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1.
PLoS One ; 17(6): e0263905, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35648754

RESUMO

Monascus is a filamentous fungus that is widely used for producing Monascus pigments in the food industry in Southeast Asia. While the development of bioinformatics has helped elucidate the molecular mechanism underlying metabolic engineering of secondary metabolite biosynthesis, the biological information on the metabolic engineering of the morphology of Monascus remains unclear. In this study, the whole genome of M. purpureus CSU-M183 strain was sequenced using combined single-molecule real-time DNA sequencing and next-generation sequencing platforms. The length of the genome assembly was 23.75 Mb in size with a GC content of 49.13%, 69 genomic contigs and encoded 7305 putative predicted genes. In addition, we identified the secondary metabolite biosynthetic gene clusters and the chitin synthesis pathway in the genome of the high pigment-producing M. purpureus CSU-M183 strain. Furthermore, it is shown that the expression levels of most Monascus pigment and citrinin clusters located genes were significantly enhanced via atmospheric room temperature plasma mutagenesis. The results provide a basis for understanding the secondary metabolite biosynthesis, and constructing the metabolic engineering of the morphology of Monascus.


Assuntos
Citrinina , Monascus , Quitina/metabolismo , Citrinina/metabolismo , Monascus/genética , Monascus/metabolismo , Família Multigênica , Sequenciamento Completo do Genoma
2.
Plant Sci ; 321: 111310, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35696910

RESUMO

A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414-423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al., Biosci. Biotechnol. Biochem., 79, 1296-1304, 2015), using an Escherichia coli expression system and characterized for its structure and mechanism of action. The crystal structure of EaLysM exhibited an almost identical fold (ßααß) to that of PrLysM2. From isothermal titration calorimetry and nuclear magnetic resonance, the binding mode and affinities of EaLysM for chitooligosaccharides (GlcNAc)n (3, 4, 5, and 6) were found to be comparable to those of PrLysM2. The LysM module in EaChiA is likely to bind (GlcNAc)n almost independently through CH-π stacking of a Tyr residue with the pyranose ring. The (GlcNAc)n-binding mode of LysMs in the LysM-chitinase conjugates from fern plants appears to differ from that of plant LysMs acting in chitin- or Nod-signal perception, in which multiple LysMs cooperatively act on (GlcNAc)n. Phylogenetic analysis suggested that LysM-GH18 conjugates of fern plants formed a monophyletic group and had been separated earlier than forming the clade of fungal chitinases with LysMs.


Assuntos
Quitinases , Gleiquênias , Quitina/química , Quitina/metabolismo , Quitinases/genética , Quitinases/metabolismo , Gleiquênias/genética , Gleiquênias/metabolismo , Filogenia
3.
Pestic Biochem Physiol ; 184: 105129, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35715067

RESUMO

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/química
4.
Proc Natl Acad Sci U S A ; 119(24): e2120853119, 2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35675426

RESUMO

Muscle attachment sites (MASs, apodemes) in insects and other arthropods involve specialized epithelial cells, called tendon cells or tenocytes, that adhere to apical extracellular matrices containing chitin. Here, we have uncovered a function for chitin deacetylases (CDAs) in arthropod locomotion and muscle attachment using a double-stranded RNA-mediated gene-silencing approach targeted toward specific CDA isoforms in the red flour beetle, Tribolium castaneum (Tc). Depletion of TcCDA1 or the alternatively spliced TcCDA2 isoform, TcCDA2a, resulted in internal tendon cuticle breakage at the femur-tibia joint, muscle detachment from both internal and external tendon cells, and defective locomotion. TcCDA deficiency did not affect early muscle development and myofiber growth toward the cuticular MASs but instead resulted in aborted microtubule development, loss of hemiadherens junctions, and abnormal morphology of tendon cells, all features consistent with a loss of tension within and between cells. Moreover, simultaneous depletion of TcCDA1 or TcCDA2a and the zona pellucida domain protein, TcDumpy, prevented the internal tendon cuticle break, further supporting a role for force-dependent interactions between muscle and tendon cells. We propose that in T. castaneum, the absence of N-acetylglucosamine deacetylation within chitin leads to a loss of microtubule organization and reduced membrane contacts at MASs in the femur, which adversely affect musculoskeletal connectivity, force transmission, and physical mobility.


