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1.
Nat Commun ; 15(1): 4733, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830951

RESUMO

Polymyxins are gram-negative antibiotics that target lipid A, the conserved membrane anchor of lipopolysaccharide in the outer membrane. Despite their clinical importance, the molecular mechanisms underpinning polymyxin activity remain unresolved. Here, we use surface plasmon resonance to kinetically interrogate interactions between polymyxins and lipid A and derive a phenomenological model. Our analyses suggest a lipid A-catalyzed, three-state mechanism for polymyxins: transient binding, membrane insertion, and super-stoichiometric cluster accumulation with a long residence time. Accumulation also occurs for brevicidine, another lipid A-targeting antibacterial molecule. Lipid A modifications that impart polymyxin resistance and a non-bactericidal polymyxin derivative exhibit binding that does not evolve into long-lived species. We propose that transient binding to lipid A permeabilizes the outer membrane and cluster accumulation enables the bactericidal activity of polymyxins. These findings could establish a blueprint for discovery of lipid A-targeting antibiotics and provide a generalizable approach to study interactions with the gram-negative outer membrane.


Assuntos
Antibacterianos , Lipídeo A , Polimixina B , Ressonância de Plasmônio de Superfície , Polimixina B/farmacologia , Polimixina B/metabolismo , Lipídeo A/metabolismo , Lipídeo A/química , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Testes de Sensibilidade Microbiana , Membrana Externa Bacteriana/metabolismo , Membrana Externa Bacteriana/efeitos dos fármacos , Cinética
2.
Nat Commun ; 15(1): 4494, 2024 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-38802368

RESUMO

Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.


Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Prótons , Staphylococcus aureus , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Staphylococcus aureus/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/química , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Antibacterianos/química , Modelos Moleculares , Transporte Biológico , Sítios de Ligação , Ligação de Hidrogênio , Conformação Proteica
3.
Recent Pat Biotechnol ; 18(4): 273-287, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38817008

RESUMO

Actinomycetes are present in various terrestrial and aquatic habitats, predominantly in the soil rhizosphere, encompassing marine and freshwater ecosystems. These microorganisms exhibit characteristics that resemble both bacteria and fungi. Numerous actinomycetes exhibit a mycelial existence and undergo significant morphological transformations. These bacteria are widely recognized as biotechnologically significant microorganisms utilized for the production of secondary metabolites. In all, over 45% of all bioactive microbial metabolites are produced by actinomycetes, which are responsible for producing around 10,000 of them. The majority of actinomycetes exhibit substantial saprophytic characteristics in their natural environment, enabling them to effectively decompose a diverse range of plant and animal waste materials during the process of decomposition. Additionally, these organisms possess a sophisticated secondary metabolic system, which enables them to synthesize almost two-thirds of all naturally occurring antibiotics. Moreover, they can create a diverse array of chemical compounds with medical or agricultural applications, including anticancer, antiparasitic, and antibacterial agents. This review aims to provide an overview of the prominent biotechnological domains in which actinobacteria and their metabolites demonstrate noteworthy applicability. The graphical abstract provides a preview of the primary sections covered in this review. This paper presents a comprehensive examination of the biotechnological applications and metabolites of actinobacteria, highlighting their potential for patent innovations.


Assuntos
Actinobacteria , Bioprospecção , Patentes como Assunto , Actinobacteria/metabolismo , Bioprospecção/métodos , Biotecnologia/métodos , Metabolismo Secundário , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/biossíntese , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Microbiologia do Solo
4.
Gut Microbes ; 16(1): 2356275, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38797999

RESUMO

Multidrug-resistant microorganisms have become a major public health concern around the world. The gut microbiome is a gold mine for bioactive compounds that protect the human body from pathogens. We used a multi-omics approach that integrated whole-genome sequencing (WGS) of 74 commensal gut microbiome isolates with metabolome analysis to discover their metabolic interaction with Salmonella and other antibiotic-resistant pathogens. We evaluated differences in the functional potential of these selected isolates based on WGS annotation profiles. Furthermore, the top altered metabolites in co-culture supernatants of selected commensal gut microbiome isolates were identified including a series of dipeptides and examined for their ability to prevent the growth of various antibiotic-resistant bacteria. Our results provide compelling evidence that the gut microbiome produces metabolites, including the compound class of dipeptides that can potentially be applied for anti-infection medication, especially against antibiotic-resistant pathogens. Our established pipeline for the discovery and validation of bioactive metabolites from the gut microbiome as novel candidates for multidrug-resistant infections represents a new avenue for the discovery of antimicrobial lead structures.


