RESUMO
Previous studies have demonstrated the presence of chitinase in Bacillus velezensis through extensive genomic sequencing and experimental analyses. However, the detailed structure, functional roles, and antifungal activity of these chitinases remain poorly characterized. In this study, genomic screening identified three genes-chiA, chiB, and lpmo10-associated with chitinase degradation in B. velezensis S161. These genes encode chitinases ChiA and ChiB, and lytic polysaccharide monooxygenase LPMO10. Both ChiA and ChiB contain two CBM50 binding domains and one catalytic domain, whereas LPMO10 includes a signal peptide and a single catalytic domain. The chitinases ChiA, its truncated variant ChiA2, and ChiB were heterologously expressed in Escherichia coli. The purified enzymes efficiently degraded colloidal chitin and inhibited the spore germination of Penicillium digitatum. Notably, even after losing one CBM50 domain, the resultant enzyme, consisting of the remaining CBM50 domain and the catalytic domain, maintained its colloidal chitin hydrolysis and antifungal activity, indicating commendable stability. These results underscore the role of B. velezensis chitinases in suppressing plant pathogenic fungi and provide a solid foundation for developing and applying chitinase-based biocontrol strategies.
Assuntos
Antifúngicos , Bacillus , Quitinases , Penicillium , Antifúngicos/farmacologia , Antifúngicos/química , Bacillus/enzimologia , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Quitina/química , Quitinases/química , Quitinases/farmacologia , Escherichia coli , Penicillium/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologiaRESUMO
In this study, secretable Vip3Ag4 protein was encapsulated in Bacillus megaterium and used for quantitative bioassays, in order to determine the UV photoprotective capacity of the cell, for preventing inactivation of the insecticidal activity of the protein. The non-encapsulated and purified protein was exposed to the UV light showing a LC50 of 518 ng/cm2 against Spodoptera littoralis larvae, whereas the exposed encapsulated protein exhibited 479 ng/cm2. In addition to the capability to accumulate Vip3 proteins for the development of novel insecticidal formulates, the B. megaterium cell has demonstrated to provide moderate protection against the deleterious action of UV light.
Assuntos
Bacillus megaterium , Proteínas de Bactérias , Inseticidas , Spodoptera , Raios Ultravioleta , Bacillus megaterium/efeitos dos fármacos , Animais , Spodoptera/efeitos dos fármacos , Inseticidas/farmacologia , Proteínas de Bactérias/farmacologia , Larva/efeitos dos fármacosRESUMO
Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive1. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species1,2. Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly3,4. Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria.
Assuntos
Antibiose , Proteínas de Bactérias , Toxinas Bacterianas , Streptomyces , Antibacterianos/biossíntese , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibiose/efeitos dos fármacos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/ultraestrutura , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/farmacologia , Microscopia Crioeletrônica , Lectinas/química , Lectinas/genética , Lectinas/metabolismo , Lectinas/ultraestrutura , Testes de Sensibilidade Microbiana , Modelos Moleculares , Streptomyces/química , Streptomyces/efeitos dos fármacos , Streptomyces/genética , Streptomyces/crescimento & desenvolvimento , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Streptomyces griseus/efeitos dos fármacos , Streptomyces griseus/genética , Streptomyces griseus/crescimento & desenvolvimento , Streptomyces griseus/metabolismoRESUMO
Fungal resistance to commonly used medicines is a growing public health threat, and there is a dire need to develop new classes of antifungals. We previously described a peptide produced by Enterococcus faecalis, EntV, that restricts Candida albicans to a benign form rather than having direct fungicidal activity. Moreover, we showed that one 12-amino acid (aa) alpha helix of this peptide retained full activity, with partial activity down to the 10aa alpha helix. Using these peptides as a starting point, the current investigation sought to identify the critical features necessary for antifungal activity and to screen for new variants with enhanced activity using both biofilm and C. elegans infection assays. First, the short peptides were screened for residues with critical activity by generating alanine substitutions. Based on this information, we used synthetic molecular evolution (SME) to rationally vary the specific residues of