Assuntos
Amidoidrolases , Proteínas de Insetos , Músculos , Tribolium , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Quitina/metabolismo , Extremidades/fisiologia , Fêmur , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Locomoção , Desenvolvimento Muscular , Músculos/enzimologia , Músculos/fisiologia , Tribolium/enzimologia , Tribolium/fisiologia
5.
Insect Biochem Mol Biol ; 145: 103783, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35525402

RESUMO

The biogenesis of chitin, a major structural polysaccharide found in the cuticle and peritrophic matrix, is crucial for insect growth and development. Chitin synthase, a membrane-integral ß-glycosyltransferase, has been identified as the core of the chitin biogenesis machinery. However, a yet unknown number of auxiliary proteins appear to assist in chitin biosynthesis, whose precise function remains elusive. Here, we identified a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), in the fruit fly Drosophila melanogaster, as a chitin biogenesis-associated protein. The physical interaction between DmSERCA and epidermal chitin synthase (Krotzkopf verkehrt, Kkv) was demonstrated and analyzed using split-ubiquitin membrane yeast two-hybrid, bimolecular fluorescent complementation, pull-down, and immunoprecipitation assays. The interaction involves N-terminal regions (aa 48-81 and aa 247-33) and C-terminal regions (aa 743-783 and aa 824-859) of DmSERCA and two N-terminal regions (aa 121-179 and aa 369-539) of Kkv, all of which are predicted be transmembrane helices. While tissue-specific knock-down of DmSERCA in the epidermis caused larval and pupal lethality, the knock-down of DmSERCA in wings resulted in smaller and crinkled wings, a significant decrease in chitin deposition, and the loss of chitin lamellar structure. Although DmSERCA is well-known for its role in muscular contraction, this study reveals a novel role in chitin synthesis, contributing to our knowledge on the machinery of chitin biogenesis.


Assuntos
Quitina Sintase , Drosophila , Sequência de Aminoácidos , Animais , Quitina/metabolismo , Quitina Sintase/genética , Quitina Sintase/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/metabolismo
6.
Nat Commun ; 13(1): 2397, 2022 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-35577789

RESUMO

The U-box type ubiquitin ligase PUB44 positively regulates pattern-triggered immunity in rice. Here, we identify PBI1, a protein that interacts with PUB44. Crystal structure analysis indicates that PBI1 forms a four-helix bundle structure. PBI1 also interacts with WRKY45, a master transcriptional activator of rice immunity, and negatively regulates its activity. PBI1 is degraded upon perception of chitin, and this is suppressed by silencing of PUB44 or expression of XopP, indicating that PBI1 degradation depends on PUB44. These data suggest that PBI1 suppresses WRKY45 activity when cells are in an unelicited state, and during chitin signaling, PUB44-mediated degradation of PBI1 leads to activation of WRKY45. In addition, chitin-induced MAP kinase activation is required for WRKY45 activation and PBI1 degradation. These results demonstrate that chitin-induced activation of WRKY45 is regulated by the cooperation between MAP kinase-mediated phosphorylation and PUB44-mediated PBI1 degradation.


Assuntos
Oryza , Quitina/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Oryza/metabolismo , Doenças das Plantas , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Protein Sci ; 31(6): e4327, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35634776

