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1.
PLoS One ; 16(10): e0258052, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34634061

RESUMO

The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, is a major corn pest of significant economic importance in the United States. The continuous need to control this corn maize pest and the development of field-evolved resistance toward all existing transgenic maize (Zea mays L.) expressing Bacillus thuringiensis (Bt) insecticidal proteins against WCR has prompted the development of new insect-protected crops expressing distinct structural classes of insecticidal proteins. In this current study, we describe the crystal structure and functional characterization of Mpp75Aa1.1, which represents the first corn rootworm (CRW) active insecticidal protein member of the ETX_MTX2 sub-family of beta-pore forming proteins (ß-PFPs), and provides new and effective protection against WCR feeding. The Mpp75Aa1.1 crystal structure was solved at 1.94 Å resolution. The Mpp75Aa1.1 is processed at its carboxyl-terminus by WCR midgut proteases, forms an oligomer, and specifically interacts with putative membrane-associated binding partners on the midgut apical microvilli to cause cellular tissue damage resulting in insect death. Alanine substitution of the surface-exposed amino acids W206, Y212, and G217 within the Mpp75Aa1.1 putative receptor binding domain I demonstrates that at least these three amino acids are required for WCR activity. The distinctive spatial arrangement of these amino acids suggests that they are part of a receptor binding epitope, which may be unique to Mpp75Aa1.1 and not present in other ETX_MTX2 proteins that do not have WCR activity. Overall, this work establishes that Mpp75Aa1.1 shares a mode of action consistent with traditional WCR-active Bt proteins despite significant structural differences.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Inseticidas/farmacologia , Controle Biológico de Vetores/métodos , Plantas Geneticamente Modificadas , Zea mays , Animais , Proteínas de Bactérias/genética , Besouros/efeitos dos fármacos , Resistência a Inseticidas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Zea mays/genética , Zea mays/metabolismo
2.
Molecules ; 26(20)2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34684855

RESUMO

Vitexin is a C-glucoside flavone that exhibits a wide range of pharmaceutical activities. However, the poor solubility of vitexin limits its applications. To resolve this limitation, two glycoside hydrolases (GHs) and four glycosyltransferases (GTs) were assayed for glycosylation activity toward vitexin. The results showed that BtGT_16345 from the Bacillus thuringiensis GA A07 strain possessed the highest glycosylation activity, catalyzing the conversion of vitexin into new compounds, vitexin-4'-O-ß-glucoside (1) and vitexin-5-O-ß-glucoside (2), which showed greater aqueous solubility than vitexin. To our knowledge, this is the first report of vitexin glycosylation. Based on the multiple bioactivities of vitexin, the two highly soluble vitexin derivatives might have high potential for pharmacological usage in the future.


Assuntos
Apigenina/metabolismo , Glucosídeos/metabolismo , Bacillus thuringiensis/metabolismo , Catálise , Flavonas/metabolismo , Glicosilação , Glicosiltransferases/metabolismo , Isoflavonas/metabolismo , Solubilidade
3.
J Invertebr Pathol ; 185: 107657, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34487747

RESUMO

Bacillus thuringiensis (Bt) has been used globally as a biopesticide for effective and environmentally friendly pest control. Research has intensified following the development of resistance by lepidopteran species to Bt insecticidal crystal proteins. Discovering new Bt strains with novel toxin properties which can overcome resistance is one of the strategies to improve pesticide sustainability. The genome of the Bacillus thuringiensis LTS290 strain was sequenced and assembled in 252 contigs containing a total of 6,391,328 bp. The novel cry79Aa1 gene from this strain was identified and cloned. Cry79Aa1 contains 729 amino acid residues and a molecular mass of 84.8 kDa by SDS-PAGE analysis. Cry79Aa1 was found to be active against the lepidopteran larvae of Spodoptera exigua, Helicoverpa armigera, and Plutella xylostella with LC50 values of 13.627 µg/mL, 42.8 µg/mL, and 38.086 µg/mL, respectively. However, Cry79Aa1 protein showed almost no insecticidal activity against Leguminivora glycinivorella, although some degree of growth retardation was observed.


Assuntos
Toxinas de Bacillus thuringiensis/genética , Bacillus thuringiensis/genética , Endotoxinas/genética , Proteínas Hemolisinas/genética , Mariposas/efeitos dos fármacos , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Controle de Insetos , Mariposas/crescimento & desenvolvimento , Controle Biológico de Vetores , Spodoptera/efeitos dos fármacos , Spodoptera/crescimento & desenvolvimento
4.
Arch Microbiol ; 203(9): 5387-5396, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34390357

