and eDNA-dependent growth were inversely related to each other. Lastly, the general stress regulator RpoS was required for neither quorum-sensing dependent nor chitin-dependent regulation of natural competence in V. parahaemolyticus.
Assuntos
Quitinases , Vibrio cholerae , Vibrio parahaemolyticus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Quitina/metabolismo , Quitinases/genética , Quitinases/metabolismo , Regulação Bacteriana da Expressão Gênica , Vibrio cholerae/genética , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismoRESUMO
Callosobruchus maculatus is the main pest cowpea (Vigna unguiculata). Given its relevance as an insect pest, studies have focused in finding toxic compounds which could prevent its predatory action towards the seeds. Clitoria fairchildiana is a native Amazon species, whose seeds are refractory to insect predation. This characteristic was the basis of our interest in evaluating the toxicity of its seed proteins to C. maculatus larvae. Seed proteins were fractioned, according to their solubility, to albumins (F1), globulins (F2), kaphyrins (F3), glutelins (F4), linked kaphyrins (F5) and cross-linked glutelins (F6). The fractionated proteins were quantified, analysed by tricine-SDS-PAGE and inserted into the diet of this insect pest in order to evaluate their insecticidal potential. The most toxic fraction to C. maculatus, the propanol soluble F3, was submitted to molecular exclusion chromatography and all of the peaks obtained, F3P1, F3P2, F3P3, caused a reduction of larval mass, especially F3P1, seen as a major ~12 kDa electrophoretic band. This protein was identified as a vicilin-like protein by mass spectrometry and BLAST analysis. The alignment of the Cfvic (C. fairchildiana vicilin) peptides with a V. unguiculata vicilin sequence, revealed that Cfvic has at least five peptides (ALLTLVNPDGR, AILTLVNPDGR, NFLAGGKDNV, ISDINSAMDR, NFLAGEK) which lined up with two chitin binding sites (ChBS). This finding was corroborated by chitin affinity chromatography and molecular docking of chitin-binding domains for N-Acetyl-D-glucosamine and by the reduction of Cfvic chitin affinity after chemical modification of its Lys residues. In conclusion, Cfvic is a 12 kDa vicilin-like protein, highly toxic to C. maculatus, acting as an insect toxin through its ability to bind to chitin structures present in the insect midgut.
Assuntos
Clitoria , Besouros , Animais , Quitina/metabolismo , Clitoria/metabolismo , Besouros/metabolismo , Cotilédone/metabolismo , Glutens/análise , Glutens/metabolismo , Larva/metabolismo , Simulação de Acoplamento Molecular , Proteínas de Armazenamento de Sementes , Sementes/químicaRESUMO
Chitooligosaccharides (COS) have a great potential to be used by pharmaceutical industry due to their many biological activities. The use of enzymes to produce them is very advantageous, however it still faces many challenges, such as discovering new strains capable to produce enzymes that are able to generate bioactive oligosaccharides. In the present study a purification protein protocol was performed to purify chitosanases produced by Bacillus toyonensis CCT 7899 for further chitosan hydrolysis. The produced chitooligosaccharides were characterized by mass spectroscopy (MS) and their antiedematogenic effect was investigated through carrageenan-induced paw edema model. The animals were treated previously to inflammation by intragastric route with COS at 30, 300 and 600 mg/kg. The purification protocol showed a good performance for the chitosanases purification using 0.20 M NaCl solution to elute it, with a 9.54-fold purification factor. The treatment with COS promoted a decrease of paw edema at all evaluated times and the AUC0-4h, proving that COS produced showed activity in acute inflammation like commercial anti-inflammatory Dexamethasone (corticosteroid). Therefore, the strategy used to purification was successfully applied and it was possible to generate bioactive oligosaccharides with potential pharmacological use.