RESUMO

N-acetylglucosamine containing compounds acting as pathogenic or symbiotic signals are perceived by plant-specific Lysin Motif Receptor-Like Kinases (LysM-RLKs). The molecular mechanisms of this perception are not fully understood, notably those of lipo-chitooligosaccharides (LCOs) produced during root endosymbioses with nitrogen-fixing bacteria or arbuscular mycorrhizal fungi. In Medicago truncatula, we previously identified the LysM-RLK LYR3 (MtLYR3) as a specific LCO-binding protein. We also showed that the absence of LCO binding to LYR3 of the non-mycorrhizal Lupinus angustifolius, (LanLYR3), was related to LysM3, which differs from that of MtLYR3 by several amino acids and, particularly, by a critical tyrosine residue absent in LanLYR3. Here, we aimed to define the LCO binding site of MtLYR3 by using molecular modelling and simulation approaches, combined with site-directed mutagenesis and LCO binding experiments. 3D models of MtLYR3 and LanLYR3 ectodomains were built, and homology modelling and molecular dynamics (MD) simulations were performed. Molecular docking and MD simulation on the LysM3 identified potential key residues for LCO binding. We highlighted by steered MD simulations that in addition to the critical tyrosine, two other residues were important for LCO binding in MtLYR3. Substitution of these residues in LanLYR3-LysM3 by those of MtLYR3-LysM3 allowed the recovery of high-affinity LCO binding in experimental radioligand-binding assays. An analysis of selective constraints revealed that the critical tyrosine has experienced positive selection pressure and is absent in some LYR3 proteins. These findings now pave the way to uncover the functional significance of this specific evolutionary pattern.


Assuntos
Quitina , Medicago truncatula , Quitina/metabolismo , Quitosana , Medicago truncatula/genética , Simulação de Acoplamento Molecular , Oligossacarídeos , Tirosina/metabolismo
8.
Commun Biol ; 5(1): 518, 2022 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-35641660

RESUMO

Microbial lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of crystalline polysaccharides including chitin and cellulose. The discovery of a large assortment of LPMO-like proteins widely distributed in insect genomes suggests that they could be involved in assisting chitin degradation in the exoskeleton, tracheae and peritrophic matrix during development. However, the physiological functions of insect LPMO-like proteins are still undetermined. To investigate the functions of insect LPMO15 subgroup I-like proteins (LPMO15-1s), two evolutionarily distant species, Tribolium castaneum and Locusta migratoria, were chosen. Depletion by RNAi of T. castaneum TcLPMO15-1 caused molting arrest at all developmental stages, whereas depletion of the L. migratoria LmLPMO15-1, prevented only adult eclosion. In both species, LPMO15-1-deficient animals were unable to shed their exuviae and died. TEM analysis revealed failure of turnover of the chitinous cuticle, which is critical for completion of molting. Purified recombinant LPMO15-1-like protein from Ostrinia furnacalis (rOfLPMO15-1) exhibited oxidative cleavage activity and substrate preference for chitin. These results reveal the physiological importance of catalytically active LPMO15-1-like proteins from distant insect species and provide new insight into the enzymatic mechanism of cuticular chitin turnover during molting.


Assuntos
Quitina , Oxigenases de Função Mista , Animais , Quitina/metabolismo , Carboidratos da Dieta , Insetos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Muda , Polissacarídeos/metabolismo
9.
Fish Shellfish Immunol ; 126: 271-282, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35609762

RESUMO

Peritrophins are peritrophic membrane (PM) proteins that can interact with chitin fibers via chitin-binding domains. Peritrophins have essential roles in providing porosity and strength to the PM that lines the shrimp midgut. Acute hepatopancreatic necrosis disease (AHPND), caused by strains of V. parahaemolyticus, is known to initially colonize the shrimp stomach and simultaneously disrupt its structural barriers (e.g., cuticle or epithelial tissues) to reach the hepatopancreas. Although stomach and hepatopancreas were identified as target tissues involved in AHPND pathogenesis, our results indicated that peritrophin in peritrophic membrane has a crucial role in determining not only colonization of AHPND-causing bacteria but also their tissue distribution. As the interaction between LvPeritrophin (LvPT) and WSSV (white spot syndrome virus) is not well understood, we noted that LvPT expression was upregulated in shrimp stomach challenged with either WSSV or AHPND. In an in vitro pathogen binding assay, there was strong binding of recombinant LvPT WSSV and AHPND-causing V. parahaemolyticus, and various bacteria. Furthermore, dsRNA-mediated LvPT silencing inhibited WSSV gene expression and viral genome replication. However, downregulation of LvPT gene expression increased copies of AHPND-causing bacteria in shrimp digestive tract, and facilitated bacterial colonization in stomach. In conclusion, we speculated that LvPT might regulate bacterial colonization during AHPND, whereas in WSSV infection, LvPT silencing favored the host. Although recombinant LvPT had strong binding with WSSV, the precise role of LvPT in WSSV infection needs further investigation. These findings increased our understanding of host-pathogen interactions in AHPND and WSSV infection that can be applied in shrimp aquaculture for developing effective antibacterial and antiviral strategies.