RESUMO

Keratinases are a group of proteases of great industrial significance. To take full advantage of Bacillus species as an inherent superior microbial producer of proteases, we performed the ribosome engineering to improve the keratinase synthesis capacity of the wild-type Bacillus thuringiensis by inducing streptomycin resistance. Mutant Bt(Str-O) was identified as a stable keratinase overproducer. Comparative characterization of the two strains revealed that, although the resistance to Streptomycin increased by eight-fold in MIC, the mutant's resistance to other commonly used antibiotics was not affected. Furthermore, the mutant exhibited an enhanced keratinase synthesis (1.5-fold) when cultured in a liquid LB medium. In the whole feather degradation experiment, the mutant could secret twofold keratinase into the medium, reaching 640 U/mL per 107 CFU. By contrast, no significant differences were found in the scanning electron microscopic analysis and spore formation experiment. To understand the genetic factors causing these phenotypic changes, we cloned and analyzed the rpsL gene. No mutation was observed. We subsequently determined the genome sequences of the two strains. Comparing the rpsL gene revealed that the emergence of streptomycin resistance was not necessarily dependent on the mutation(s) in the generally recognized "hotspot." Genome-wide analysis showed that the phenotypic changes of the mutant were the collective consequence of the genetic variations occurring in the regulatory regions and the non-coding RNA genes. This study demonstrated the importance of genetic changes in regulatory regions and the effectiveness of irrational ribosome engineering in creating prokaryotic microbial mutants without sufficient genetic information.


Assuntos
Bacillus thuringiensis , Estreptomicina , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Mutação , Peptídeo Hidrolases/genética , Sequências Reguladoras de Ácido Nucleico , Estreptomicina/farmacologia
5.
Curr Microbiol ; 78(9): 3483-3493, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34272975

RESUMO

Lon protease, an intracellular protease, plays a key role in cell homeostasis in bacteria and is involved in numerous physiological processes. In this work, we aimed to study the impact of Lon on the production of endotoxins and stress response in Bacillus thuringiensis, which is an important bioinsecticide alternative for toxic chemicals. For this purpose, lon gene was cloned into a multi-copy vector with its original promoter and transcriptional terminator and expressed in B. thuringiensis serovar israelensis ATCC 35,646. Our results showed that the recombinant lon gene transcribed and translated efficiently and the resulting protein was active. Although the sporulation efficiency of the recombinant strain was found to be reduced and its mobility impaired, overexpression of the lon gene triggered the production of endotoxin. Together with increased biofilm formation, recombinant strain exhibited significantly better adaptation to osmotic and heat shock stresses and UV exposure compared to wild type and the control strain with empty plasmid. This study suggested a possible link between Lon protease and the production of insecticide and stress response in B. thuringiensis and provides a platform for future studies focusing on enhancing bio-insecticidal production using this bacterium.


Assuntos
Bacillus thuringiensis , Protease La , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/genética , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Plasmídeos , Protease La/genética
6.
Arch Insect Biochem Physiol ; 108(2): e21834, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34288075

RESUMO

The Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae), is a major pest of potato plants worldwide and is notorious for its ability to develop resistance to insecticides. Cry3 toxins synthesized by Bacillus thuringiensis ssp. tenebrionis have been used successfully to manage this pest. Resistance to Cry toxins is a concerning problem for many insect pests; therefore, it is important to determine the mechanisms by which insects acquire resistance to these toxins. Cadherin-like and ABC transporter proteins have been implicated in the mode of action of Cry toxins as mutations in these genes render lepidopterans resistant to them; however, clear consensus does not exist on whether these proteins also play a role in Cry3 toxin activity and/or development of resistance in coleopterans. In the current study, we identified the L. decemlineata orthologues of the cadherin (LdCAD) and the ABCB transporter (LdABCB1) that have been implicated in the mode of action of Cry toxins in other coleopterans. Suppression of LdABCB1 via RNA interference reduced toxin-related larval mortality, whereas partial silencing of LdCAD did not. Our results suggest that the ABCB is involved in the mode of action of Cry3Aa toxins; however, no evidence was found to support the role of cadherin as a receptor of Cry3Aa in L. decemlineata.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Toxinas de Bacillus thuringiensis/farmacologia , Besouros , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Caderinas/genética , Caderinas/metabolismo , Besouros/efeitos dos fármacos , Besouros/metabolismo , Besouros/microbiologia , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/metabolismo , Larva/microbiologia , Controle Biológico de Vetores , Interferência de RNA
7.
Toxins (Basel) ; 13(5)2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34065247

RESUMO

Laboratory selection for resistance of field populations is a well-known and useful tool to understand the potential of insect populations to evolve resistance to insecticides. It provides us with estimates of the frequency of resistance alleles and allows us to study the mechanisms by which insects developed resistance to shed light on the mode of action and optimize resistance management strategies. Here, a field population of Mythimna separata was subjected to laboratory selection with either Vip3Aa, Cry1Ab, or Cry1F insecticidal proteins from Bacillus thuringiensis. The population rapidly evolved resistance to Vip3Aa reaching, after eight generations, a level of >3061-fold resistance, compared with the unselected insects. In contrast, the same population did not respond to selection with Cry1Ab or Cry1F. The Vip3Aa resistant population did not show cross resistance to either Cry1Ab or Cry1F. Radiolabeled Vip3Aa was tested for binding to brush border membrane vesicles from larvae from the susceptible and resistant insects. The results did not show any qualitative or quantitative difference between both insect samples. Our data, along with previous results obtained with other Vip3Aa-resistant populations from other insect species, suggest that altered binding to midgut membrane receptors is not the main mechanism of resistance to Vip3Aa.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Inseticidas/farmacologia , Mariposas/fisiologia , Animais , Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Resistência a Inseticidas , Inseticidas/isolamento & purificação , Larva , Ligação Proteica
8.
Pest Manag Sci ; 77(10): 4593-4606, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34092019