Assuntos
Bacillus , Quitosana , Animais , Bacillus/metabolismo , Quitina/metabolismo , Quitosana/química , Edema/induzido quimicamente , Edema/tratamento farmacológico , Glicosídeo Hidrolases/metabolismo , Inflamação , Oligossacarídeos/metabolismoRESUMO
Large volumes of fruit and vegetable production are lost during postharvest handling due to attacks by necrotrophic fungi. One of the promising alternatives proposed for the control of postharvest diseases is the induction of natural defense responses, which can be activated by recognizing molecules present in pathogens, such as chitin. Chitin is one of the most important components of the fungal cell wall and is recognized through plant membrane receptors. These receptors belong to the receptor-like kinase (RLK) family, which possesses a transmembrane domain and/or receptor-like protein (RLP) that requires binding to another RLK receptor to recognize chitin. In addition, these receptors have extracellular LysM motifs that participate in the perception of chitin oligosaccharides. These receptors have been widely studied in Arabidopsis thaliana (A. thaliana) and Oryza sativa (O. sativa); however, it is not clear how the molecular recognition and plant defense mechanisms of chitin oligosaccharides occur in other plant species or fruits. This review includes recent findings on the molecular recognition of chitin oligosaccharides and how they activate defense mechanisms in plants. In addition, we highlight some of the current advances in chitin perception in horticultural crops.
Assuntos
Quitina/metabolismo , Produtos Agrícolas/microbiologia , Resistência à Doença , Polissacarídeos Fúngicos/metabolismo , Horticultura , Interações Hospedeiro-Patógeno , Biomarcadores , Produtos Agrícolas/imunologia , Resistência à Doença/imunologia , Interações Hospedeiro-Patógeno/imunologia , Doenças das Plantas/microbiologia , Receptores de Superfície Celular/metabolismo , Transdução de SinaisRESUMO
Aedes aegypti is a mosquito vector of arboviruses such as dengue, chikungunya, zika and yellow fever that cause important public health diseases. The incidence and gravity of these diseases justifies the search for effective measures to reduce the presence of this vector in the environment. Bioinsecticides are an effective alternative method for insect control, with added ecological benefits such as biodegradability. The current study demonstrates that a chitinolytic enzyme complex produced by the fungus Trichoderma asperellum can disrupt cuticle formation in the L3 larvae phase of A. aegypti, suggesting such biolarvicidal action could be used for mosquito control. T. asperellum was exposed to chitin from different sources. This induction of cell wall degrading enzymes, including chitinase, N-acetylglucosaminidase and ß-1,3-glucanase. Groups of 20 L3 larvae of A. aegypti were exposed to varying concentrations of chitinolytic enzymes induced with commercial chitin (CWDE) and larvae cell wall degrading enzymes (L-CWDE). After 72 h of exposure to the CWDE, 100% of larvae were killed. The same percent mortality was observed after 48 h of exposure to L-CWDE at half the CWDE enzyme mixture concentration. Exoskeleton deterioration was further observed by scanning and electron microscopy. Our findings indicate that L-CWDE produced by T. asperellum reflect chitinolytic enzymes with greater specificity for L3 larval biomolecules. This specificity is characterized by the high percentage of mortality compared with CWDE treatments and also by abrupt changes in patterns of the cellular structures visualized by scanning and transmission electron microscopy. These mixtures of chitinolytic enzymes could be candidates, as adjuvant or synergistic molecules, to replace conventional chemical insecticides currently in use.
