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Medicinal plants belonging to the genus Berberis may be considered an interesting source of drugs to counteract the problem of antimicrobial multiresistance. The important properties associated with this genus are mainly due to the presence of berberine, an alkaloid with a benzyltetrahydroisoquinoline structure. Berberine is active against both Gram-negative and Gram-positive bacteria, influencing DNA duplication, RNA transcription, protein synthesis, and the integrity of the cell surface structure. Countless studies have shown the enhancement of these beneficial effects following the synthesis of different berberine analogues. Recently, a possible interaction between berberine derivatives and the FtsZ protein was predicted through molecular docking simulations. FtsZ is a highly conserved protein essential for the first step of cell division in bacteria. The importance of FtsZ for the growth of numerous bacterial species and its high conservation make it a perfect candidate for the development of broad-spectrum inhibitors. In this work, we investigate the inhibition mechanisms of the recombinant FtsZ of Escherichia coli by different N-arylmethyl benzodioxolethylamines as berberine simplified analogues appropriately designed to evaluate the effect of structural changes on the interaction with the enzyme. All the compounds determine the inhibition of FtsZ GTPase activity by different mechanisms. The tertiary amine 1c proved to be the best competitive inhibitor, as it causes a remarkable increase in FtsZ Km (at 40 µM) and a drastic reduction in its assembly capabilities. Moreover, a fluorescence spectroscopic analysis carried out on 1c demonstrated its strong interaction with FtsZ (Kd = 26.6 nM). The in vitro results were in agreement with docking simulation studies.
Asunto(s)
Berberina , Proteínas del Citoesqueleto , Proteínas del Citoesqueleto/metabolismo , Simulación del Acoplamiento Molecular , Berberina/química , Escherichia coli/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Recombinantes/metabolismo , Antibacterianos/farmacologíaRESUMEN
Antibiotics are commonly used to treat pathogenic bacteria, but their prolonged use contributes to the development and spread of drug-resistant microorganisms raising the challenge to find new alternative drugs. Antimicrobial peptides (AMPs) are small/medium molecules ranging 10-60 residues synthesized by all living organisms and playing important roles in the defense systems. These features, together with the inability of microorganisms to develop resistance against the majority of AMPs, suggest that these molecules might represent effective alternatives to classical antibiotics. Because of their high biodiversity, with over one million described species, and their ability to live in hostile environments, insects represent the largest source of these molecules. However, production of insect AMPs in native forms is challenging. In this work we investigate a defensin-like antimicrobial peptide identified in the Hermetia illucens insect through a combination of transcriptomics and bioinformatics approaches. The C-15867 AMP was produced by recombinant DNA technology as a glutathione S-transferase (GST) fusion peptide and purified by affinity chromatography. The free peptide was then obtained by thrombin proteolysis and structurally characterized by mass spectrometry and circular dichroism analyses. The antibacterial activity of the C-15867 peptide was evaluated in vivo by determination of the minimum inhibitory concentration (MIC). Finally, crystal violet assays and SEM analyses suggested disruption of the cell membrane architecture and pore formation with leaking of cytosolic material.
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Several alkylating agents that either occur in the environment or are self-produced can cause DNA-damaging injuries in bacterial cells. Therefore, all microorganisms have developed repair systems that are able to counteract DNA alkylation damage. The adaptive response to alkylation stress in Escherichia coli consists of the Ada operon, which has been widely described; however, the homologous system in Mycobacterium tuberculosis (MTB) has been shown to have a different genetic organization but it is still largely unknown. In order to describe the defense system of MTB, we first investigated the proteins involved in the repair mechanism in the homologous non-pathogenic mycobacterium M. smegmatis. Ogt, Ada-AlkA and FadE8 proteins were recombinantly produced, purified and characterized. The biological role of Ogt was examined using proteomic experiments to identify its protein partners in vivo under stress conditions. Our results suggested the formation of a functional complex between Ogt and Ada-AlkA, which was confirmed both in silico by docking calculations and by gel filtration chromatography. We propose that this stable association allows the complex to fulfill the biological roles exerted by Ada in the homologous E. coli system. Finally, FadE8 was demonstrated to be structurally and functionally related to its E. coli homologous, AidB.