RESUMO

BACKGROUND: Broad use of insecticidal Cry proteins from Bacillus thuringiensis in biopesticides and transgenic crops has resulted in cases of practical field resistance, highlighting the need for novel approaches to insect control. Previously we described an anti-Cry1Ab idiotypic-antibody (B12-scFv) displaying toxicity against rice leafroller (Cnaphalocrocis medinalis) larvae, supporting the potential of antibodies for pest control. The goal of the present study was to generate insecticidal antibodies against diamondback moth (Plutella xylostella) larvae. RESULTS: Four genetically engineered antibodies (GEAbs) were designed in silico from B12-scFv using three-dimensional (3D) structure and docking predictions to alkaline phosphatase (ALP) as a Cry1Ac receptor in P. xylostella. Among these GEAbs, the GEAb-dVL antibody consisting of two light chains had overlapping binding sites with Cry1A and Cry1B proteins and displayed high binding affinity to P. xylostella midgut brush border membrane (BBM) proteins. Proteins in BBM identified by pull-down assays as binding to GEAb-dVL included an ABC transporter and V-ATPase subunit A protein. Despite lacking the α-helical structures in Cry1A that are responsible for pore formation, ingestion of GEAb-dVL disrupted the P. xylostella larval midgut epithelium and resulted in toxicity. Apoptotic genes were activated in gut cells upon treatment with GEAb-dVL . CONCLUSION: This study describes the first insecticidal GEAb targeting P. xylostella by mimicking Cry proteins. Data support that GEAb-dVL toxicity is associated to activation of intracellular cell death pathways, in contrast to pore-formation associated toxicity of Cry proteins. This work provides a foundation for the design of novel insecticidal antibodies for insect control. © 2021 Society of Chemical Industry.


Assuntos
Bacillus thuringiensis , Inseticidas , Mariposas , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Larva/metabolismo , Mariposas/metabolismo , Ligação Proteica
9.
Biochem J ; 478(13): 2589-2600, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34129679

RESUMO

The ATP binding cassette (ABC) transporters are membrane proteins that can act as putative receptors for Cry proteins from Bacillus thuringiensis (Bt) in the midgut of different insects. For the beet armyworm, Spodoptera exigua, ABCC2 and ABCC3 have been found to interact with Cry1A proteins, the main insecticidal proteins used in Bt crops, as well as Bt-based pesticides. The ABCC2 has shown to have specific binding towards Cry1Ac and is involved in the toxic process of Cry1A proteins, but the role of this transporter and how it relates with the Cry1A proteins is still unknown. Here, we have characterized the interactions between the SeABCC2 and the main proteins that bind to the receptor. By labeling the Cry1Aa protein, we have found that virtually all of the binding is in an oligomeric state, a conformation that allowed higher levels of specific binding that could not be achieved by the monomeric protein on its own. Furthermore, we have observed that Cry1A proteins can hetero-oligomerize in the presence of the transporter, which is reflected in an increase in binding and toxicity to SeABCC2-expressing cells. This synergism can be one of the reasons why B. thuringiensis co-expresses different Cry1 proteins that can apparently have similar binding preferences. The results from in vitro competition and ex vivo competition showed that Cry1Aa, Cry1Ab and Cry1Ac share functional binding sites. By using Cry1Ab-Cry1Ac chimeras, the presence of domain I from Cry1A proteins was revealed to be critical for oligomer formation.


Assuntos
Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/química , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas de Insetos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Spodoptera/metabolismo , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Sobrevivência Celular/genética , Endotoxinas/química , Endotoxinas/genética , Proteínas Hemolisinas/química , Proteínas Hemolisinas/genética , Proteínas de Insetos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Mutação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Células Sf9 , Spodoptera/citologia , Spodoptera/genética
10.
Int J Radiat Biol ; 97(9): 1299-1315, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34032553