Assuntos
Penaeidae , Vibrio parahaemolyticus , Vírus da Síndrome da Mancha Branca 1 , Animais , Quitina/metabolismo , Hepatopâncreas/metabolismo , Interações Hospedeiro-Patógeno , Penaeidae/microbiologia , Vibrio parahaemolyticus/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologia
10.
Appl Environ Microbiol ; 88(11): e0064522, 2022 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-35604230

RESUMO

Phytopathogens represent a large agricultural challenge. The use of chemical pesticides is harmful to the environment, animals, and humans. Therefore, new sustainable and biological alternatives are urgently needed. The insect-pathogenic bacterium Photorhabdus luminescens, already used in combination with entomopathogenic nematodes (EPNs) as a biocontrol agent, is characterized by two different phenotypic cell forms, called primary (1°) and secondary (2°). The 1° cells are symbiotic with EPNs and are used for biocontrol, and the 2° cells are unable to undergo symbiosis with EPNs, remain in the soil after insect infection, and specifically interact with plant roots. A previous RNA sequencing (RNAseq) analysis showed that genes encoding the exochitinase Chi2A and chitin binding protein (CBP) are highly upregulated in 2° cells exposed to plant root exudates. Here, we investigate Chi2A and CBP functions and demonstrate that both are necessary for P. luminescens 2° cells to inhibit the growth of the phytopathogenic fungus Fusarium graminearum. We provide evidence that Chi2A digests chitin and thereby inhibits fungal growth. Furthermore, we show that 2° cells specifically colonize fungal hyphae as one of the first mechanisms to protect plants from fungal phytopathogens. Finally, soil pot bioassays proved plant protection from F. graminearum by 2° cells, where Chi2A and CPB were essential for this process. This work gives molecular insights into the new applicability of P. luminescens as a plant-growth-promoting and plant-protecting organism in agriculture. IMPORTANCE The enteric enterobacterium Photorhabdus luminescens is already being used as a bioinsecticide since it is highly pathogenic toward a broad range of insects. However, the bacteria exist in two phenotypically different cell types, called 1° and 2° cells. Whereas only 1° cells are symbiotic with their nematode partner to infect insects, 2° cells were shown to remain in the soil after an insect infection cycle. It was demonstrated that 2° cells specifically interact with plant roots. Here, we show that the bacteria are beneficial for the plants by protecting them from phytopathogenic fungi. Specific colonization of the fungus mycelium as well as chitin-degrading activity mediated by the chitin binding protein (CBP) and the chitinase Chi2A are essential for this process. Our data give evidence for the novel future applicability of P. luminescens as a plant-growth-promoting organism and biopesticide.


Assuntos
Nematoides , Photorhabdus , Animais , Quitina/metabolismo , Fusarium , Insetos/microbiologia , Photorhabdus/genética , Solo , Simbiose
11.
Molecules ; 27(10)2022 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-35630540