Assuntos
Aedes/efeitos dos fármacos , Hypocreales/enzimologia , Inseticidas , Larva/efeitos dos fármacos , Animais , Parede Celular/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/farmacologia , Inseticidas/química , Inseticidas/farmacologiaRESUMO
The first line of the Arthropods defense against infections is the hard-structured exoskeleton, a physical barrier, usually rich in insoluble chitin. For entomopathogenic fungi that actively penetrate the host body, an arsenal of hydrolytic enzymes (as chitinases and N-acetylglucosaminidases), that break down chitin, is essential. Notably, twenty-one putative chitinase genes have been identified in the genome of Metarhizium anisopliae, a generalist entomopathogenic fungus. As a multigenic family, with enzymes that, presumably, perform redundant functions, the main goal is to understand the singularity of each one of such genes and to discover their precise role in the fungal life cycle. Specially chitinases that can act as virulence determinants are of interest since these enzymes can lead to more efficient biocontrol agents. Here we explored a horizontally acquired chitinase from M. anisopliae, named chiMaD1. The deletion of this gene did not lead to phenotypic alterations or diminished supernatant's chitinolytic activity. Surprisingly, chiMaD1 deletion enhanced M. anisopliae virulence to the cattle tick (Rhipicephalus microplus) larvae and engorged females, while did not alter the virulence to the mealworm larvae (Tenebrio molitor). These results add up to recent reports of deleted genes that enhanced entomopathogenic virulence, showing the complexity of host-pathogen interactions.
Assuntos
Quitinases/genética , Proteínas Fúngicas/genética , Metarhizium/patogenicidade , Rhipicephalus/microbiologia , Animais , Quitina/metabolismo , Quitinases/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Transferência Genética Horizontal , Interações Hospedeiro-Patógeno , Larva/microbiologia , Metarhizium/classificação , Metarhizium/enzimologia , Metarhizium/genética , Controle Biológico de Vetores , Filogenia , Tenebrio/microbiologia , VirulênciaRESUMO
The cowpea weevil (Callosobruchus maculatus) is the main pest that attacks cowpea (Vigna unguiculata) seeds during storage, causing nutritional and economic losses in the cowpea crop. Thus, studies aiming to identify resistant cowpea cultivars have been developed. Chitin-binding proteins (CBP), such vicilins and chitinases, have been detected in seeds and related with the toxicity to insects. In this work, we investigated the presence of chitin-binding proteins in the partially resistant cowpea cv. BRS Xiquexique and evaluated their toxicity towards cowpea weevil. The CBP fraction was isolated by chitin affinity chromatography. CBP fraction showed, through 15% SDS PAGE, protein bands with varying molecular masses, mainly below 55â¯kDa. Proteins present in CBP fraction were identified by Western blotting and mass spectrometry analysis, as vicilins and chitinases. CBP fraction, at 5%, was able to interfere with the development of cowpea weevil, decreasing larval mass and length. A CBV (chitin-binding vicilin) fraction isolated from CBP fraction was toxic, at 2.0%, to C. maculatus, decreasing larval mass and length in 64.3% and 33.23%, respectively. These results suggest that chitin binding proteins, such vicilins and chitinases, may be related to the resistance of cowpea cv. BRS Xiquexique to the infestation by C. maculatus.
Assuntos
Besouros , Vigna , Gorgulhos , Animais , Proteínas de Transporte , Quitina/metabolismo , Besouros/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Vigna/metabolismo , Gorgulhos/metabolismoRESUMO
BACKGROUND: The thermodimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis. Although poorly studied, paracoccin (PCN) from Paracoccidioides brasiliensis has been shown to harbor lectinic, enzymatic, and immunomodulatory properties that affect disease development. METHODS: Mutants of P. brasiliensis overexpressing PCN (ov-PCN) were constructed by Agrobacterium tumefaciens-mediated transformation. ov-PCN strains were analyzed and inoculated intranasally or intravenously to mice. Fungal burden, lung pathology, and survival were monitored to evaluate virulence. Electron microscopy was used to evaluate the size of chito-oligomer particles released by ov-PCN or wild-type strains to growth media. RESULTS: ov-PCN strains revealed no differences in cell growth and viability, although PCN overexpression favored cell separation, chitin processing that results in the release of smaller chito-oligomer particles, and enhanced virulence. Our data show that PCN triggers a critical effect in the cell wall biogenesis through the chitinase activity resulting from overexpression of PCN. As such, PCN overexpression aggravates the disease caused by P. brasiliensis. CONCLUSIONS: Our data are consistent with a model in which PCN modulates the cell wall architecture via its chitinase activity. These findings highlight the potential for exploiting PCN function in future therapeutic approaches.