RESUMO

PURPOSE: Combining gamma irradiation and nanotechnology has become one of the most promising new approaches for area-wide (AW) pest management in recent years. The laboratory trials were conducted to determine the combining effects of BT-AgNPs and gamma irradiation for controlling P. gossypiella. Radio-sensitivity of male pupae at different doses of gamma radiation and the effectiveness of biosynthesized silver nanoparticles using Bacillus thuringiensis on larval instar were assayed. Additionally, the ultrastructure changes on the alimentary canal of 4th instar larvae were studied to evaluate the impact of the combined approach at a cellular level. MATERIALS AND METHODS: Laboratory- rearing technique was used for rearing Pectinophora gossypiella. The irradiation process was achieved at Co60 - Gamma Chamber (4000 A). Alanine dosimeters were used for measuring the average absorbed dose and dose mapping. Preparation of Silver nanoparticles (AgNPs) using Bacillus thuringiensis (Bt) and their characterization has been investigated. The treated 4th instar larvae by gamma irradiation or ∕and BT-AgNPs were dissected under the stereo microscope. The alimentary canal was obtained anatomically and Transmission Electron Microscope) was used in examining the stained sections. RESULTS: Based on the nonhatching eggs produced by irradiated males' pupae, the values of effective doses were calculated. The effective doses ranged from 16 to 291 Gy for the ED25 - ED75. The sterility index reached 74.1% when irradiated with males by 291 Gy crossed with nonirradiated females and the adult emergence decreased to be 35.3%. The insecticidal potential of Bt-AgNPs on the 2nd and 4th larval instars was dose-dependent and its LC50 toxicity value was 0. 3 and 0. 4 mg/ml, respectively. The lethal concentration LC50 of the 2nd instar larvae increased the larval and pupal mortality to 55% and 44.4%, respectively, and reduced the adult emergence to be 55.6%. The combining effects of Bt-AgNPs with 291 Gy induced 100% pupae mortality and there was no adult emergence in F1 generation. Such effects also severed the ultrastructure deformity of the midgut of the 4th instar larvae after the two-day post-treatment. CONCLUSIONS: The combining effects are recommended as an effective IPM program to control P. gossypiella by releasing sterile males (derived from pupae irradiated with 291 Gy) crossing with the normal females in the field, and reducing the fertility of the population to 31.2%. Subsequently, the resulted larvae treated with LC50 of Bt-AgNPs prevented the adult emergence and stopped the life cycle of P. gossypiella.


Assuntos
Bacillus thuringiensis/metabolismo , Raios gama , Nanopartículas Metálicas , Mariposas/efeitos dos fármacos , Mariposas/efeitos da radiação , Prata/química , Prata/farmacologia , Animais , Larva/efeitos dos fármacos , Larva/efeitos da radiação , Controle de Pragas , Tolerância a Radiação/efeitos dos fármacos
11.
Toxins (Basel) ; 13(3)2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33809820

RESUMO

Concerns about resistance development to conventional insecticides in diamondback moth (DBM) Plutella xylostella (L.), the most destructive pest of Brassica vegetables, have stimulated interest in alternative pest management strategies. The toxicity of Bacillus thuringiensis subsp. aizawai (Bt GO33A) combined with chlorantraniliprole (Chl) has not been documented. Here, we examined single and combined toxicity of chlorantraniliprole and Bt to assess the levels of resistance in four DBM strains. Additionally, enzyme activities were tested in field-original highly resistant (FOH-DBM), Bt-resistant (Bt-DBM), chlorantraniliprole-resistant (CL-DBM), and Bt + chlorantraniliprole-resistant (BtC-DBM) strains. The Bt product had the highest toxicity to all four DBM strains followed by the mixture of insecticides (Bt + Chl) and chlorantraniliprole. Synergism between Bt and chlorantraniliprole was observed; the combination of Bt + (Bt + Chl) (1:1, LC50:LC50) was the most toxic, showing a synergistic effect against all four DBM strains with a poison ratio of 1.35, 1.29, 1.27, and 1.25. Glutathione S-transferase (GST) and carboxyl-esterase (CarE) activities showed positive correlations with chlorantraniliprole resistance, but no correlation was observed with resistance to Bt and Bt + Chl insecticides. Expression of genes coding for PxGST, CarE, AChE, and MFO using qRT-PCR showed that the PxGST and MFO were significantly overexpressed in Bt-DBM. However, AChE and CarE showed no difference in the four DBM strains. Mixtures of Bt with chlorantraniliprole exhibited synergistic effects and may aid the design of new combinations of pesticides to delay resistance in DBM strains substantially.


