RESUMO
GroEL is a chaperonin that helps other proteins fold correctly. However, alternative activities, such as acting as an insect toxin, have also been discovered. This work evaluates the chaperonin and insecticidal activity of different GroEL proteins from entomopathogenic nematodes on G. mellonella. The ability to synergize with the ExoA toxin of Pseudomonas aeruginosa was also investigated. The GroELXn protein showed the highest insecticidal activity among the different GroELs. In addition, it was able to significantly activate the phenoloxidase system of the target insects. This could tell us about the mechanism by which it exerts its toxicity on insects. GroEL proteins can enhance the toxic activity of the ExoA toxin, which could be related to its chaperonin activity. However, there is a significant difference in the synergistic effect that is more related to its alternative activity as an insecticidal toxin.
Assuntos
Inseticidas , Mariposas , Nematoides , Animais , Inseticidas/toxicidade , Inseticidas/metabolismo , Chaperonina 60/metabolismo , Chaperonina 60/farmacologia , Insetos/metabolismo , Bactérias/metabolismo , Larva/metabolismoRESUMO
Bacillus thuringiensis (Bt) produces different insecticidal proteins effective for pest control. Among them, Cry insecticidal proteins have been used in transgenic plants for the control of insect pests. However, evolution of resistance by insects endangers this technology. Previous work showed that the lepidopteran insect Plutella xylostella PxHsp90 chaperone enhanced the toxicity of Bt Cry1A protoxins by protecting them from degradation by the larval gut proteases and by enhancing binding of the protoxin to its receptors present in larval midgut cells. In this work, we show that PxHsp70 chaperone also protects Cry1Ab protoxin from gut proteases degradation, enhancing Cry1Ab toxicity. We also show that both PxHsp70 and PxHsp90 chaperones act cooperatively, increasing toxicity and the binding of Cry1Ab439D mutant, affected in binding to midgut receptors, to cadherin receptor. Also, insect chaperones recovered toxicity of Cry1Ac protein to a Cry1Ac-highly resistant P. xylostella population, NO-QAGE, that has a disruptive mutation in an ABCC2 transporter linked to Cry1Ac resistance. These data show that Bt hijacked an important cellular function for enhancing its infection capability, making use of insect cellular chaperones for enhancing Cry toxicity and for lowering the evolution of insect resistance to these toxins.
Assuntos
Bacillus thuringiensis , Inseticidas , Animais , Bacillus thuringiensis/genética , Insetos , Larva/genética , Chaperonas Moleculares , Proteínas de Choque Térmico HSP90/genética , Peptídeo Hidrolases , Proteínas de Choque Térmico HSP70/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidadeRESUMO
The increase in antimicrobial resistance has raised questions about how to use these drugs safely, especially in veterinary medicine, animal nutrition, and agriculture. Escherichia coli is an important human and animal pathogen that frequently contains plasmids carrying antibiotic resistance genes. Extra chromosomal elements are required for various functions or conditions in microorganisms. Several phage-like plasmids have been identified, which are important in antibiotic resistance. In this work, the molecular characterization of the pBOq-IncQ (4.5 kb) and pBOq-95LK (95 kb) plasmids found in the E. coli strain BOq 01, a multidrug resistant bacteria isolated from a poultry farm, are considered. Plasmid pBOq-IncQ belongs to the incQ incompatibility plasmid family and is involved in sulfonamide resistance. Plasmid pBOq-95LK is a lytic phage-like plasmid that is involved in the lysis of the E. coli BOq 01 strain and carries a bleomycin resistance gene and a strain cured of this plasmid shows bleomycin sensitivity. Induction of the lytic cycle indicates that this phage-like plasmid is an active phage. This type of plasmid has been reported to acquire genes such as mcr-1, which codes for colistin resistance and bacterial persistence and is a significant public health threat. A genome comparison, a pangenomic and phylogenomic analysis with other phage-like plasmids reported in the literature were performed to understand better the evolution of this kind of plasmid in bacteria and its potential importance in antibiotic resistance.
RESUMO
Bacteria of the genera Xenorhabdus and Photorhabdus are symbionts of entomopathogenic nematodes. Despite their close phylogenetic relationship, they show differences in their pathogenicity and virulence mechanisms in target insects. These differences were explored by the analysis of the pangenome, as it provides a framework for characterizing and defining the gene repertoire. We performed the first pangenome analysis of 91 strains of Xenorhabdus and Photorhabdus; the analysis showed that the Photorhabdus genus has a higher number of genes associated with pathogenicity. However, biological tests showed that whole cells of X. nematophila SC 0516 were more virulent than those of P. luminescens HIM3 when both were injected into G. mellonella larvae. In addition, we cloned and expressed the GroEL proteins of both bacteria, as this protein has been previously indicated to show insecticidal activity in the genus Xenorhabdus. Among these proteins, Cpn60-Xn was found to be the most toxic at all concentrations tested, with an LC50 value of 102.34 ng/larva. Sequence analysis suggested that the Cpn60-Xn toxin was homologous to Cpn60-Pl; however, Cpn60-Xn contained thirty-five differentially substituted amino acid residues that could be responsible for its insecticidal activity.