Assuntos
Antibacterianos , Bactérias , Microbioma Gastrointestinal , Microbioma Gastrointestinal/efeitos dos fármacos , Humanos , Bactérias/classificação , Bactérias/metabolismo , Bactérias/isolamento & purificação , Bactérias/genética , Bactérias/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Simbiose , Metaboloma , Sequenciamento Completo do Genoma , Farmacorresistência Bacteriana Múltipla , Salmonella/efeitos dos fármacos , Salmonella/metabolismo , Salmonella/genética , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia
5.
Microb Biotechnol ; 17(5): e14487, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38801351

RESUMO

Pseudomonas aeruginosa is a notorious multidrug-resistant pathogen that poses a serious and growing threat to the worldwide public health. The expression of resistance determinants is exquisitely modulated by the abundant regulatory proteins and the intricate signal sensing and transduction systems in this pathogen. Downregulation of antibiotic influx porin proteins and upregulation of antibiotic efflux pump systems owing to mutational changes in their regulators or the presence of distinct inducing molecular signals represent two of the most efficient mechanisms that restrict intracellular antibiotic accumulation and enable P. aeruginosa to resist multiple antibiotics. Treatment of P. aeruginosa infections is extremely challenging due to the highly inducible mechanism of antibiotic resistance. This review comprehensively summarizes the regulatory networks of the major porin proteins (OprD and OprH) and efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY) that play critical roles in antibiotic influx and efflux in P. aeruginosa. It also discusses promising therapeutic approaches using safe and efficient adjuvants to enhance the efficacy of conventional antibiotics to combat multidrug-resistant P. aeruginosa by controlling the expression levels of porins and efflux pumps. This review not only highlights the complexity of the regulatory network that induces antibiotic resistance in P. aeruginosa but also provides important therapeutic implications in targeting the inducible mechanism of resistance.


Assuntos
Antibacterianos , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/genética , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Porinas/metabolismo , Porinas/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico
6.
Microbiol Spectr ; 12(6): e0354623, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38695664

RESUMO

Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases. Previously, we cloned and characterized the biochemical properties of the M23 catalytic domain of the EnpA (EnpACD) protein from Enterococcus faecalis. Unlike other enzymes within the M23 family, EnpACD demonstrates broad specificity. However, its activity is constrained under low ionic strength conditions. In this study, we present the engineering of three chimeric enzymes comprising EnpACD fused with three distinct SH3b cell wall-binding domains. These chimeras exhibit enhanced tolerance to environmental conditions and sustained activity in bovine and human serum. Furthermore, our findings demonstrate that the addition of SH3b domains influences the activity of the chimeric enzymes, thereby expanding their potential applications in combating antimicrobial resistance.IMPORTANCEThese studies demonstrate that the addition of the SH3b-binding domain to the EnpACD results in generation of chimeras with a broader tolerance to ionic strength and pH values, enabling them to remain active over a wider range of conditions. Such approach offers a relatively straightforward method for obtaining antibacterial enzymes with tailored properties and emphasizes the potential for proteins' engineering with enhanced functionality, contributing to the ongoing efforts to address antimicrobial resistance effectively.


Assuntos
Antibacterianos , Proteínas de Bactérias , Enterococcus faecalis , Engenharia de Proteínas , Concentração Osmolar , Enterococcus faecalis/genética , Enterococcus faecalis/enzimologia , Enterococcus faecalis/efeitos dos fármacos , Humanos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Animais , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , Bovinos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/química , Parede Celular/metabolismo , Parede Celular/genética , Domínio Catalítico/genética , Farmacorresistência Bacteriana/genética
7.
Microb Pathog ; 192: 106680, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38729380

RESUMO

Biocontrol of phytopathogens involving the use of bioactive compounds produced by lactic acid bacteria (LAB), is a promising approach to manage many diseases in agriculture. In this study, a lactic acid bacterium designated YB1 was isolated from fermented olives and selected for its antagonistic activity against Verticillium dahliae (V. dahliae) and Agrobacterium tumefaciens (A. tumefaciens). Based on the 16S rRNA gene nucleotide sequence analysis (1565 pb, accession number: OR714267), the new isolate YB1 bacterium was assigned as Leuconostoc mesenteroides YB1 (OR714267) strain. This bacterium produces an active peptide "bacteriocin" called BacYB1, which was purified in four steps. Matrix-assisted lasers desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) based approach was performed to identify and characterize BacYB1. The exact mass was 5470.75 Da, and the analysis of the N-terminal sequence (VTRASGASTPPGTASPFKTL) of BacYB1 revealed no significant similarity to currently available antimicrobial peptides. The BacYB1 displayed a bactericidal mode of action against A. tumefaciens. The potentiel role of BacYB1 to supress the growth of A. tumefaciens was confirmed by live-dead cells viability assay. In pot experiments, the biocontrol efficacy of BacYB1 against V. dahliae wilt on young olive trees was studied. The percentage of dead plants (PDP) and the final mean symptomes severity (FMS) of plants articifialy infected by V. dahliae and treated with the pre-purified peptide BacYB1 (preventive and curative treatments) were significantly inferior to untreated plants. Biochemical analysis of leaves of the plants has shown that polyophenols contents were highly detected in plants infected by V. dahliae and the highest contents of chlorophyl a, b and total chlorophyll were recorded in plants treated with the combination of BacYB1 with the biofertilisant Humivital. BacYB1 presents a promising alternative for the control of Verticillium wilt and crown gall diseases.