Assuntos
Bacillus thuringiensis/metabolismo , Brassica/parasitologia , Resistência a Inseticidas , Inseticidas/farmacologia , Mariposas/efeitos dos fármacos , Mariposas/microbiologia , Controle Biológico de Vetores , ortoaminobenzoatos/farmacologia , Acetilcolinesterase/genética , Acetilcolinesterase/metabolismo , Animais , Bacillus thuringiensis/genética , Carboxilesterase/genética , Carboxilesterase/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Regulação Enzimológica da Expressão Gênica , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Resistência a Inseticidas/genética , Mariposas/enzimologia , Mariposas/genética
12.
PLoS One ; 16(4): e0249699, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33831084

RESUMO

Plants have developed various mechanisms to respond specifically to each biotrophic attack. It has been shown that the electrical signals emitted by plants are associated with herbivory stress responses and can lead to the activation of multiple defences. Bt cotton is a genetically modified pest-resistant plant that produces an insecticide from Bacillus thuringiensis (Bt) to control Lepidopteran species. Surprisingly, there is no study-yet, that characterizes the signalling mechanisms in transgenic cotton plants attacked by non-target insects, such as aphids. In this study, we characterized the production of electrical signals on Bt and non-Bt cotton plants infested with Aphis gossypii and, in addition, we characterized the dispersal behaviour of aphids to correlate this behaviour to plant signalling responses. Electrical signalling of the plants was recorded with an extracellular measurement technique. Impressively, our results showed that both Bt and non-Bt cotton varieties, when attacked by A. gossypii, emitted potential variation-type electrical signals and clearly showed the presence of distinct responses regarding their perception and the behaviour of aphids, with evidence of delay, in terms of signal amount, and almost twice the amount of Cry1F protein was observed on Bt cotton plants at the highest density of insects/plant. We present in our article some hypotheses that are based on plant physiology and insect behaviour to explain the responses found on Bt cotton plants under aphid stress.


Assuntos
Afídeos/microbiologia , Bacillus thuringiensis/metabolismo , Gossypium/microbiologia , Gossypium/parasitologia , Estresse Fisiológico/fisiologia , Animais , Gossypium/genética , Herbivoria/fisiologia , Insetos/microbiologia , Inseticidas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Plantas Geneticamente Modificadas/parasitologia , Transdução de Sinais/genética , Estresse Fisiológico/genética
13.
mBio ; 12(2)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758089

RESUMO

ß-Lactams are a class of antibiotics that target the synthesis of peptidoglycan, an essential component of the cell wall. ß-Lactams inhibit the function of penicillin-binding proteins (PBPs), which form the cross-links between strands of peptidoglycan. Resistance to ß-lactams complicates the treatment of bacterial infections. In recent years, the spread of ß-lactam resistance has increased with growing intensity. Resistance is often conferred by ß-lactamases, which inactivate ß-lactams, or the expression of alternative ß-lactam-resistant PBPs. σP is an extracytoplasmic function (ECF) σ factor that controls ß-lactam resistance in the species Bacillus thuringiensis, Bacillus cereus, and Bacillus anthracis σP is normally held inactive by the anti-σ factor RsiP. σP is activated by ß-lactams that trigger the proteolytic destruction of RsiP. Here, we identify the penicillin-binding protein PbpP and demonstrate its essential role in the activation of σP Our data show that PbpP is required for σP activation and RsiP degradation. Our data suggest that PbpP acts as a ß-lactam sensor since the binding of a subset of ß-lactams to PbpP is required for σP activation. We find that PbpP likely directly or indirectly controls site 1 cleavage of RsiP, which results in the degradation of RsiP and, thus, σP activation. σP activation results in increased expression of ß-lactamases and, thus, increased ß-lactam resistance. This work is the first report of a PBP acting as a sensor for ß-lactams and controlling the activation of an ECF σ factor.IMPORTANCE The bacterial cell envelope is the target for numerous antibiotics. Many antibiotics target the synthesis of peptidoglycan, which is a central metabolic pathway essential for bacterial survival. One of the most important classes of antibiotics has been ß-lactams, which inhibit the transpeptidase activity of penicillin-binding proteins to decrease the cross-linking of peptidoglycan and the strength of the cell wall. While ß-lactam antibiotics have historically proven to be effective, resistance to ß-lactams is a growing problem. The ECF σ factor σP is required for ß-lactam resistance in B. thuringiensis and close relatives, including B. anthracis Here, we provide insight into the mechanism of activation of σP by ß-lactams.


Assuntos
Antibacterianos/farmacologia , Bacillus thuringiensis/efeitos dos fármacos , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Fator sigma/genética , Fator sigma/metabolismo , beta-Lactamas/farmacologia , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Proteínas de Ligação às Penicilinas/classificação , Resistência beta-Lactâmica , beta-Lactamases/metabolismo
14.
FEMS Microbiol Lett ; 368(5)2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33720297

RESUMO

Bacillus thuringiensis has been widely used as a biological control agent against insect pests. Additionally, nematicidal strains have been under investigation. In this report, 310 native strains of B. thuringiensis against Caenorhabditis elegans were tested. Only the LBIT-596 and LBIT-107 strains showed significant mortality. LC50s of spore-crystal complexes were estimated at 37.18 and 31.89 µg/mL for LBIT-596 and LBIT-107 strains, respectively, while LC50s of partially purified crystals was estimated at 23.76 and 20.25 µg/mL for LBIT-596 and LBIT-107, respectively. The flagellin gene sequence and plasmid patterns indicated that LBIT-596 and LBIT-107 are not related to each other. Sequences from internal regions of a cry5B and a cyt1A genes were found in the LBIT-596 strain, while a cry21A, a cry14A and a cyt1A genes were found in the LBIT-107 strain. Genome sequence of the LBIT-107 strain showed new cry genes, along with other virulence factors, hence, total nematicidal activity of the LBIT-107 strain may be the result of a multifactorial effect. The highlight of this contribution is that translocation of spore-crystal suspensions of LBIT-107 into tomato plants inoculated at their rhizosphere decreased up to 90% the number of galls of Meloidogyne incognita, perhaps the most important nematode pest in the world.