the 10aa variant in combination to generate a library that was screened to identify variants with more potent antifungal activity than the parent template. Five gain-of-function peptides were identified. Additionally, chemical modifications to the peptides to increase stability, including substitutions of D-amino acids and hydrocarbon stapling, were investigated. The most promising peptides were additionally tested in mouse models of oropharyngeal and systemic candidiasis where their efficacy in preventing infection was demonstrated. The expectation is that these discoveries will contribute to the development of new therapeutics in the fight against antimicrobial resistant fungi. IMPORTANCE: Since the early 1980s, the incidence of disseminated life-threatening fungal infections has been on the rise. Worldwide, Candida and Cryptococcus species are among the most common agents causing these infections. Simultaneously, with this rise of clinical incidence, there has also been an increased prevalence of antifungal resistance, making treatment of these infections very difficult. For example, there are now strains of Candida auris that are resistant to all three classes of currently used antifungal drugs. In this study, we report on a strategy that allows for the development of novel antifungal agents by using synthetic molecular evolution. These discoveries demonstrate that the enhancement of antifungal activity from naturally occurring peptides is possible and can result in clinically relevant agents that have efficacy in multiple in vivo models as well as the potential for broad-spectrum activity.
Assuntos
Antifúngicos , Biofilmes , Caenorhabditis elegans , Candida albicans , Candidíase , Enterococcus faecalis , Testes de Sensibilidade Microbiana , Antifúngicos/farmacologia , Antifúngicos/química , Animais , Camundongos , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Biofilmes/efeitos dos fármacos , Candidíase/tratamento farmacológico , Candidíase/microbiologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/genética , Caenorhabditis elegans/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Modelos Animais de Doenças , Peptídeos/farmacologia , Peptídeos/genética , Peptídeos/químicaRESUMO
The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC50 equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T0 and T1 generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of ß-pore-forming toxins (ß-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC50 for CBW larvae and the significant accumulation of these toxins in the flower buds of both T0 and T1 plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.
Assuntos
Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Gossypium , Proteínas Hemolisinas , Larva , Plantas Geneticamente Modificadas , Gorgulhos , Gossypium/genética , Gossypium/parasitologia , Animais , Gorgulhos/genética , Plantas Geneticamente Modificadas/genética , Endotoxinas/genética , Endotoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/farmacologia , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/farmacologia , Larva/efeitos dos fármacos , Bacillus thuringiensis/genética , Controle Biológico de VetoresRESUMO
Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.
Assuntos
Compostos Azabicíclicos , Carbapenêmicos , Ceftazidima , Enterobacteriaceae , Enterobacteriaceae/genética , Carbapenêmicos/farmacologia , Tigeciclina/farmacologia , Sistemas de Distribuição no Hospital , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Combinação de Medicamentos , beta-Lactamases/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Cefalosporinas/farmacologia , Klebsiella pneumoniae/genética , Genótipo , Testes de Sensibilidade MicrobianaRESUMO
Bacillus thuringiensis (Bt) is widely used to produce biological pesticides. However, its persistence is limited because of ultraviolet (UV) rays. In our previous study, we found that exogenous intermediates of the urea cycle were beneficial to Bt for survival under UV stress. To further explore the effect of the urea cycle on the resistance mechanism of Bt, the rocF/argG gene, encoding arginase and argininosuccinate synthase, respectively, were knocked out and recovered in this study. After the target genes were removed, respectively, the urea cycle in the tested Bt was inhibited to varying degrees. The UV stress test showed that the urea cycle disorder could reduce the resistance of Bt under UV stress. Meanwhile, the antioxidant enzyme activities of Bt were also decreased to varying degrees due to the knockout of the target genes. All of these results revealed that the urea cycle can metabolically regulate the stress resistance of Bt.