Assuntos
Agrobacterium tumefaciens , Bacteriocinas , Leuconostoc mesenteroides , Olea , Doenças das Plantas , RNA Ribossômico 16S , Agrobacterium tumefaciens/metabolismo , Bacteriocinas/farmacologia , Bacteriocinas/metabolismo , Olea/microbiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , RNA Ribossômico 16S/genética , Leuconostoc mesenteroides/metabolismo , Leuconostoc mesenteroides/genética , Agentes de Controle Biológico/metabolismo , Agentes de Controle Biológico/farmacologia , Verticillium/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Antibiose , Filogenia , Antibacterianos/farmacologia , Antibacterianos/metabolismo
8.
Microb Pathog ; 192: 106707, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38777241

RESUMO

Bacterial wilt of tomato caused by Ralstonia solanacearum is a critical soilborne disease that drastically reduces yield. In the current study, an endophytic strain NEAU-CP5 with strong antagonistic activity against R. solanacearum was isolated from tomato seeds and characterized. The strain was identified as Bacillus velezensis based on 16S rRNA gene and whole genome sequence analysis. NEAU-CP5 can secrete amylase, protease, and cellulase, and also produce known antibacterial metabolites, including cyclo (leucylprolyl), cyclo (phenylalanyl-prolyl), cyclo (Pro-Gly), 3-benzyl-2,5-piperazinedione, pentadecanoic acid, eicosane, 2-methyoic acid, isovaleric acid, dibuty phthalate, and esters of fatty acids (HFDU), which may be responsible for its strong antibacterial activity. Fourteen gene clusters associated with antibacterial properties were also identified in the whole genome sequence of NEAU-CP5. Pot experiment demonstrated that the application of 108 CFU/mL NEAU-CP5 on tomato plants significantly reduced the incidence of tomato bacterial wilt by 68.36 ± 1.67 %. NEAU-CP5 also increased the activity of defense-related enzymes (CAT, POD, PPO, SOD, and PAL) in tomato plants. This is the first report of an effective control of bacterial wilt on tomato plants by B. velezensis and highlights the potential of NEAU-CP5 as a potential biocontrol agent for the management of tomato bacterial wilt.


Assuntos
Bacillus , Filogenia , Doenças das Plantas , RNA Ribossômico 16S , Ralstonia solanacearum , Sementes , Solanum lycopersicum , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Ralstonia solanacearum/genética , Bacillus/isolamento & purificação , Bacillus/genética , Bacillus/metabolismo , Bacillus/classificação , Sementes/microbiologia , RNA Ribossômico 16S/genética , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Endófitos/isolamento & purificação , Endófitos/genética , Endófitos/metabolismo , Genoma Bacteriano , Sequenciamento Completo do Genoma , Antibiose , Família Multigênica , Amilases/metabolismo , Amilases/genética , DNA Bacteriano/genética
9.
Talanta ; 276: 126231, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38788376

RESUMO

Extracellular polymeric substances (EPS), which were an important fraction of natural organic matter (NOM), played an important role in various environmental processes. However, the heterogeneity, complexity, and dynamics of EPS make their interactions with antibiotics elusive. Using advanced multispectral technology, this study examined how EPS interacts with different concentrations of tetracycline (TC) in the soil system. Our results demonstrated that protein-like (C1), fulvic-like (C2), and humic-like (C3) fractions were identified from EPS. Two-dimensional synchronous correlation spectroscopy (2D-SF-COS) indicated that the protein-like fraction gave faster responses than the fulvic-like fraction during the TC binding process. The sequence of structural changes in EPS due to TC binding was revealed by two-dimensional Fourier Transformation Infrared correlation spectroscopy (2D-FTIR-COS) as follows: 1550 > 1660 > 1395 > 1240 > 1087 cm-1. It is noteworthy that the sensitivity of the amide group to TC has been preserved, with its intensity gradually increasing to become the primary binding site for TC. The integration of hetero-2DCOS maps with moving window 2D correlation spectroscopy (MW2DCOS) provided a unique insight into understanding the correlation between EPS fractions and functional groups during the TC binding process. Moreover, molecular docking (MD) discovered that the extracellular proteins would provide plenty of binding sites with TC through salt bridges, hydrogen bonds, and π-π base-stacking forces. With these results, systematic investigations of the dynamic changes in EPS components under different concentrations of antibiotic exposure demonstrated the advanced capabilities of multispectral technology in examining intricate interactions with EPS in the soil environment.