RESUMO
Bacillus thuringiensis insecticidal Cry toxins break down larval midgut-cells after forming pores. The 3D-structures of Cry4Ba and Cry5Ba revealed a trimeric-oligomer after cleavage of helices α-1 and α-2a, where helix α-3 is extended and made contacts with adjacent monomers. Molecular dynamic simulations of Cry1Ab-oligomer model based on Cry4Ba-coordinates showed that E101 forms a salt-bridge with R99 from neighbor monomer. An additional salt bridge was identified in the trimeric-Cry5Ba, located at the extended helix α-3 in the region corresponding to the α-2b and α-3 loop. Both salt-bridges were analyzed by site directed mutagenesis. Single-point mutations in the Lepidoptera-specific Cry1Ab and Cry1Fa toxins were affected in toxicity, while reversed double-point mutant partially recovered the phenotype, consistent with a critical role of these salt-bridges. The single-point mutations in the salt-bridge at the extended helix α-3 of the nematicidal Cry5Ba were also non-toxic. The incorporation of this additional salt bridge into the nontoxic Cry1Ab-R99E mutant partially restored oligomerization and toxicity, supporting that the loop between α-2b and α-3 forms part of an extended helix α-3 upon oligomerization of Cry1 toxins. Overall, these results highlight the role in toxicity of salt-bridge formation between helices α-3 of adjacent monomers supporting a conformational change in helix α-3.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/química , Endotoxinas/química , Proteínas Hemolisinas/química , Sequência de Aminoácidos , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica em alfa-Hélice , Multimerização Proteica , Alinhamento de SequênciaRESUMO
Bacillus thuringiensis Cry toxins are currently used for pest control in transgenic crops but evolution of resistance by the insect pests threatens the use of this technology. The Cry1AbMod toxin was engineered to lack the alpha helix-1 of the parental Cry1Ab toxin and was shown to counter resistance to Cry1Ab and Cry1Ac toxins in different insect species including the fall armyworm Spodoptera frugiperda. In addition, Cry1AbMod showed enhanced toxicity to Cry1Ab-susceptible S. frugiperda populations. To gain insights into the mechanisms of this Cry1AbMod-enhanced toxicity, we isolated the Cry1AbMod toxin binding proteins from S. frugiperda brush border membrane vesicles (BBMV), which were identified by pull-down assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The LC-MS/MS results indicated that Cry1AbMod toxin could bind to four classes of aminopeptidase (N1, N3, N4 y N5) and actin, with the highest amino acid sequence coverage acquired for APN 1 and APN4. In addition to these proteins, we found other proteins not previously described as Cry toxin binding proteins. This is the first report that suggests the interaction between Cry1AbMod and APN in S. frugiperda.
Assuntos
Proteínas de Bactérias/metabolismo , Antígenos CD13/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/metabolismo , Spodoptera/enzimologia , Actinas/química , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Antígenos CD13/química , Antígenos CD13/isolamento & purificação , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insetos/química , Proteínas de Insetos/isolamento & purificação , Microvilosidades/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin.IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.