Assuntos
Bacillus thuringiensis , Bacillus thuringiensis/genética , Ureia , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologiaRESUMO
BACKGROUND: Practical resistance of Helicoverpa zea to Cry proteins has become widespread in the US, making Vip3Aa the only effective Bacillus thuringiensis (Bt) protein for controlling this pest. Understanding the genetic basis of Vip3Aa resistance in H. zea is essential in sustaining the long-term efficacy of Vip3Aa. The objectives of this study were to characterize the inheritance of Vip3Aa resistance in four distinct field-derived H. zea strains (M1-RR, AC4-RR, R2-RR and R15-RR), and to test for shared genetic basis among these strains and a previously characterized Texas resistant strain (LT#70-RR). RESULTS: Maternal effects and sex linkage were absent, and the effective dominance level (DML) was 0.0 across Vip3Aa39 concentrations ranging from 1.0 to 31.6 µg cm-2, in all H. zea resistant strains. Mendelian monogenic model tests indicated that Vip3Aa resistance in each of the four strains was controlled by a single gene. However, interstrain complementation tests indicated that three distinct genetic loci are involved in Vip3Aa resistance in the five resistant H. zea strains: one shared by M1-RR and LT#70-RR; another shared by R2-RR and R15-RR; and a distinct one for AC4-RR. CONCLUSION: Results of this study indicate that Vip3Aa resistance in all H. zea strains was controlled by a single, recessive and autosomal gene. However, there were three distinct genetic loci associated with Vip3Aa resistance in the five resistant H. zea strains. The information generated from this study is valuable for exploring mechanisms of Vip3Aa resistance, monitoring the evolution of Vip3Aa resistance, and devising effective strategies for managing Vip3Aa resistance in H. zea. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.
Assuntos
Proteínas de Bactérias , Resistência a Medicamentos , Mariposas , Mariposas/efeitos dos fármacos , Mariposas/genética , Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Resistência a Medicamentos/genética , Controle de Pragas/métodos , Dose Letal Mediana , Teste de Complementação Genética , Genes Recessivos/genética , AnimaisRESUMO
BACKGROUND: With the increasing incidence of pest resistance to transgenic crops producing Bacillus thuringiensis (Bt) proteins in the field, elucidating the molecular basis of resistance is important for monitoring, delaying and countering pest resistance. Previous work revealed that mutation or down-regulated expression of the cadherin gene (PgCad1) is associated with pink bollworm (Pectinophora gossypiella) resistance to Cry1Ac, and 20 mutant PgCad1 alleles (r1-r20) were characterized. Here, we tested the hypothesis that the ABC transporter PgABCC2 is a functional receptor for the Bt toxin Cry1Ac and that a mutation is associated with resistance. RESULTS: We identified and characterized the first resistance allele (rC2) of PgABCC2 in the laboratory-selected Cry1Ac-resistant strain AQ-C2 of pink bollworm. The rC2 allele had a one-base deletion in exon20, resulting in a frameshift and the introduction of a premature stop codon. This resulting PgABCC2 protein had a truncated C-terminus, including the loss of the NBD2 domain. AQ-C2 exhibited 20.2-fold greater resistance to Cry1Ac than the susceptible strain, and its inheritance of Cry1Ac resistance was recessive and genetically linked to PgABCC2. When produced in cultured insect cells, recombinant wild-type and rC2 mutant PgABCC2 proteins localized within the cell plasma membrane, although substantial cytoplasmic retention was also observed for the mutant protein, while the mutant PgABCC2 caused a 13.9-fold decrease in Cry1Ac toxicity versus the wild-type PgABCC2. CONCLUSIONS: PgABCC2 is a functional receptor of Cry1Ac and the loss of its carboxyl terminus (including its NBD2 domain) confers low-level resistance to Cry1Ac in both larvae and in cultured cells. © 2024 Society of Chemical Industry.