Assuntos
Escherichia coli , Matriz Extracelular de Substâncias Poliméricas , Simulação de Acoplamento Molecular , Tetraciclina , Tetraciclina/química , Tetraciclina/metabolismo , Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Matriz Extracelular de Substâncias Poliméricas/metabolismo , Matriz Extracelular de Substâncias Poliméricas/química , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Sítios de Ligação , Espectroscopia de Infravermelho com Transformada de Fourier
10.
J Microbiol ; 62(4): 261-275, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38816673

RESUMO

Lipopolysaccharide (LPS) is a critical component of the extracellular leaflet within the bacterial outer membrane, forming an effective physical barrier against environmental threats in Gram-negative bacteria. After LPS is synthesized and matured in the bacterial cytoplasm and the inner membrane (IM), LPS is inserted into the outer membrane (OM) through the ATP-driven LPS transport (Lpt) pathway, which is an energy-intensive process. A trans-envelope complex that contains seven Lpt proteins (LptA-LptG) is crucial for extracting LPS from the IM and transporting it across the periplasm to the OM. The last step in LPS transport involves the mediation of the LptDE complex, facilitating the insertion of LPS into the outer leaflet of the OM. As the Lpt system plays an essential role in maintaining the impermeability of the OM via LPS decoration, the interactions between these interconnected subunits, which are meticulously regulated, may be potential targets for the development of new antibiotics to combat multidrug-resistant Gram-negative bacteria. In this review, we aimed to provide an overview of current research concerning the structural interactions within the Lpt system and their implications to clarify the function and regulation of LPS transport in the overall process of OM biogenesis. Additionally, we explored studies on the development of therapeutic inhibitors of LPS transport, the factors that limit success, and future prospects.


Assuntos
Antibacterianos , Bactérias Gram-Negativas , Lipopolissacarídeos , Lipopolissacarídeos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Antibacterianos/química , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Transporte Biológico , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/química , Membrana Externa Bacteriana/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla
11.
Microbiol Spectr ; 12(6): e0427823, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38712967

RESUMO

Within the realm of Gram-negative bacteria, bacteriocins are secreted almost everywhere, and the most representative are colicin and pyocin, which are secreted by Escherichia coli and Pseudomonas aeruginosa, respectively. Signal peptides at the amino terminus of bacteriocins or ABC transporters can secrete bacteriocins, which then enter bacteria through cell membrane receptors and exert toxicity. In general, the bactericidal spectrum is usually narrow, killing only the kin or closely related species. Our previous research indicates that YPK_0952 is an effector of the third Type VI secretion system (T6SS-3) in Yersinia pseudotuberculosis. Next, we sought to determine its identity and characterize its toxicity. We found that YPK_0952 (a pyocin-like effector) can achieve intra-species and inter-species competitive advantages through both contact-dependent and contact-independent mechanisms mediated by the T6SS-3 while enhancing the intestinal colonization capacity of Y. pseudotuberculosis. We further identified YPK_0952 as a DNase dependent on Mg2+, Ni2+, Mn2+, and Co2+ bivalent metal ions, and the homologous immune protein YPK_0953 can inhibit its activity. In summary, YPK_0952 exerts toxicity by degrading nucleic acids from competing cells, and YPK_0953 prevents self-attack in Y. pseudotuberculosis.IMPORTANCEBacteriocins secreted by Gram-negative bacteria generally enter cells through specific interactions on the cell surface, resulting in a narrow bactericidal spectrum. First, we identified a new pyocin-like effector protein, YPK_0952, in the third Type VI secretion system (T6SS-3) of Yersinia pseudotuberculosis. YPK_0952 is secreted by T6SS-3 and can exert DNase activity through contact-dependent and contact-independent entry into nearby cells of the same and other species (e.g., Escherichia coli) to help Y. pseudotuberculosis to exert a competitive advantage and promote intestinal colonization. This discovery lays the foundation for an in-depth study of the different effector protein types within the T6SS and their complexity in competing interactions. At the same time, this study provides a new development for the toolbox of toxin/immune pairs for studying Gram-negative bacteriocin translocation.