Assuntos
Parede Celular/metabolismo , Quitina/metabolismo , Proteínas Fúngicas/fisiologia , Lectinas/fisiologia , Paracoccidioides/patogenicidade , Animais , Citocinas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Paracoccidioidomicose/imunologia , Fagocitose , VirulênciaRESUMO
Natural transformation is a broadly conserved mechanism of horizontal gene transfer (HGT) in bacteria that can shape their evolution through the acquisition of genes that promote virulence, antibiotic resistance, and other traits. Recent work has established that neighbor predation via type VI secretion systems, bacteriocins, and virulent phages plays an important role in promoting HGT. Here, we demonstrate that in chitin estuary microcosms, Vibrio cholerae K139 lysogens exhibit prophage-dependent neighbor predation of nonlysogens to enhance HGT. Through predation of nonlysogens, K139 lysogens also have a fitness advantage under these microcosm conditions. The ecological strategy revealed by our work provides a better understanding of the evolutionary mechanisms used by bacteria to adapt in their natural setting and contributes to our understanding of the selective pressures that may drive prophage maintenance in bacterial genomes.IMPORTANCE Prophages are nearly ubiquitous in bacterial species. These integrated phage elements have previously been implicated in horizontal gene transfer (HGT) largely through their ability to carry out transduction (generalized or specialized). Here, we show that prophage-encoded viral particles promote neighbor predation leading to enhanced HGT by natural transformation in the waterborne pathogen Vibrio cholerae Our findings contribute to a comprehensive understanding of the dynamic forces involved in prophage maintenance which ultimately drive the evolution of naturally competent bacteria in their natural environment.
Assuntos
Prófagos/genética , Vibrio cholerae/genética , Vibrio cholerae/virologia , Animais , Quitina/metabolismo , Transferência Genética Horizontal , Comportamento Predatório , Prófagos/crescimento & desenvolvimento , Vibrio cholerae/patogenicidade , VirulênciaRESUMO
Epigenetic mechanisms such as histone acetylation and deacetylation participate in regulation of the genes involved in encystation of Entamoeba invadens. However, the histones and target residues involved, and whether the acetylation and deacetylation of the histones leads to the regulation of gene expression associated with the encystation of this parasite, remain unknown. In this study, we found that E. invadens histone H4 is acetylated in both stages of the parasite and is more highly acetylated during the trophozoite stage than in the cyst. Histone hyperacetylation induced by Trichostatin A negatively affects the encystation of E. invadens, and this inhibition is associated with the downregulation of the expression of genes implicated in the synthesis of chitin, polyamines, gamma-aminobutyric acid pathways and cyst wall proteins, all of which are important in the formation of cysts. Finally, in silico analysis and activity assays suggest that a class I histone deacetylase (EiHDAC3) could be involved in control of the expression of a subset of genes that are important in several pathways during encystation. Therefore, the identification of enzymes that acetylate and/or deacetylate histones that control encystation in E. invadens could be a promising therapeutic target for preventing transmission of other amoebic parasites such as E. histolytica, the causative agent of amoebiasis in humans.