Assuntos
Toxinas de Bacillus thuringiensis , Proteínas de Bactérias , Endotoxinas , Proteínas Hemolisinas , Resistência a Inseticidas , Mariposas , Mutação , Animais , Toxinas de Bacillus thuringiensis/farmacologia , Resistência a Inseticidas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/metabolismo , Endotoxinas/farmacologia , Endotoxinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/genética , Mariposas/genética , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Inseticidas/farmacologiaRESUMO
Inhibition of quorum sensing is considered to be an effective strategy of control and treatment of a wide range of acute and persistent infections. Pseudomonas aeruginosa is an opportunistic bacterium with a high adaptation potential that contributes to healthcare-associated infections. In the present study, the effects of the synthesized hybrid structures bearing sterically hindered phenolic and heterocyclic moieties in a single scaffold on the production of virulence factors by P. aeruginosa were determined. It has been shown that the obtained compounds significantly reduce both pyocyanin and alginate production and stimulate the biosynthesis of siderophores in vitro, which may be attributed to their iron-chelating properties. The results of docking-based inverse high-throughput virtual screening indicate that transcription regulator LasR and Cu-transporter OPRC could be potential molecular targets for these compounds. Investigation of the impact small molecules exert on the molecular mechanisms of the production of bacterial virulence factors may pave the way for the design and development of novel antibacterial agents.
Assuntos
Pseudomonas aeruginosa , Fatores de Virulência , Transativadores/farmacologia , Percepção de Quorum , Piocianina , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Antibacterianos/farmacologia , Antibacterianos/química , BiofilmesRESUMO
The peritrophic matrix (or peritrophic membrane, PM) is present in most insects where it acts as a barrier to mechanical insults and pathogens, as well as a facilitator of digestive processes. The PM is formed by the binding of structural PM proteins, referred to as peritrophins, to chitin fibrils and spans the entire midgut in lepidopterans. To investigate the role of peritrophins in a highly polyphagous lepidopteran pest, namely the cotton leafworm (Spodoptera littoralis), we generated Insect Intestinal Mucin (IIM-) and non-mucin Peritrophin (PER-) mutant strains via CRISPR/Cas9 mutagenesis. Both strains exhibited deformed PMs and retarded developmental rates. Bioassays conducted with Bacillus thuringiensis (Bt) and nucleopolyhedrovirus (SpliNPV) formulations showed that both the IIM- and PER- mutant larvae were more susceptible to these bioinsecticides compared to the wild-type (WT) larvae with intact PM. Interestingly, the provision of chitin-binding agent Calcofluor (CF) in the diet lowered the toxicity of Bt formulations in both WT and IIM- larvae and the protective effect of CF was significantly lower in PER- larvae. This suggested that the interaction of CF with PER is responsible for Bt resistance mediated by CF. In contrast, the provision of CF caused increased susceptibility to SpliNPV in both mutants and WT larvae. The study showed the importance of peritrophins in the defense against pathogens in S. littoralis and revealed novel insights into CF-mediated resistance to Cry toxin.
Assuntos
Bacillus thuringiensis , Mariposas , Nucleopoliedrovírus , Animais , Bacillus thuringiensis/metabolismo , Spodoptera/metabolismo , Nucleopoliedrovírus/metabolismo , Mariposas/metabolismo , Larva/metabolismo , Endotoxinas/farmacologia , Quitina/metabolismo , Proteínas de Bactérias/farmacologia , Proteínas Hemolisinas/farmacologiaRESUMO
A variety of coordinated host-cell responses are activated as defense mechanisms against pore-forming toxins (PFTs). Bacillus thuringiensis (Bt) is a worldwide used biopesticide whose efficacy and precise application methods limits its use to replace synthetic pesticides in agricultural settings. Here, we analyzed the intestinal defense mechanisms of two lepidopteran insect pests after intoxication with sublethal dose of Bt PFTs to find out potential functional genes. We show that larval intestinal epithelium was initially damaged by the PFTs and that larval survival was observed after intestinal epithelium regeneration. Further analyses showed that the intestinal regeneration caused by Cry9A protein is regulated through c-Jun NH (2) terminal kinase (JNK) and Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. JAK/STAT signaling regulates intestinal regeneration through proliferation and differentiation of intestinal stem cells to defend three different Bt proteins including Cry9A, Cry1F or Vip3A in both insect pests, Chilo suppressalis and Spodoptera frugiperda. Consequently, a nano-biopesticide was designed to improve pesticidal efficacy based on the combination of Stat double stranded RNA (dsRNA)-nanoparticles and Bt strain. This formulation controlled insect pests with better effect suggesting its potential use to reduce the use of synthetic pesticides in agricultural settings for pest control.