Assuntos
Antinematódeos/metabolismo , Bacillus thuringiensis/metabolismo , Agentes de Controle Biológico/metabolismo , Caenorhabditis elegans/microbiologia , Doenças das Plantas/terapia , Tylenchoidea/microbiologia , Animais , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/genética , Endotoxinas/genética , Flagelina/genética , Proteínas Hemolisinas/genética , Lycopersicon esculentum/parasitologia , Doenças das Plantas/parasitologia , Plasmídeos/genética , Fatores de Virulência/genética
15.
Int J Mol Sci ; 22(5)2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33668147

RESUMO

Bacillus thuringiensis, commonly referred to as Bt, is an object of the lasting interest of microbiologists due to its highly effective insecticidal properties, which make Bt a prominent source of biologicals. To categorize the exuberance of Bt strains discovered, serotyping assays are utilized in which flagellin serves as a primary seroreactive molecule. Despite its convenience, this approach is not indicative of Bt strains' phenotypes, neither it reflects actual phylogenetic relationships within the species. In this respect, comparative genomic and proteomic techniques appear more informative, but their use in Bt strain classification remains limited. In the present work, we used a bottom-up proteomic approach based on fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) coupled with liquid chromatography/tandem mass spectrometry(LC-MS/MS) protein identification to assess which stage of Bt culture, vegetative or spore, would be more informative for strain characterization. To this end, the proteomic differences for the israelensis-attributed strains were assessed to compare sporulating cultures of the virulent derivative to the avirulent one as well as to the vegetative stage virulent bacteria. Using the same approach, virulent spores of the israelensis strain were also compared to the spores of strains belonging to two other major Bt serovars, namely darmstadiensis and thuringiensis. The identified proteins were analyzed regarding the presence of the respective genes in the 104 Bt genome assemblies available at open access with serovar attributions specified. Of 21 proteins identified, 15 were found to be encoded in all the present assemblies at 67% identity threshold, including several virulence factors. Notable, individual phylogenies of these core genes conferred neither the serotyping nor the flagellin-based phylogeny but corroborated the reconstruction based on phylogenomics approaches in terms of tree topology similarity. In its turn, the distribution of accessory protein genes was not confined to the existing serovars. The obtained results indicate that neither gene presence nor the core gene sequence may serve as distinctive bases for the serovar attribution, undermining the notion that the serotyping system reflects strains' phenotypic or genetic similarity. We also provide a set of loci, which fit in with the phylogenomics data plausibly and thus may serve for draft phylogeny estimation of the novel strains.


Assuntos
Bacillus thuringiensis/classificação , Proteínas de Bactérias/metabolismo , Flagelina/metabolismo , Proteoma/metabolismo , Sorotipagem/métodos , Fatores de Virulência/metabolismo , Virulência , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/patogenicidade , Proteínas de Bactérias/genética , Cromatografia Líquida , Flagelina/genética , Filogenia , Proteoma/análise , Espectrometria de Massas em Tandem , Fatores de Virulência/genética
16.
PLoS One ; 16(2): e0246696, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33591990

RESUMO

Maize with enhanced ß-carotene production was engineered to counteract pervasive vitamin A deficiency in developing countries. Second-generation biofortified crops are being developed with additional traits that confer pest resistance. These include crops that can produce Bacillus thuringiensis Berliner (Bt) insecticidal proteins. Currently, it is unknown whether ß-carotene can confer fitness benefits through to insect pests, specifically through altering Ostrinia nubilalis foraging behaviour or development in the presence of Bt insecticidal toxin. Therefore the effects of dietary ß-carotene plus Bt insecticidal protein on feeding behaviour, mortality, and physiology in early and late instars of O. nubilalis larvae were investigated. The results of two-choice experiments showed that irrespective of ß-carotene presence, at day five 68%-90% of neonates and 69%-77% of fifth-instar larvae avoided diets with Cry1A protein. Over 65% of neonate larvae preferred to feed on diets with ß-carotene alone compared to 39% of fifth-instar larvae. Higher mortality (65%-97%) in neonates fed diets supplemented with ß-carotene alone and in combination with Bt protein was found, whereas <36% mortality was observed when fed diets without supplemented ß-carotene or Bt protein. Diets with both ß-carotene and Bt protein extended 25 days the larval developmental duration from neonate to fifth instar (compared to Bt diets) but did not impair larval or pupal weight. Juvenile hormone and 20-hydroxyecdysone regulate insect development and their levels were at least 3-fold higher in larvae fed diets with ß-carotene for 3 days. Overall, these results suggest that the effects of ß-carotene and Bt protein on O. nubilalis is dependent on larval developmental stage. This study is one of the first that provides insight on how the interaction of novel traits may modulate crop susceptibility to insect pests. This understanding will in turn inform the development of crop protection strategies with greater efficacy.