Assuntos
Entamoeba , Histona Desacetilases/metabolismo , Animais , Quitina/metabolismo , Entamoeba/enzimologia , Humanos , Processamento de Proteína Pós-Traducional , Trofozoítos/enzimologiaRESUMO
The chitinases have extensive biotechnological potential but have been little exploited commercially due to the low number of good chitinolytic microorganisms. The purpose of this study was to identify a chitinolytic fungal and optimize its production using solid state fermentation (SSF) and agroindustry substrate, to evaluate different chitin sources for chitinase production, to evaluate different solvents for the extraction of enzymes produced during fermentation process, and to determine the nematicide effect of enzymatic extract and biological control of Meloidogyne javanica and Meloidogyne incognita nematodes. The fungus was previously isolated from bedbugs of Tibraca limbativentris Stal (Hemiptera: Pentatomidae) and selected among 51 isolated fungal as the largest producer of chitinolytic enzymes in SSF. The isolate UFSMQ40 has been identified as Trichoderma koningiopsis by the amplification of tef1 gene fragments. The greatest chitinase production (10.76 U gds-1) occurred with wheat bran substrate at 55% moisture, 15% colloidal chitin, 100% of corn steep liquor, and two discs of inoculum at 30 °C for 72 h. Considering the enzymatic inducers, the best chitinase production by the isolated fungus was achieved using chitin in colloidal, powder, and flakes. The usage of 1:15 g/mL of sodium citrate-phosphate buffer was the best ratio for chitinase extraction of SSF. The Trichoderma koningiopsis UFSMQ40 showed high mortality of M. javanica and M. incognita when applied to treatments with enzymatic filtrated and the suspension of conidia.
Assuntos
Quitina/metabolismo , Quitinases/biossíntese , Fermentação , Hypocreales/enzimologia , Animais , Percevejos-de-Cama/microbiologia , Agentes de Controle Biológico , Biotecnologia , Fibras na Dieta , Nematoides/efeitos dos fármacos , Esporos Fúngicos/metabolismo , Temperatura , Zea maysRESUMO
Poor solubility is the main drawback of the direct industrial exploitation of chitin, the second most abundant biopolymer after cellulose. Chemical methods are conventional to solubilize chitin from natural sources. Enzymatic hydrolysis of soluble chitinous substrates is a promising approach to obtain value-added by-products, such as N-acetylglucosamine units or low molecular weight chito-oligomers. Protein display on the bacterial membrane remains attractive to produce active enzymes anchored to a biological surface. The Lpp-OmpA system, a gene fusion of the Lpp signal sequence with the OmpA transmembrane region, represents the traditional system for targeting enzymes to the E. coli surface. EhCHT1, the amoebic chitinase, exhibits an efficient endochitinolytic activity and significant biochemical features, such as stability over a wide range of pH values. Using an extended Lpp-OmpA system as a protein carrier, we engineered E. coli to express the catalytic domain of EhCHT1 on the surface and assess the endochitinase activity as a trait. Engineered bacteria showed a consistent hydrolytic rate over a typical substrate, suggesting that the displayed enzyme has operational stability. This study supports the potential of biomembrane-associated biocatalysts as a reliable technology for the hydrolysis of soluble chitinous substrates.
Assuntos
Amoeba/enzimologia , Domínio Catalítico , Quitinases/genética , Quitinases/metabolismo , Escherichia coli/genética , Engenharia Genética , Quitina/metabolismo , Quitinases/química , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrólise , SolubilidadeRESUMO
Vicilins are related to cowpea seed resistance toward Callosobruchus maculatus due to their ability to bind to chitinous structures lining larval midgut. However, this binding mechanism is not fully understood. Here, we identified chitin binding sites and investigated how in vitro and in silico chemical modifications interfere with vicilin chitin binding and insect toxicity. In vitro assays showed that unmodified vicilin strongly binds to chitin matrices, mainly with acetylated chitin. Chemical modifications of specific amino acids (tryptophan, lysine, tyrosine), as well as glutaraldehyde cross-linking, decreased the evaluated parameters. In silico analyses identified at least one chitin binding site in vicilin monomer, the region between Arg208 and Lys216, which bears the sequence REGIRELMK and forms an α helix, exposed in the 3D structure. In silico modifications of Lys223 (acetylated at its terminal nitrogen) and Trp316 (iodinated to 7-iodine-L-tryptophan or oxidized to ß-oxy-indolylalanine) decreased vicilin chitin binding affinity. Glucose, sucrose, and N-acetylglucosamine also interfered with vicilin chitin binding affinity.