RESUMO

Salinity is one of the major abiotic stresses limiting crop growth and productivity worldwide. Salt stress during germination degenerates crop establishment and declines yield in wheat, therefore alleviating the damage of salt stress to wheat seedlings is crucial. Chitooligosaccharide (COS) was grafted with γ-aminobutyric acid based on the idea of bioactive molecular splicing, and the differences in salt resistance before and after grafting were compared. The expected derivative was successfully synthesized and exhibited better salt resistance-inducing activity than the raw materials. By activating antioxidant enzymes such as superoxide dismutases (SOD), catalase (CAT) and phenylalanine ammonia-lyase (PAL) and subsequently eliminating reactive oxygen species (ROS) in a timely manner, the rate of O-2 production and H2O2 content of wheat seedlings were reduced, and the dynamic balance of free radical metabolism in the plant body was maintained. A significantly reduced MDA content, reduced relative permeability of the cell membrane, and decreased degree of damage to the cell membrane were observed. A significant increase in the content of soluble sugar, maintenance of osmotic regulation and the stability of the cell membrane structure, effective reduction in the salt stress-induced damage to wheat, and the induction of wheat seedling growth were also observed, thereby improving the salt tolerance of wheat seedlings.


Assuntos
Plântula , Triticum , Quitina/metabolismo , Quitosana , Peróxido de Hidrogênio/farmacologia , Oligossacarídeos , Triticum/metabolismo , Ácido gama-Aminobutírico/metabolismo
12.
Sci Rep ; 12(1): 8830, 2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35614185

RESUMO

Chitin-glucan (CG), an insoluble dietary fiber, has been shown to improve cardiometabolic disorders associated with obesity in mice. Its effects in healthy subjects has recently been studied, revealing its interaction with the gut microbiota. In this double-blind, randomized, cross-over, twice 3-week exploratory study, we investigated the impacts of CG on the cardiometabolic profile and gut microbiota composition and functions in 15 subjects at cardiometabolic risk. They consumed as a supplement 4.5 g of CG daily or maltodextrin as control. Before and after interventions, fasting and postprandial metabolic parameters and exhaled gases (hydrogen [H2] and methane [CH4]) were evaluated. Gut microbiota composition (16S rRNA gene sequencing analysis), fecal concentrations of bile acids, long- and short-chain fatty acids (LCFA, SCFA), zonulin, calprotectin and lipopolysaccharide binding protein (LBP) were analyzed. Compared to control, CG supplementation increased exhaled H2 following an enriched-fiber breakfast ingestion and decreased postprandial glycemia and triglyceridemia response to a standardized test meal challenge served at lunch. Of note, the decrease in postprandial glycemia was only observed in subjects with higher exhaled H2, assessed upon lactulose breath test performed at inclusion. CG decreased a family belonging to Actinobacteria phylum and increased 3 bacterial taxa: Erysipelotrichaceae UCG.003, Ruminococcaceae UCG.005 and Eubacterium ventriosum group. Fecal metabolites, inflammatory and intestinal permeability markers did not differ between groups. In conclusion, we showed that CG supplementation modified the gut microbiota composition and improved postprandial glycemic response, an early determinant of cardiometabolic risk. Our results also suggest breath H2 production as a non-invasive parameter of interest for predicting the effectiveness of dietary fiber intervention.


Assuntos
Doenças Cardiovasculares , Microbioma Gastrointestinal , Animais , Bactérias , Glicemia/análise , Quitina/metabolismo , Fibras na Dieta/análise , Suplementos Nutricionais , Fezes/microbiologia , Glucanos/metabolismo , Humanos , Camundongos , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética
13.
Mol Biol Rep ; 49(5): 4141-4148, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35474055

RESUMO

BACKGROUND: Bacillus velezensis possesses numerous chitinolytic enzymes; however, not much is known about the role of its chitinase molecules. METHODS AND RESULTS: In this study, the chiA gene, which encodes a novel domain structure possessing family 18 chitinase from B. velezensis RB.IBE29, was expressed successfully in Escherichia coli BL21-CodonPlus (DE3)-RIPL using the pColdII expression vector. The recombinant protein, rBvChiA, was purified using the HisTrap FF column. Purified rBvChiA showed hydrolytic activity against insoluble chitin and bound to chitinous substrates. In addition, the purified recombinant enzyme displayed remarkable inhibition effects on the spore germination of Fusarium falciforme and the egg hatch of root-knot nematodes (Meloidogyne spp.), which are the main causes of black pepper diseases in the Central Highlands region, Vietnam. CONCLUSION: The current work results might enable further studies to develop novel chitinase A and strain RB.IBE29 as a natural fungicide and nematicide for sustainable black pepper production and other crops in the Central Highlands, Vietnam. This is the second report describing chitinase from B. velezensis based on the experimental data.