Assuntos
Toxinas de Bacillus thuringiensis/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Mariposas/fisiologia , beta Caroteno/farmacologia , Animais , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/genética , Endotoxinas/metabolismo , Comportamento Alimentar/fisiologia , Proteínas Hemolisinas/metabolismo , Resistência a Inseticidas/efeitos dos fármacos , Inseticidas/farmacologia , Larva/fisiologia , Lepidópteros/metabolismo , Mariposas/metabolismo , Controle Biológico de Vetores/métodos , Plantas Geneticamente Modificadas/metabolismo , Pupa/metabolismo , Zea mays/genética , Zea mays/metabolismo , beta Caroteno/metabolismo
17.
Biochim Biophys Acta Proteins Proteom ; 1869(6): 140634, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33636413

RESUMO

One proposed toxic mechanism of Bacillus thuringiensis Cry δ-endotoxins involves pore formation in target membranes by the α4-α5 transmembrane hairpin constituting their pore-forming domain. Here, nine selected charged and uncharged polar residues in the pore-lining α4 of the Cry4Aa mosquito-active toxin were substituted with Ala. All mutant toxins, i.e., D169A, R171A, Q173A, H178A, Y179A, H180A, Q182A, N183A and E187A, were over-expressed in Escherichia coli as 130-kDa protoxin inclusions at levels comparable to the wild-type toxin. Bioassays against Aedes aegypti larvae revealed that only H178A and H180A mutants displayed a drastic reduction in biotoxicity, albeit almost complete insolubility observed for H178A, but not for H180A inclusions. Further mutagenic analysis showed that replacements of His180 with charged (Arg, Lys, Asp, Glu), small uncharged polar (Ser, Cys) or small non-polar (Gly, Val) residues severely impaired the biotoxicity, unlike substitutions with relatively large uncharged (Asn, Gln, Leu) or aromatic (Phe, Tyr, Trp) residues. Similar to the trypsin-activated wild-type toxin, both bio-active and -inactive H180 mutants were still capable of releasing entrapped calcein from lipid vesicles and producing cation-selective channels with ~130-pS maximum conductance. Analysis of the Cry4Aa structure revealed the existence of a hydrophobic cavity near the critical His180 side-chain. Analysis of simulated structures revealed that His180-to-smaller residue conversions create a gap disrupting such cavity's hydrophobicity and hence structural arrangements of the α4-α5 hairpin. Altogether, our data disclose a critical involvement in Cry4Aa-biotoxicity of His180 exclusively present in the lumen-facing α4 for providing proper environment for the α4-α5 hairpin prior to membrane-inserted pore formation.


Assuntos
Aedes/crescimento & desenvolvimento , Toxinas de Bacillus thuringiensis/química , Toxinas de Bacillus thuringiensis/toxicidade , Bacillus thuringiensis/metabolismo , Endotoxinas/química , Endotoxinas/toxicidade , Proteínas Hemolisinas/química , Proteínas Hemolisinas/toxicidade , Histidina/genética , Aedes/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Bacillus thuringiensis/química , Bacillus thuringiensis/genética , Toxinas de Bacillus thuringiensis/genética , Endotoxinas/genética , Fluoresceínas/metabolismo , Proteínas Hemolisinas/genética , Interações Hidrofóbicas e Hidrofílicas , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Modelos Moleculares , Simulação de Dinâmica Molecular , Domínios Proteicos , Estrutura Secundária de Proteína
18.
Ecotoxicol Environ Saf ; 212: 111967, 2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33524911

RESUMO

Non-target effects of genetically engineered (GE) plants on aquatic Daphnia magna have been studied by feeding the species with different maize materials containing insecticidal Cry proteins from Bacillus thuringiensis (Bt). The results of those studies were often difficult to interpret, because only one GE plant was compared to one related non-GE control. In such a setting, effects of the Cry proteins cannot be distinguished from plant background effects, in particular when the test species is nutritionally stressed. In the present study, we tested the suitability of three different maize materials, i.e., flour, leaves and pollen, from five diverse non-GE maize lines (including EXP 258, a breeding line that is closely related to a SmartStax Bt maize) as exclusive food sources for D. magna. The parameters recorded included survival, sublethal endpoints such as body size, number of moltings to first offspring, time to first offspring, number of individuals in first clutch, total number of clutches, total number of offspring, average number of offspring per clutch, and population measures such as net reproductive rate R0, generation time T and intrinsic rate of increase rm. The results showed that D. magna can survive, grow and reproduce when fed only maize materials, although the performance was poorer than when fed algae, which indicates nutritional stress. Large differences in life table and population parameters of D. magna were observed among the different maize lines. Our results suggest that confounding effects caused by nutritional stress and plant background might explain some of the conflicting results previously published on the effects of Bt crops on D. magna. Using 95% confidence intervals for the means of the five maize lines for all measured parameters of D. magna performance in our study, we captured the natural range of variation. This information is useful for the interpretation of observed differences in D. magna performance between a GE plant and its non-GE comparator as it helps judging whether observed effects are of biological relevance. If differences between a GE and comparator line are observed and their biological relevance needs to be assessed in future risk assessments of GE maize, 1) the data on natural variation of the different parameters generated by previous studies can be informative (e.g. data from our study for maize fed D. magna); 2) for additional experiments the inclusion of multiple unrelated non-GE comparators should be considered; In addition, it should be taken into account that nutritional stress can affect the outcome of the study.