Assuntos
Bacillus thuringiensis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/toxicidade , Endotoxinas/química , Endotoxinas/toxicidade , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidade , Inseticidas/química , Inseticidas/toxicidade , Aedes/efeitos dos fármacos , Animais , Toxinas de Bacillus thuringiensis , Estabilidade de Medicamentos , Modelos Moleculares , Estrutura Molecular , Mutação PuntualRESUMO
Bacillus thuringiensis Cry2Ab toxin has been used in combination with Cry1Ac for resistance management on the Bt-cotton that is widely planted worldwide. However, little is known regarding Cry2Ab mode of action. Particularly, there is a gap of knowledge on the identification of insect midgut proteins that bind Cry2Ab and mediate toxicity. In the case of Cry1Ab toxin, a transmembrane cadherin protein and glycosyl-phosphatidylinositol (GPI) anchored proteins like aminopeptidase-N1 (APN1) or alkaline-phosphatase (ALP) from Manduca sexta, have been shown to be important for oligomer formation and insertion into the membrane. Binding competition experiments showed that Cry2Ab toxin does not share binding sites with Cry1Ab toxin in M. sexta brush border membrane vesicles (BBMV). Also, that Cry2Ab shows reduced binding to the Cry1Ab binding molecules cadherin, APN1 or ALP. Finally, ligand blot experiments and protein sequence by LC-MS/MS identified APN2 isoform as a Cry2Ab binding protein. Cloning and expression of APN2 confirmed that APN2 is a Cry2Ab binding protein.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Antígenos CD13/química , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/química , Manduca/enzimologia , Receptores de Superfície Celular/química , Sequência de Aminoácidos , Animais , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Antígenos CD13/isolamento & purificação , Antígenos CD13/metabolismo , Endotoxinas/química , Proteínas Hemolisinas/química , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/metabolismo , Resistência a Inseticidas , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Ligantes , Manduca/genética , Receptores de Superfície Celular/isolamento & purificação , Receptores de Superfície Celular/metabolismoRESUMO
Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.
Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Endotoxinas/genética , Evolução Molecular , Proteínas Hemolisinas/genética , Mariposas/metabolismo , Controle Biológico de Vetores , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Endotoxinas/química , Endotoxinas/metabolismo , Engenharia Genética/métodos , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Inseticidas/metabolismoRESUMO
A novel peptide named Pg8 was purified from the venom of the South African scorpion Parabuthus granulatus and its primary structure was determined. It contains 63 amino acid residues tightly folded by 4 disulfide bridges. The gene coding for this peptide was cloned from a cDNA library. By recursive PCR strategy a hybrid gene was constructed having a factor X recognition site for proteolysis and a modified sequence for preferential codon usage of E. coli. A pQE30 molecular vector already contained a His-tag was used for expression. This construction was expressed in BL21 and Origami strains. The fusion protein from inclusion bodies was separated by HPLC (yield approximately 5mg/L) and properly folded in vitro. Lethality tests showed that the recombinant peptide was toxic and was used to immunize mice. A volume of 0.25ml of the anti-serum produced was capable of protecting up to 3 LD(50) doses of pure toxin Pg8 but also, and more importantly, the entire soluble venom.
Assuntos
Antivenenos/genética , Venenos de Escorpião/genética , Venenos de Escorpião/imunologia , Escorpiões/genética , Sequência de Aminoácidos , Animais , Anticorpos/análise , Antivenenos/imunologia , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Feminino , Genes/genética , Genes/imunologia , Camundongos , Dados de Sequência Molecular , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Venenos de Escorpião/enzimologiaRESUMO
Plant cell walls undergo dynamic changes in response to different environmental stress conditions. In response to water deficit, two related proline-rich glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell walls in Phaseolus vulgaris (Covarrubias et al. in Plant Physiol 107:1119-1128, 1995). In this work, we show that p33 and p36 are able to form a 240 kDa oligomer, which is found in the cell wall soluble fraction. We present evidence indicating that the highest accumulation of these proteins in response to water deficit occurs in the growing regions of common bean seedlings, particularly in the phloem tissues. These proteins were detected in P. vulgaris cell suspension cultures, where the p33/p36 ratio was higher under hyperosmotic conditions than in bean seedlings subjected to the same treatment. The results support a role for these proteins during the plant cell response to changes in its water status, and suggest that cell wall modifications are induced in active growing cells of common bean in response to water limitation.
Assuntos
Parede Celular/metabolismo , Phaseolus/metabolismo , Floema/fisiologia , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Água/metabolismo , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Phaseolus/citologia , Phaseolus/crescimento & desenvolvimento , Proteínas de Plantas/isolamento & purificação , Abastecimento de ÁguaRESUMO
The venom of the scorpion Tityus costatus contains peptides toxic to humans but scarce information on their structure and function is available. Here, we report the separation of 50 different components by high performance liquid chromatography and the identification of approximately 90 distinct components by mass spectrometry analysis, with molecular weights varying from 413 to 45482 atomic mass units. Four peptides were fully sequenced: (i) a butantoxin-like peptide that blocks Shaker K+ channel; (ii) an insect toxin-like peptide; (iii) a scorpine-like peptide, and a short heptapeptide of unknown function. Fifteen peptides were directly sequenced at the N-terminal region, among which are components toxic to mice. A cDNA library was constructed and 13 clones were isolated and sequenced. Some of these peptides and genes are similar to other known scorpion toxins. Based on these results, stings by scorpions of the species Tityus costatus should be taken with caution by medical doctors.