Assuntos
Bacillus , Quitinases , Sequência de Aminoácidos , Bacillus/genética , Quitina/metabolismo , Quitinases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo
14.
PLoS One ; 17(4): e0265969, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35363796

RESUMO

The tissue of insects, pests, and fungi has a chitin layer followed by protein in the cell membrane. The complete biodegradation of chitin and protein-present in the waste requires the action of two enzymes, namely chitinase, and protease. Combining chitinase and protease in a single protein/enzyme will serve as a bifunctional enzyme that can efficiently degrade the chitin and protein-rich biomass. The present study was aimed to fuse these two enzymes to produce a single protein and study the kinetics of the recombinant fusion protein. A chitinase and alkaline protease genes were isolated, cloned, and expressed successfully as a fusion product in heterologous host Escherichia coli. The two native genes were successfully fused in E.coli by using flexible glycine-serine (G4S)2 linker (GGGGS, GS linker). The recombinant fusion protein in E.coli showed hydrolyzed chitin and protein on chitin and bovine serum albumin agar plates confirming the successful cloning and expression of chitinase and protease enzymes in a single fusion protein. The common pUC18-T7 mini vector with the ompA signal sequence helps the extracellular expression of fusion protein efficiently. The native gel electrophoresis revealed a molecular mass of purified protein as 92.0 kDa. The fusion protein's maximal chitinase and protease activity occurred at pH 5.0 and 8.0 and 30 0C, respectively resembling the individual enzymes'. In the kinetic studies of the fusion protein, it was observed that the presence of metal ions such as Cu2+, Na2+, and Ca2+; significantly enhanced the enzyme activities while organic solvents oxidants and chemicals have drastically affected the activities of both the enzymes in the fusion protein. No such fusion protein has been produced in a heterologous host yet. The reports on fusion protein with biomass-degrading capacity are also scarce. This is probably the first report of a bifunctional chitinase/protease expressed in E. coli.


Assuntos
Quitinases , Escherichia coli , Quitina/metabolismo , Quitinases/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo
15.
Protein Sci ; 31(5): e4289, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35481637

RESUMO

The Plasmodium ookinete uses chitinase activity to penetrate the acellular, chitin-containing peritrophic matrix to invade the mosquito vector. Plasmodium ookinetes from different parasite clades secrete two structurally distinct forms of chitinase, one, a short form lacking a C-terminal putative chitin-binding domain (CBD), the other, a long form with both proenzyme and C-terminal putative chitin-binding domains. Here, we structurally and functionally characterize the three cysteines in the short chitinase of the human-infecting malaria parasite, P. falciparum testing the hypothesis that one unpaired cysteine would not contribute to chitinase-specific enzymatic activity which would identify this residue as potentially involved in intermolecular disulfide bonding and heteromultimeric invasion complex formation as previously described. To test this hypothesis, we produced and characterized recombinant wild-type and cysteine-mutation PfCHT1 proteins in E. coli and used biophysical and enzymatic approaches to examine their enzymatic activities and chitin-binding affinities. The cysteine-203 PfCHT1 mutation had no effect on chitinolytic and chitin-binding functions. The cysteine-220 and cysteine-230 mutants were enzymatically inactive and did not bind to chitin. The artificial intelligence-based protein prediction algorithm, AlphaFold, correctly identified the involvement of cys-220 and cys-230 in the intramolecular disulfide linkages key to maintaining properly folded chitinase structural integrity. AlphaFold predicted that cys-203 cysteine is surface exposed and thus involved in intermolecular protein-protein interaction. Production of the cys-to-ser 203 PfCHT1 mutant facilitated recombinant protein production. Future cellular and biochemical studies are needed to further understand details of Plasmodium ookinete mosquito midgut invasion.