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Acil-CoA Deshidrogenasa/química , Proteínas Bacterianas/química , Reparación del ADN , ADN Bacteriano/genética , Metiltransferasas/química , Mycobacterium smegmatis/genética , Acil-CoA Deshidrogenasa/genética , Acil-CoA Deshidrogenasa/metabolismo , Alquilantes/farmacología , Alquilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cromosomas Bacterianos/química , Clonación Molecular , Daño del ADN , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Metiltransferasas/genética , Metiltransferasas/metabolismo , Simulación del Acoplamiento Molecular , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteómica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Biofilms consist of a complex microbial community adhering to biotic or abiotic surfaces and enclosed within a protein/polysaccharide self-produced matrix. The formation of this structure represents the most important adaptive mechanism that leads to antibacterial resistance, and therefore, closely connected to pathogenicity. Antimicrobial peptides (AMPs) could represent attractive candidates for the design of new antibiotics because of their specific characteristics. AMPs show a broad activity spectrum, a relative selectivity towards their targets (microbial membranes), the ability to act on both proliferative and quiescent cells, a rapid mechanism of action, and above all, a low propensity for developing resistance. This article investigates the effect at subMIC concentrations of Temporin-L (TL) on biofilm formation in Pseudomonas fluorescens (P. fluorescens) both in static and dynamic conditions, showing that TL displays antibiofilm properties. Biofilm formation in static conditions was analyzed by the Crystal Violet assay. Investigation of biofilms in dynamic conditions was performed in a commercial microfluidic device consisting of a microflow chamber to simulate real flow conditions in the human body. Biofilm morphology was examined using Confocal Laser Scanning Microscopy and quantified via image analysis. The investigation of TL effects on P. fluorescens showed that when subMIC concentrations of this peptide were added during bacterial growth, TL exerted antibiofilm activity, impairing biofilm formation both in static and dynamic conditions. Moreover, TL also affects mature biofilm as confocal microscopy analyses showed that a large portion of preformed biofilm architecture was clearly perturbed by the peptide addition with a significative decrease of all the biofilm surface properties and the overall biomass. Finally, in these conditions, TL did not affect bacterial cells as the live/dead cell ratio remained unchanged without any increase in damaged cells, confirming an actual antibiofilm activity of the peptide.
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Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Polisacáridos Bacterianos/química , Pseudomonas fluorescens/efectos de los fármacos , Antibacterianos/farmacología , Adhesión Bacteriana/efectos de los fármacos , Biomasa , Farmacorresistencia Bacteriana/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Microfluídica , Microscopía Confocal , Polímeros/química , Resistencia al Corte , Estrés Mecánico , Propiedades de SuperficieRESUMEN
Prothoracicotropic hormone (PTTH) is a neuropeptide that triggers a cascade of events within the prothoracic gland (PG) cells, leading to the activation of all the crucial enzymes involved in ecdysone biosynthesis, the main insect steroid hormone. Studies concerning ecdysteroidogenesis predicted PTTH action using brain extract (BE), consisting in a complex mixture in which some components positively or negatively interfere with PTTH-stimulated ecdysteroidogenesis. Consequently, the integration of these opposing factors in steroidogenic tissues leads to a complex secretory pattern. A recombinant form of prothoracicotropic hormone (rPTTH) from the tobacco budworm Heliothis virescens (F.) (Lepidoptera: Noctuidae) was expressed and purified to perform in vitro tests in a standard and repeatable manner. A characterization of rPTTH primary and secondary structures was performed. The ability of rPTTH and H. virescens BE to stimulate ecdysteroidogenesis was investigated on the third day of fifth larval stage. rPTTH activity was compared with the BE mixture by enzyme immunoassay and western blot, revealing that they equally stimulate the production of significant amount of ecdysone, through a transduction cascade that includes the TOR pathway, by the phosphorylation of 4E binding protein (4E-BP) and S6 kinase (S6K), the main targets of TOR protein. The results of these experiments suggest the importance of obtaining a functional pure hormone to perform further studies, not depending on the crude brain extract, composed by different elements and susceptible to different uncontrollable variables.
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Ecdisteroides/biosíntesis , Hormonas de Insectos/farmacología , Mariposas Nocturnas/metabolismo , Extractos de Tejidos/farmacología , Animales , Encéfalo , Hormonas de Insectos/aislamiento & purificación , Mariposas Nocturnas/efectos de los fármacosRESUMEN
PRUNE is a member of the DHH (Asp-His-His) phosphoesterase protein superfamily of molecules important for cell motility, and implicated in cancer progression. Here we investigated multiple families from Oman, India, Iran and Italy with individuals affected by a new autosomal recessive neurodevelopmental and degenerative disorder in which the cardinal features include primary microcephaly and profound global developmental delay. Our genetic studies identified biallelic mutations of PRUNE1 as responsible. Our functional assays of disease-associated variant alleles revealed impaired microtubule polymerization, as well as cell migration and proliferation properties, of mutant PRUNE. Additionally, our studies also highlight a potential new role for PRUNE during microtubule polymerization, which is essential for the cytoskeletal rearrangements that occur during cellular division and proliferation. Together these studies define PRUNE as a molecule fundamental for normal human cortical development and define cellular and clinical consequences associated with PRUNE mutation.