Assuntos
Proteínas de Bactérias , Piocinas , Sistemas de Secreção Tipo VI , Infecções por Yersinia pseudotuberculosis , Yersinia pseudotuberculosis , Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/genética , Sistemas de Secreção Tipo VI/metabolismo , Sistemas de Secreção Tipo VI/genética , Piocinas/metabolismo , Infecções por Yersinia pseudotuberculosis/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Animais , Camundongos , Humanos , Bacteriocinas/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo
12.
Arch Microbiol ; 206(6): 268, 2024 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-38762847

RESUMO

Actinomycetes, a diverse group of bacteria with filamentous growth characteristics, have long captivated researchers and biochemists for their prolific production of secondary metabolites. Among the myriad roles played by actinomycete secondary metabolites, their historical significance in the field of biocontrol stands out prominently. The fascinating journey begins with the discovery of antibiotics, where renowned compounds like streptomycin, tetracycline, and erythromycin revolutionized medicine and agriculture. The history of biocontrol traces its roots back to the early twentieth century, when scientists recognized the potential of naturally occurring agents to combat pests and diseases. The emergence of synthetic pesticides in the mid-twentieth century temporarily overshadowed interest in biocontrol. However, with growing environmental concerns and the realization of the negative ecological impacts of chemical pesticides, the pendulum swung back towards exploring sustainable alternatives. Beyond their historical role as antibiotics, actinomycete-produced secondary metabolites encompass a rich repertoire with biopesticide potential. The classification of these compounds based on chemical structure and mode of action is highlighted, demonstrating their versatility against both plant pathogens and insect pests. Additionally, this review provides in-depth insights into how endophytic actinomycete strains play a pivotal role in biocontrol strategies. Case studies elucidate their effectiveness in inhibiting Spodoptera spp. and nematodes through the production of bioactive compounds. By unraveling the multifunctional roles of endophytic actinomycetes, this review contributes compelling narrative knowledge to the field of sustainable agriculture, emphasizing the potential of these microbial allies in crafting effective, environmentally friendly biocontrol strategies for combating agricultural pests.


Assuntos
Actinobacteria , Agricultura , Controle Biológico de Vetores , Actinobacteria/metabolismo , Animais , Agentes de Controle Biológico/metabolismo , Metabolismo Secundário , Doenças das Plantas/prevenção & controle , Doenças das Plantas/microbiologia , Doenças das Plantas/parasitologia , Praguicidas/metabolismo , Spodoptera/microbiologia , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Nematoides/microbiologia , Endófitos/metabolismo
13.
J Hazard Mater ; 472: 134555, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38728864

RESUMO

This study aimed to isolate marine bacteria to investigate their stress response, inhibition mechanisms, and degradation processes under high-load conditions of salinity and enrofloxacin (ENR). The results demonstrated that marine bacteria exhibited efficient pollutant removal efficiency even under high ENR stress (up to 10 mg/L), with chemical oxygen demand (COD), total phosphorus (TP), total nitrogen (TN) and ENR removal efficiencies reaching approximately 88%, 83%, 61%, and 73%, respectively. The predominant families of marine bacteria were Bacillaceae (50.46%), Alcanivoracaceae (32.30%), and Rhodobacteraceae (13.36%). They responded to ENR removal by altering cell membrane properties, stimulating the activity of xenobiotic-metabolizing enzymes and antioxidant systems, and mitigating ENR stress through the secretion of extracellular polymeric substance (EPS). The marine bacteria exhibited robust adaptability to environmental factors and effective detoxification of ENR, simultaneously removing carbon, nitrogen, phosphorus, and antibiotics from the wastewater. The attapulgite carrier enhanced the bacteria's resistance to the environment. When treating actual mariculture wastewater, the removal efficiencies of COD and TN exceeded 80%, TP removal efficiency exceeded 90%, and ENR removal efficiency approached 100%, significantly higher than reported values in similar salinity reactors. Combining the constructed physical and mathematical models of tolerant bacterial, this study will promote the practical implementation of marine bacterial-based biotechnologies in high-loading saline wastewater treatment.