Assuntos
Bacillus thuringiensis , Praguicidas , Animais , Bacillus thuringiensis/genética , Janus Quinases/genética , Tirosina , Endotoxinas/genética , Insetos , Spodoptera/genética , Larva , Praguicidas/farmacologia , Regeneração , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/genética , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/genética , Plantas Geneticamente Modificadas , Controle Biológico de Vetores/métodosRESUMO
Bacillus thuringiensis Cry9 proteins show high insecticidal activity against different lepidopteran pests. Cry9 could be a valuable alternative to Cry1 proteins because it showed a synergistic effect with no cross-resistance. However, the pore-formation region of the Cry9 proteins is still unclear. In this study, nine mutations of certain Cry9Aa helices α3 and α4 residues resulted in a complete loss of insecticidal activity against the rice pest Chilo suppressalis; however, the protein stability and receptor binding ability of these mutants were not affected. Among these mutants, Cry9Aa-D121R, Cry9Aa-D125R, Cry9Aa-D163R, Cry9Aa-E165R, and Cry9Aa-D167R are unable to form oligomers in vitro, while the oligomers formed by Cry9Aa-R156D, Cry9Aa-R158D, and Cry9Aa-R160D are unstable and failed to insert into the membrane. These data confirmed that helices α3 and α4 of Cry9Aa are involved in oligomerization, membrane insertion, and toxicity. The knowledge of Cry9 pore-forming action may promote its application as an alternative to Cry1 insecticidal proteins.
Assuntos
Bacillus thuringiensis , Inseticidas , Animais , Bacillus thuringiensis/química , Inseticidas/química , Endotoxinas/genética , Endotoxinas/toxicidade , Endotoxinas/química , Domínios Proteicos , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/farmacologia , Proteínas de Bactérias/toxicidade , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/toxicidade , Proteínas Hemolisinas/química , Larva/metabolismoRESUMO
Sound has been shown to impact microbial behaviors. However, our understanding of the chemical and molecular mechanisms underlying these microbial responses to acoustic vibration is limited. In this study, we used untargeted metabolomics analysis to investigate the effects of 100-Hz acoustic vibration on the intra- and extracellular hydrophobic metabolites of P. aeruginosa PAO1. Our findings revealed increased levels of fatty acids and their derivatives, quinolones, and N-acylethanolamines upon sound exposure, while rhamnolipids (RLs) showed decreased levels. Further quantitative real-time polymerase chain reaction experiments showed slight downregulation of the rhlA gene (1.3-fold) and upregulation of fabY (1.5-fold), fadE (1.7-fold), and pqsA (1.4-fold) genes, which are associated with RL, fatty acid, and quinolone biosynthesis. However, no alterations in the genes related to the rpoS regulators or quorum-sensing networks were observed. Supplementing sodium oleate to P. aeruginosa cultures to simulate the effects of sound resulted in increased tolerance of P. aeruginosa in the presence of sound at 48 h, suggesting a potential novel response-tolerance correlation. In contrast, adding RL, which went against the response direction, did not affect its growth. Overall, these findings provide potential implications for the control and manipulation of virulence and bacterial characteristics for medical and industrial applications.