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Encéfalo/crecimiento & desarrollo , Proteínas Portadoras/genética , Discapacidades del Desarrollo/genética , Microcefalia/genética , Adolescente , Diferenciación Celular/genética , Movimiento Celular/genética , Corteza Cerebral/crecimiento & desarrollo , Niño , Preescolar , Citoesqueleto/genética , Citoesqueleto/ultraestructura , Femenino , Genes Recesivos , Trastornos Heredodegenerativos del Sistema Nervioso/genética , Humanos , Lactante , Masculino , Microtúbulos/genética , Microtúbulos/ultraestructura , Mutación/genética , Linaje , Monoéster Fosfórico Hidrolasas , Adulto JovenRESUMEN
The development of new drug delivery systems for targeted chemotherapy release in cancer cells represents a very promising tool. In this contest, protein-based nanocages have considerable potential as drug delivery devices. Notably, ferritin has emerged as an excellent candidate due to its unique architecture, surface properties and high biocompatibility. A promising strategy might then involve ferritin cargos for specifical release of AntiMicrobial Peptides endowed with anticancer activity to cancer cells. In this paper, we encapsulated the TRIL analogue of Temporin-L peptide within a ferritin nanocage and evaluated the cargo biological properties. The results demonstrated a reduced haemolytic activity of the peptide and a selective cytotoxicity activity on cancer cells likely mediated by oxidative stress while having no effects on non-tumoral cells. The combination of the properties of ferritin with TRIL, might open up the way to the development of novel peptide delivery systems for future pharmaceutical applications.
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Ferritinas , Péptidos , Ferritinas/química , Péptidos/farmacología , Péptidos/química , Sistemas de Liberación de Medicamentos/métodosRESUMEN
For their easy and high-yield recombinant production, their high stability in a wide range of physico-chemical conditions and their characteristic hollow structure, ferritins (Fts) are considered useful scaffolds to encapsulate bioactive molecules. Notably, for the absence of immunogenicity and the selective interaction with tumor cells, the nanocages constituted by the heavy chain of the human variant of ferritin (hHFt) are optimal candidates for the delivery of anti-cancer drugs. hHFt nanocages can be disassembled and reassembled in vitro to allow the loading of cargo molecules, however the currently available protocols present some relevant drawbacks. Indeed, protein disassembly is achieved by exposure to extreme pH (either acidic or alkaline), followed by incubation at neutral pH to allow reassembly, but the final protein recovery and homogeneity are not satisfactory. Moreover, the exposure to extreme pH may affect the structure of the molecule to be loaded. In this paper, we report an alternative, efficient and reproducible procedure to reversibly disassemble hHFt under mild pH conditions. We demonstrate that a small amount of sodium dodecyl sulfate (SDS) is sufficient to disassemble the nanocage, which quantitatively reassembles upon SDS removal. Electron microscopy and X-ray crystallography show that the reassembled protein is identical to the untreated one. The newly developed procedure was used to encapsulate two small molecules. When compared to the existing disassembly/reassembly procedures, our approach can be applied in a wide range of pH values and temperatures, is compatible with a larger number of cargos and allows a higher protein recovery.
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Introduction: The increase in bacterial strains resistant to conventional antibiotics is an alarming problem for human health and could lead to pandemics in the future. Among bacterial pathogens responsible for a large variety of severe infections there is Pseudomonas aeruginosa. Therefore, there is an urgent need for new molecules with antimicrobial activity or that can act as adjuvants of antibiotics already in use. In this scenario, antimicrobial peptides (AMPs) hold great promise. Recently, we characterized a frog-skin AMP derived from esculentin-1a, namely Esc(1-21)-1c, endowed with antipseudomonal activity without being cytotoxic to human cells. Methods: The combinatorial effect of the peptide and antibiotics was investigated through the checkerboard assay, differential proteomic and transcriptional analysis. Results: Here, we found that Esc(1-21)-1c can synergistically inhibit the growth of P. aeruginosa cells with three different antibiotics, including tetracycline. We therefore investigated the underlying mechanism implemented by the peptide using a differential proteomic approach. The data revealed a significant decrease in the production of three proteins belonging to the MexAB-OprM efflux pump upon treatment with sub-inhibitory concentration of Esc(1-21)-1c. Down-regulation of these proteins was confirmed by transcriptional analysis and direct measurement of their relative levels in bacterial cells by tandem mass spectrometry analysis in multiple reaction monitoring scan mode. Conclusion: These evidences suggest that treatment with Esc(1-21)-1c in combination with antibiotics would increase the intracellular drug content making bacteria more susceptible to the antibiotic. Overall, these results highlight the importance of characterizing new molecules able to synergize with conventional antibiotics, paving the way for the development of alternative therapeutic strategies based on AMP/antibiotic formulations to counteract the emergence of resistant bacterial strains and increase the use of "old" antibiotics in medical practice.