Assuntos
Antibacterianos , Enrofloxacina , Nitrogênio , Fósforo , Águas Residuárias , Poluentes Químicos da Água , Águas Residuárias/química , Enrofloxacina/metabolismo , Poluentes Químicos da Água/metabolismo , Antibacterianos/metabolismo , Fósforo/metabolismo , Fósforo/química , Nitrogênio/metabolismo , Biodegradação Ambiental , Bactérias/metabolismo , Aquicultura , Eliminação de Resíduos Líquidos/métodos
14.
J Phys Chem B ; 128(20): 4911-4921, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38736363

RESUMO

To combat surging multidrug-resistant Gram-negative bacterial infections, better strategies to improve the efficacy of existing drugs are critical. Because the dual membrane cell envelope is the first line of defense for these bacteria, it is crucial to understand the permeation properties of the drugs through it. Our recent study shows that isosmotic conditions prevent drug permeation inside Gram-negative bacteria, Escherichia coli, while hypoosmotic stress enhances the process. Here, we unravel the reason behind such differential drug penetration. Specifically, we dissect the roles of electrostatic screening and low membrane permeability in the penetration failure of drugs under osmotically balanced conditions. We compare the transport of a quaternary ammonium compound malachite green in the presence of an electrolyte (NaCl) and a wide variety of commonly used organic osmolytes, e.g., sucrose, proline, glycerol, sorbitol, and urea. These osmolytes of different membrane permeability (i.e., nonpermeable sucrose and NaCl, freely permeable urea and glycerol, and partially permeable proline and sorbitol) clarify the role of osmotic stress in cell envelope permeability. The results showcase that under balanced osmotic conditions, drug molecules fail to penetrate inside E. coli cells because of low membrane permeabilities and not because of electrostatic screening imposed by the osmolytes. Contribution of the electrostatic interactions, however, cannot be completely overruled as at osmotically imbalanced conditions, drug transport across the bacterial subcellular compartments is found to be dependent on the osmolytes used.


Assuntos
Permeabilidade da Membrana Celular , Escherichia coli , Pressão Osmótica , Eletricidade Estática , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Transporte Biológico , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Membrana Celular/metabolismo , Membrana Celular/química
15.
Biotechnol J ; 19(5): e2400039, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38797723

RESUMO

Industrial production of bioactive compounds from actinobacteria, such as erythromycin and its derivatives, faces challenges in achieving optimal yields. To this end, the Design-Build-Test-Learn (DBTL) framework, a systematic metabolic engineering approach, was employed to enhance erythromycin production in Saccharopolyspora erythraea (S. erythraea) E3 strain. A genetically modified strain, S. erythraea E3-CymRP21-dcas9-sucC (S. erythraea CS), was developed by suppressing the sucC gene using an inducible promoter and dcas9 protein. The strain exhibited improved erythromycin synthesis, attributed to enhanced precursor synthesis and increased NADPH availability. Transcriptomic and metabolomic analyses revealed altered central carbon metabolism, amino acid metabolism, energy metabolism, and co-factor/vitamin metabolism in CS. Augmented amino acid metabolism led to nitrogen depletion, potentially causing cellular autolysis during later fermentation stages. By refining the fermentation process through ammonium sulfate supplementation, erythromycin yield reached 1125.66 mg L-1, a 43.5% increase. The results demonstrate the power of the DBTL methodology in optimizing erythromycin production, shedding light on its potential for revolutionizing antibiotic manufacturing in response to the global challenge of antibiotic resistance.


Assuntos
Eritromicina , Fermentação , Engenharia Metabólica , Saccharopolyspora , Eritromicina/biossíntese , Engenharia Metabólica/métodos , Saccharopolyspora/genética , Saccharopolyspora/metabolismo , Antibacterianos/biossíntese , Antibacterianos/metabolismo
16.
Appl Environ Microbiol ; 90(5): e0057224, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38700332

RESUMO

Multi-resistant bacteria are a rapidly emerging threat to modern medicine. It is thus essential to identify and validate novel antibacterial targets that promise high robustness against resistance-mediating mutations. This can be achieved by simultaneously targeting several conserved function-determining protein-protein interactions in enzyme complexes from prokaryotic primary metabolism. Here, we selected two evolutionary related glutamine amidotransferase complexes, aminodeoxychorismate synthase and anthranilate synthase, that are required for the biosynthesis of folate and tryptophan in most prokaryotic organisms. Both enzymes rely on the interplay of a glutaminase and a synthase subunit that is conferred by a highly conserved subunit interface. Consequently, inhibiting subunit association in both enzymes by one competing bispecific inhibitor has the potential to suppress bacterial proliferation. We comprehensively verified two conserved interface hot-spot residues as potential inhibitor-binding sites in vitro by demonstrating their crucial role in subunit association and enzymatic activity. For in vivo target validation, we generated genomically modified Escherichia coli strains in which subunit association was disrupted by modifying these central interface residues. The growth of such strains was drastically retarded on liquid and solid minimal medium due to a lack of folate and tryptophan. Remarkably, the bacteriostatic effect was observed even in the presence of heat-inactivated human plasma, demonstrating that accessible host metabolite concentrations do not compensate for the lack of folate and tryptophan within the tested bacterial cells. We conclude that a potential inhibitor targeting both enzyme complexes will be effective against a broad spectrum of pathogens and offer increased resilience against antibiotic resistance. IMPORTANCE: Antibiotics are indispensable for the treatment of bacterial infections in human and veterinary medicine and are thus a major pillar of modern medicine. However, the exposure of bacteria to antibiotics generates an unintentional selective pressure on bacterial assemblies that over time promotes the development or acquisition of resistance mechanisms, allowing pathogens to escape the treatment. In that manner, humanity is in an ever-lasting race with pathogens to come up with new treatment options before resistances emerge. In general, antibiotics with novel modes of action require more complex pathogen adaptations as compared to chemical derivates of existing entities, thus delaying the emergence of resistance. In this contribution, we use modified Escherichia coli strains to validate two novel targets required for folate and tryptophan biosynthesis that can potentially be targeted by one and the same bispecific protein-protein interaction inhibitor and promise increased robustness against bacterial resistances.