Assuntos
Daphnia/fisiologia , Plantas Geneticamente Modificadas/fisiologia , Zea mays/fisiologia , Animais , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Produtos Agrícolas/metabolismo , Daphnia/efeitos dos fármacos , Endotoxinas/metabolismo , Farinha , Proteínas Hemolisinas/genética , Inseticidas/farmacologia , Melhoramento Vegetal , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/toxicidade , Pólen , Medição de Risco , Zea mays/metabolismo
19.
Microbiol Mol Biol Rev ; 85(1)2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33504654

RESUMO

Extensive use of chemical insecticides adversely affects both environment and human health. One of the most popular biological pest control alternatives is bioinsecticides based on Bacillus thuringiensis This entomopathogenic bacterium produces different protein types which are toxic to several insect, mite, and nematode species. Currently, insecticidal proteins belonging to the Cry and Vip3 groups are widely used to control insect pests both in formulated sprays and in transgenic crops. However, the benefits of B. thuringiensis-based products are threatened by insect resistance evolution. Numerous studies have highlighted that mutations in genes coding for surrogate receptors are responsible for conferring resistance to B. thuringiensis Nevertheless, other mechanisms may also contribute to the reduction of the effectiveness of B. thuringiensis-based products for managing insect pests and even to the acquisition of resistance. Here, we review the relevant literature reporting how invertebrates (mainly insects and Caenorhabditis elegans) respond to exposure to B. thuringiensis as either whole bacteria, spores, and/or its pesticidal proteins.


Assuntos
Toxinas de Bacillus thuringiensis/metabolismo , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Caenorhabditis elegans/microbiologia , Endotoxinas/metabolismo , Proteínas Hemolisinas/metabolismo , Insetos/microbiologia , Animais , Bacillus thuringiensis/genética , Inseticidas/metabolismo , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Ácaros/microbiologia , Controle Biológico de Vetores , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
20.
Pest Manag Sci ; 77(3): 1492-1501, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33145907

RESUMO

BACKGROUND: The fall armyworm Spodoptera frugiperda is a major agricultural pest that has invaded the East Hemisphere since 2016, generating a serious threat to food security worldwide including Africa and Asia. The Cry toxins produced by Bacillus thuringiensis (Bt) have been shown to be effective against this insect pest. In different insect ABC transporters (ABCC2 or ABCC3) have been shown to be involved as receptors of some Cry1 toxins. Here we analyzed the role of SfABCC2 and SfABCC3 in the toxicity of Cry1Fa and Cry1Ab toxins in this insect pest. RESULTS: Two S. frugiperda SfABCC2 and SfABCC3 knockout strains, coding for potential functional Bt receptors, were created using CRISPR/Cas9 genome editing system. Both knockout strains showed resistance to both Cry1Fa and Cry1Ab toxins compared with the susceptible strain. SfABCC2 knockout strain showed higher resistance to both Cry toxins than SfABCC3 knockout strain, suggesting a major role of SfABCC2 in the mode of action of these Cry toxins. In addition, expression of SfABCC2 and SfABCC3 genes in Trichoplusia ni Hi5 cells also increased the susceptibility to Cry1Ab and Cry1Fa toxins, in agreement with the genome editing results. The double knockout of SfABCC2 and SfABCC3 strain was not viable in contrast to other lepidopteran species. Furthermore, we report here that SfABCC2 or SfABCC3 knockout strains increased their susceptibility to abamectin and spinosad insecticides. CONCLUSION: We provide functional evidence that in S. frugiperda these two ABCC transporters serve as receptors of Bt Cry1Fa and Cry1Ab toxins. © 2020 Society of Chemical Industry.


Assuntos
Bacillus thuringiensis , Transportadores de Cassetes de Ligação de ATP , África , Animais , Ásia , Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Endotoxinas/genética , Endotoxinas/toxicidade , Proteínas Hemolisinas/toxicidade , Larva/genética , Larva/metabolismo , Spodoptera/genética , Spodoptera/metabolismo
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