Assuntos
Quitinases , Plasmodium falciparum , Animais , Inteligência Artificial , Quitina/metabolismo , Quitinases/química , Cisteína/genética , Dissulfetos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Mosquitos Vetores , Plasmodium falciparum/genética , Proteínas de Protozoários
16.
Carbohydr Polym ; 287: 119325, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35422291

RESUMO

Intermediate-state bacteria produced in the process of bacteriostasis have always been a potential threat to public health, but they are very easy to be overlooked. As a natural and non-toxic biological antibacterial agent, chitooligosaccharide (COS) has attracted the public's attention. However, little is known about the microbial stress response during the antibacterial process of COS. In this study, the antibacterial mechanisms of COS were expounded, and the formation of sublethal and viable but nonculturable (VBNC) state were further investigated. The COS was shown to bind to the cell envelopes, and the permeability and integrity of bacterial cell membrane were damaged severely, accompanied by the increase of intracellular reactive oxygen species and decrease of adenosine triphosphate content. Flow cytometry analysis indicated that COS finally inactivated Escherichia coli through the sublethal injury process. While for Staphylococcus aureus, some cells were induced into VBNC state by COS, causing incomplete inactivation.


Assuntos
Quitosana , Infecções por Escherichia coli , Infecções Estafilocócicas , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias , Quitina/metabolismo , Quitina/farmacologia , Quitosana/metabolismo , Quitosana/farmacologia , Escherichia coli , Humanos , Viabilidade Microbiana , Oligossacarídeos , Staphylococcus aureus
17.
Int J Mol Sci ; 23(7)2022 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-35409210

RESUMO

The insect glycoside hydrolase family 20 ß-N-acetylhexosaminidases (HEXs) are key enzymes involved in chitin degradation. In this study, nine HEX genes in Bombyx mori were identified by genome-wide analysis. Bioinformatic analysis based on the transcriptome database indicated that each gene had a distinct expression pattern. qRT-PCR was performed to detect the expression pattern of the chitooligosaccharidolytic ß-N-acetylglucosaminidase (BmChiNAG). BmChiNAG was highly expressed in chitin-rich tissues, such as the epidermis. In the wing disc and epidermis, BmChiNAG has the highest expression level during the wandering stage. CRISPR/Cas9-mediated BmChiNAG deletion was used to study the function. In the BmChiNAG-knockout line, 39.2% of female heterozygotes had small and curly wings. The ultrastructure of a cross-section showed that the lack of BmChiNAG affected the stratification of the wing membrane and the formation of the correct wing vein structure. The molting process of the homozygotes was severely hindered during the larva to pupa transition. Epidermal sections showed that the endocuticle of the pupa was not degraded in the mutant. These results indicate that BmChiNAG is involved in chitin catabolism and plays an important role in the molting and wing development of the silkworm, which highlights the potential of BmChiNAG as a pest control target.


Assuntos
Bombyx , Animais , Bombyx/metabolismo , Quitina/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Larva/genética , Larva/metabolismo , Muda/genética , Pupa
18.
Pestic Biochem Physiol ; 183: 105083, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35430073