Assuntos
Pseudomonas aeruginosa , Vibração , Percepção de Quorum/genética , Virulência , Fatores de Virulência , Ácidos Graxos/farmacologia , Acústica , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , BiofilmesRESUMO
BACKGROUND: The commercialized Bt (Bacillus thuringiensis) crops accumulate Bt protein within cells, but the intracellular interactions of foreign protein with endogenous protein inevitably result in large or small unintended effects. In this study, the Bt gene Cry1Ca was linked with the sequences of extracellular secretion signal peptide and carbohydrate binding module 11 to constitute a fusion gene SP-Cry1Ca-CBM11, and the fusion gene driven by constitutive promoters was used for secreting and anchoring onto the cell wall to minimize unintended effects. RESULTS: The transient expression in tobacco leaves demonstrated that the fusion protein was anchored on cell walls. The Cry1Ca contents of five homozygous rice transformants of single-copy insertion were different and descended in the order leaf > root > stem. The maximum content of Cry1Ca was 17.55 µg g-1 in leaves of transformant 21H037. The bioassay results revealed that the transformants exhibited high resistance to lepidopteran pests. The corrected mortality of pink stem borer (Sesamia inferens) and striped stem borer (Chilo suppressalis) ranged from 96.33% to 100%, and from 83.32% to 100%, respectively, and the corrected mortality of rice leaf roller (Cnaphalocrocis medinalis) was 92.53%. Besides, the agronomic traits of the five transformants were normal and similar to that of the recipient, and the transformants were highly resistant to glyphosate at the germination and seedling stages. CONCLUSION: The fusion Bt protein was accumulated on cell walls and endowed the rice with high resistance to lepidopteran pests without unintended effects in agronomic traits. © 2023 Society of Chemical Industry.
Assuntos
Bacillus thuringiensis , Lepidópteros , Mariposas , Oryza , Animais , Lepidópteros/genética , Oryza/genética , Oryza/metabolismo , Endotoxinas/farmacologia , Toxinas de Bacillus thuringiensis/metabolismo , Toxinas de Bacillus thuringiensis/farmacologia , Proteínas de Bactérias/farmacologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Proteínas Hemolisinas/metabolismo , Bacillus thuringiensis/genética , Controle Biológico de Vetores/métodosRESUMO
Klebsiella pneumoniae (Kp), which is associated with hospital-acquired infections, is extensively drug-resistant (XDR), making treatment difficult. Understanding the genetic epidemiology of XDR-Kp can help determine its potential to be hypervirulent (hv) through the presence of siderophores. We characterized the genomes of 18 colistin-resistant XDR-Kp isolated from 14 patients with complicated tract infection at an Indian healthcare facility. The 18 organisms comprised the following sequence types (STs): ST14 (n = 9), ST147 (n = 5), ST231 (n = 2), ST2096 (n = 1), and ST25 (n = 1). Many patients in each ward were infected with the same ST, suggesting a common source of infection. Some patients had recurrent infections with multiple STs circulating in the ward, providing evidence of hospital transmission. ß-lactamase genes (blaCTX-M-1, blaSHV, and blaampH) were present in all isolates. blaNDM-1 was present in 15 isolates, blaOXA-1 in 16 isolates, blaTEM-1D in 13 isolates, and blaOXA-48 in 3 isolates. Disruption of mgrB by various insertion sequences was responsible for colistin resistance in 6 isolates. The most common K-type among isolates was K2 (n = 10). One XDR convergent hvKp ST2096 mutation (iuc+ybt+blaOXA-1+blaOXA-48) was associated with prolonged hospitalization. Convergent XDR-hvKp has outbreak potential, warranting effective antimicrobial stewardship and infection control.
Assuntos
Infecções por Klebsiella , Infecções Urinárias , Humanos , Colistina/farmacologia , Klebsiella pneumoniae , Antibacterianos/farmacologia , Infecções por Klebsiella/epidemiologia , beta-Lactamases/genética , beta-Lactamases/farmacologia , Infecções Urinárias/epidemiologia , Testes de Sensibilidade Microbiana , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologiaRESUMO
We prepared a series of cinnamoyl-containing furanones by an affordable and short synthesis. The nineteen compounds hold a variety of substituents including electron-donating, electron-withdrawing, bulky and meta-substituted phenyls, as well as heterocyclic rings. Compounds showed antibiofilm activity in S. aureus, K. pneumoniae and, more pronounced, against P. aeruginosa. The disruption of quorum sensing (QS) was tested using the violacein test and molecular docking predicted the antagonism of LasR as a plausible mechanism of action. The trimethoxylated and diene derivatives showed the best antibiofilm and anti-QS properties, thus becoming candidates for further modifications.