Assuntos
Antranilato Sintase , Antibacterianos , Escherichia coli , Antranilato Sintase/metabolismo , Antranilato Sintase/genética , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Transaminases/metabolismo , Transaminases/genética , Transaminases/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Triptofano/metabolismo , Inibidores Enzimáticos/farmacologia
17.
Proc Natl Acad Sci U S A ; 121(20): e2318855121, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38709926

RESUMO

TipA, a MerR family transcription factor from Streptomyces lividans, promotes antibiotic resistance by sequestering broad-spectrum thiopeptide-based antibiotics, thus counteracting their inhibitory effect on ribosomes. TipAS, a minimal binding motif which is expressed as an isoform of TipA, harbors a partially disordered N-terminal subdomain that folds upon binding multiple antibiotics. The extent and nature of the underlying molecular heterogeneity in TipAS that shapes its promiscuous folding-function landscape is an open question and is critical for understanding antibiotic-sequestration mechanisms. Here, combining equilibrium and time-resolved experiments, statistical modeling, and simulations, we show that the TipAS native ensemble exhibits a pre-equilibrium between binding-incompetent and binding-competent substates, with the fully folded state appearing only as an excited state under physiological conditions. The binding-competent state characterized by a partially structured N-terminal subdomain loses structure progressively in the physiological range of temperatures, swells on temperature increase, and displays slow conformational exchange across multiple conformations. Binding to the bactericidal antibiotic thiostrepton follows a combination of induced-fit and conformational-selection-like mechanisms, via partial binding and concomitant stabilization of the binding-competent substate. These ensemble features are evolutionarily conserved across orthologs from select bacteria that infect humans, underscoring the functional role of partial disorder in the native ensemble of antibiotic-sequestering proteins belonging to the MerR family.


Assuntos
Antibacterianos , Proteínas de Bactérias , Dobramento de Proteína , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Streptomyces lividans/metabolismo , Streptomyces lividans/genética , Ligação Proteica , Conformação Proteica , Modelos Moleculares , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química
18.
Curr Microbiol ; 81(7): 195, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38809483

RESUMO

The endolichenic fungi are an unexplored group of organisms for the production of bioactive secondary metabolites. The aim of the present study is to determine the antibacterial potential of endolichenic fungi isolated from genus Parmotrema. The study is continuation of our previous work, wherein a total of 73 endolichenic fungi were isolated from the lichenized fungi, which resulted in 47 species under 23 genera. All the isolated endolichenic fungi were screened for preliminary antibacterial activity. Five endolichenic fungi-Daldinia eschscholtzii, Nemania diffusa, Preussia sp., Trichoderma sp. and Xylaria feejeensis, were selected for further antibacterial activity by disc diffusion method. The zone of inhibition ranged from 14.3 ± 0.1 to 23.2 ± 0.1. The chemical composition of the selected endolichenic fungi was analysed through GC-MS, which yielded a total of 108 compounds from all the selected five endolichenic fungi. Diethyl phthalate, 1-hexadecanol, dibutyl phthalate, n-tetracosanol-1, 1-nonadecene, pyrrol[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methyl) and tetratetracontane were found to be common compounds among one or the other endolichenic fungi, which possibly were responsible for antibacterial activity. GC-MS data were further analysed through Principal Component Analysis which showed D. eschscholtzii to be with unique pattern of expression of metabolites. Compound confirmation test revealed coumaric acid to be responsible for antibacterial activity in D. eschscholtzii. So, the study proves that endolichenic fungi that inhabit lichenized fungal thalli could be a source of potential antibacterial compounds.


Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Metabolismo Secundário , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Líquens/microbiologia , Líquens/química , Bactérias/efeitos dos fármacos , Bactérias/classificação , Bactérias/metabolismo , Ascomicetos/metabolismo , Ascomicetos/química , Cromatografia Gasosa-Espectrometria de Massas
19.
J Hazard Mater ; 472: 134521, 2024 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-38718513

RESUMO

Norfloxacin (NOR) is widely used in medicine and animal husbandry, but its accumulation in the environment poses a substantial threat to ecological and human health. Traditional physical, chemical, and rudimentary biological methods often fall short in mitigating NOR contamination, necessitating innovative biological approaches. This study proposes an engineered bacterial consortium found in marine sediment as a strategy to enhance NOR degradation through inter-strain co-metabolism of diverse substrates. Strategically supplementing the engineered bacterial consortium with exogenous carbon sources and metal ions boosted the activity of key degradation enzymes like laccase, manganese peroxidase, and dehydrogenase. Iron and amino acids demonstrated synergistic effects, resulting in a remarkable 70.8% reduction in NOR levels. The innovative application of molecular docking elucidated enzyme interactions with NOR, uncovering potential biodegradation mechanisms. Quantitative assessment reinforced the efficiency of NOR degradation within the engineered bacterial consortium. Four metabolic routes are herein proposed: acetylation, defluorination, ring scission, and hydroxylation. Notably, this study discloses distinctive, co-operative metabolic pathways for NOR degradation within the specific microbial community. These findings provide new ways of understanding and investigating the bioremediation potential of NOR contaminants, which may lead to the development of more sustainable and effective environmental management strategies.


Assuntos
Biodegradação Ambiental , Simulação de Acoplamento Molecular , Norfloxacino , Norfloxacino/metabolismo , Antibacterianos/metabolismo , Antibacterianos/química , Redes e Vias Metabólicas , Bactérias/metabolismo , Sedimentos Geológicos/microbiologia , Sedimentos Geológicos/química , Consórcios Microbianos , Poluentes Químicos da Água/metabolismo , Poluentes Químicos da Água/química
20.
Int J Antimicrob Agents ; 63(6): 107171, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38588869

RESUMO

OBJECTIVES: Stenotrophomonas spp. intrinsically resistant to many ß-lactam antibiotics are found throughout the environment. CESS-1 identified in Stenotrophomonas sp. KCTC 12332 is an uncharacterized class A ß-lactamase. The goal of this study was to reveal biochemical and structural characteristics of CESS-1. METHODS: The hydrolytic activities of CESS-1 towards penicillins (penicillin G and ampicillin), cephalosporins (cephalexin, cefaclor, and cefotaxime), and carbapenems (imipenem and meropenem) was spectrophotometrically monitored. Structural information on E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin were determined by X-ray crystallography. RESULTS: CESS-1 displayed hydrolytic activities toward penicillins and cephalosporins, with negligible activity toward carbapenems. Although cefaclor, cephalexin, and ampicillin have similar structures with identical R1 side chains, the catalytic parameters of CESS-1 toward them were distinct. The kcat values for cefaclor, cephalexin, and ampicillin were 1249.6 s-1, 204.3 s-1, and 69.8 s-1, respectively, with the accompanying KM values of 287.6 µM, 236.7 µM, and 28.8 µM, respectively. CONCLUSIONS: CESS-1 was able to discriminate between cefaclor and cephalexin with a single structural difference at C3 position: -Cl (cefaclor) and -CH3 (cephalexin). Structural comparisons among three E166Q mutants of CESS-1 acylated by cefaclor, cephalexin, or ampicillin, revealed that cooperative positional changes in the R1 side chain of substrates and their interaction with the ß5-ß6 loop affect the distance between Asn170 and the deacylating water at the acyl-enzyme intermediate state. This is directly associated with the differential hydrolytic activities of CESS-1 toward the three structurally similar ß-lactam antibiotics.


Assuntos
Stenotrophomonas , beta-Lactamases , beta-Lactamases/genética , beta-Lactamases/química , beta-Lactamases/metabolismo , Especificidade por Substrato , Cristalografia por Raios X , Stenotrophomonas/genética , Stenotrophomonas/enzimologia , Stenotrophomonas/metabolismo , Stenotrophomonas/química , Hidrólise , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Carbapenêmicos/farmacologia , Carbapenêmicos/metabolismo , Cefalosporinas/metabolismo , Cefalosporinas/farmacologia , Penicilinas/metabolismo , Penicilinas/farmacologia , Cinética
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