RESUMO

Metformin, considered to be a potent AMPK activator, is widely used for clinical therapy of cancer and diabetes due to its distinct function in regulating cell energy balance and body metabolism. However, the effect of metformin-induced AMPK activation on the growth and development of insects remains largely unknown. In the present study, we focused on the role of metformin in regulating the growth and development of Hyphantria cunea, a notorious defoliator in the forestry. Firstly, we obtained the complete coding sequences of HcAMPKα2, HcAMPKß1, HcAMPKγ2 from H. cunea, which encoded a protein of 512, 281, and 680 amino acids respectively. Furthermore, the phylogenetic analysis revealed that these three subunits were highly homologous with the AMPK subunits from other lepidopteran species. According to the bioassay, we found metformin remarkably restrained the growth and development of H. cunea larvae, and caused molting delayed and body weight reduced. In addition, expressions of HcAMPKα2, HcAMPKß1, and HcAMPKγ2 were upregulated 3.30-, 5.93- and 5.92-folds at 24 h after treatment, confirming that metformin activated AMPK signaling at the transcriptional level in H. cunea larvae. Conversely, the expressions of two vital Halloween genes (HcCYP306A1 and HcCYP314A1) in the 20E synthesis pathway were remarkably suppressed by metformin. Thus, we presumed that metformin delayed larval molting probably by impeding 20E synthesis in the H. cunea larvae. Finally, we found that metformin accelerated glycogen breakdown, elevated in vivo trehalose level, promoted chitin synthesis, and upregulated transcriptions of the genes in chitin synthesis pathway. Taken together, the findings provide a new insight into the molecular mechanisms by which AMPK regulates carbohydrate metabolism and chitin synthesis in insects.


Assuntos
Metformina , Mariposas , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Quitina/metabolismo , Larva/metabolismo , Metformina/metabolismo , Metformina/farmacologia , Muda , Mariposas/genética , Filogenia
19.
Plant Physiol ; 189(2): 839-857, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35312013

RESUMO

Plant sphingolipids mostly possess 2-hydroxy fatty acids (HFA), the synthesis of which is catalyzed by FA 2-hydroxylases (FAHs). In Arabidopsis (Arabidopsis thaliana), two FAHs (FAH1 and FAH2) have been identified. However, the functions of FAHs and sphingolipids with HFAs (2-hydroxy sphingolipids) are still unknown because of the lack of Arabidopsis lines with the complete deletion of FAH1. In this study, we generated a FAH1 mutant (fah1c) using CRISPR/Cas9-based genome editing. Sphingolipid analysis of fah1c, fah2, and fah1cfah2 mutants revealed that FAH1 hydroxylates very long-chain FAs (VLCFAs), whereas the substrates of FAH2 are VLCFAs and palmitic acid. However, 2-hydroxy sphingolipids are not completely lost in the fah1cfah2 double mutant, suggesting the existence of other enzymes catalyzing the hydroxylation of sphingolipid FAs. Plasma membrane (PM) analysis and molecular dynamics simulations revealed that hydroxyl groups of sphingolipid acyl chains play a crucial role in the organization of nanodomains, which are nanoscale liquid-ordered domains mainly formed by sphingolipids and sterols in the PM, through hydrogen bonds. In the PM of the fah1cfah2 mutant, the expression levels of 26.7% of the proteins, including defense-related proteins such as the pattern recognition receptors (PRRs) brassinosteroid insensitive 1-associated receptor kinase 1 and chitin elicitor receptor kinase 1, NADPH oxidase respiratory burst oxidase homolog D (RBOHD), and heterotrimeric G proteins, were lower than that in the wild-type. In addition, reactive oxygen species (ROS) burst was suppressed in the fah1cfah2 mutant after treatment with the pathogen-associated molecular patterns flg22 and chitin. These results indicated that 2-hydroxy sphingolipids are necessary for the organization of PM nanodomains and ROS burst through RBOHD and PRRs during pattern-triggered immunity.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Quitina/metabolismo , Ácidos Graxos/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória , Esfingolipídeos/metabolismo
20.
New Phytol ; 234(5): 1606-1613, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35297054

RESUMO

CERK1 (Chitin Elicitor Receptor Kinase 1), a lysin motif-containing pattern recognition receptor (PRR), perceives chitooligosaccharides (COs) to mount immune and symbiotic responses. However, CERK1, for a relatively long time, has been regarded as a co-receptor in plant immunity, mainly due to its lack of high binding affinity to known elicitors. Recent studies demonstrated several novel carbohydrates as ligands of CERK1 in different plant species and recognized CERK1 as a key receptor in plant immunity and symbiosis. This review summarizes recent knowledge acquired on the role of CERK1 in plant-microbe interactions.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Quitina/metabolismo , Imunidade Vegetal
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