Assuntos
Lactonas , Staphylococcus aureus , Antibacterianos/farmacologia , Proteínas de Bactérias/farmacologia , Biofilmes , Lactonas/farmacologia , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa , Percepção de QuorumRESUMO
Pest insects pose a heavy burden on global agricultural industries with small molecule insecticides being predominantly used for their control. Unwanted side effects and resistance development plagues most small molecule insecticides such as the neonicotinoids, which have been reported to be harmful to honeybees. Bioinsecticides like Bacillus thuringiensis (Bt) toxins can be used as environmentally-friendly alternatives. Arachnid venoms comprise another promising source of bioinsecticides, containing a multitude of selective and potent insecticidal toxins. Unfortunately, no standardised insect models are currently available to assess the suitability of insecticidal agents under laboratory conditions. Thus, we aimed to develop a laboratory model that closely mimics field conditions by employing a leaf disk assay (LDA) for oral application of insecticidal agents in a bioassay tray format. Neonate larvae of the cotton bollworm (Helicoverpa armigera) were fed with soybean (Glycine max) leaves that were treated with different insecticidal agents. We observed dose-dependent insecticidal effects for Bt toxin and the neonicotinoid insecticide imidacloprid, with imidacloprid exhibiting a faster response. Furthermore, we identified several insecticidal arachnid venoms that were active when co-applied with sub-lethal doses of Bt toxin. We propose the H. armigera LDA as a suitable tool for assessing the insecticidal effects of insecticidal agents against lepidopterans.
Assuntos
Venenos de Artrópodes , Bacillus thuringiensis , Inseticidas , Mariposas , Neonicotinoides , Nitrocompostos , Toxinas Biológicas , Humanos , Recém-Nascido , Animais , Inseticidas/toxicidade , Glycine max , Helicoverpa armigera , Toxinas de Bacillus thuringiensis/farmacologia , Larva , Insetos , Toxinas Biológicas/farmacologia , Venenos de Artrópodes/farmacologia , Bioensaio , Folhas de Planta , Proteínas de Bactérias/farmacologia , Proteínas Hemolisinas/toxicidade , Endotoxinas , Controle Biológico de Vetores , Resistência a InseticidasRESUMO
Crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are widely used in transgenic crops to control important insect pests. Bt crops have many benefits compared with traditional broad-spectrum insecticides, including improved pest control with reduced negative impacts on off-target organisms and fewer environmental consequences. Transgenic corn and cotton producing Cry2Ab Bt toxin are used globally to control several major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Resistance to the Cry2Ab toxin and to Bt crops producing Cry2Ab is associated with mutations in the midgut ATP-binding cassette transporter ABCA2 gene in several lepidopterans. Gene-editing knockout has further shown that ABCA2 plays an important functional role in Cry2Ab intoxication. However, the precise role of ABCA2 in the mode of action of Cry2Ab has yet to be reported. Here, we used two in vitro expression systems to study the roles of the H. armigera ABCA2 (HaABCA2) protein in Cry2Ab intoxication. Cry2Ab bound to cultured Sf9 insect cells producing HaABCA2, resulting in specific and dose-dependent susceptibility to Cry2Ab. In contrast, Sf9 cells expressing recombinant mutant proteins missing at least one of the extracellular loop regions 1, 3, 4, and 6 or the intracellular loop containing nucleotide-binding domain 1 lost susceptibility to Cry2Ab, indicating these regions are important for receptor function. Consistent with these results, Xenopus laevis oocytes expressing recombinant HaABCA2 showed strong ion membrane flux in the presence of Cry2Ab, suggesting that HaABCA2 is involved in promoting pore formation during Cry2Ab intoxication. Together with previously published data, our results support HaABCA2 being an important receptor of Cry2Ab where it functions to promote intoxication in H. armigera.