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Both nanostructure and multivalency enhance the biological activities of antimicrobial peptides (AMPs), whose mechanism of action is cooperative. In addition, the efficacy of a particular AMP should benefit from a steady concentration at the local place of action and, therefore, from a slow release after a dynamic repository. In the context of emerging multi-resistant bacterial infections and the urgent need for novel and effective antimicrobial drugs, we tested these concepts through the engineering of four AMPs into supramolecular complexes as pharmacological entities. For that purpose, GWH1, T22, Pt5, and PaD, produced as GFP or human nidogen-based His-tagged fusion proteins, were engineered as self-assembling oligomeric nanoparticles ranging from 10 to 70 nm and further packaged into nanoparticle-leaking submicron granules. Since these materials slowly release functional nanoparticles during their time-sustained unpacking, they are suitable for use as drug depots in vivo. In this context, a particular AMP version (GWH1-NIDO-H6) was selected for in vivo validation in a zebrafish model of a complex bacterial infection. The GWH1-NIDO-H6-secreting protein granules are protective in zebrafish against infection by the multi-resistant bacterium Stenotrophomonas maltophilia, proving the potential of innovative formulations based on nanostructured and slowly released recombinant AMPs in the fight against bacterial infections.
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Antimicrobial peptides (AMPs) are a unique and diverse group of molecules endowed with a broad spectrum of antibiotics properties that are being considered as new alternative therapeutic agents. Most of these peptides are membrane-active molecules, killing bacteria by membrane disruption. However, recently an increasing number of AMPs was shown to enter bacterial cells and target intracellular processes fundamental for bacterial life. In this paper we investigated the mechanism of action of Maganin-2 (Mag-2), a well-known antimicrobial peptide isolated from the African clawed frog Xenopus laevis, by functional proteomic approaches. Several proteins belonging to E. coli macromolecular membrane complexes were identified as Mag-2 putative interactors. Among these, we focused our attention on BamA a membrane protein belonging to the BAM complex responsible for the folding and insertion of nascent ß-barrel Outer Membrane Proteins (OMPs) in the outer membrane. In silico predictions by molecular modelling, in vitro fluorescence binding and Light Scattering experiments carried out using a recombinant form of BamA confirmed the formation of a stable Mag-2/BamA complex and indicated a high affinity of the peptide for BamA. Functional implications of this interactions were investigated by two alternative and complementary approaches. The amount of outer membrane proteins OmpA and OmpF produced in E. coli following Mag-2 incubation were evaluated by both western blot analysis and quantitative tandem mass spectrometry in Multiple Reaction Monitoring scan mode. In both experiments a gradual decrease in outer membrane proteins production with time was observed as a consequence of Mag-2 treatment. These results suggested BamA as a possible good target for the rational design of new antibiotics since this protein is responsible for a crucial biological event of bacterial life and is absent in humans.
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The research of new therapeutic agents to fight bacterial infections has recently focused on the investigation of antimicrobial peptides (AMPs), the most common weapon that all organisms produce to prevent invasion by external pathogens. Among AMPs, the amphibian Temporins constitute a well-known family with high antibacterial properties against Gram-positive and Gram-negative bacteria. In particular, Temporin-L was shown to affect bacterial cell division by inhibiting FtsZ, a tubulin-like protein involved in the crucial step of Z-ring formation at the beginning of the division process. As FtsZ represents a leading target for new antibacterial compounds, in this paper we investigated in detail the interaction of Temporin L with Escherichia coli FtsZ and designed two TL analogues in an attempt to increase peptide-protein interactions and to better understand the structural determinants leading to FtsZ inhibition. The results demonstrated that the TL analogues improved their binding to FtsZ, originating stable protein-peptide complexes. Functional studies showed that both peptides were endowed with a high capability of inhibiting both the enzymatic and polymerization activities of the protein. Moreover, the TL analogues were able to inhibit bacterial growth at low micromolar concentrations. These observations may open up the way to the development of novel peptide or peptidomimetic drugs tailored to bind FtsZ, hampering a crucial process of bacterial life that might be proposed for future pharmaceutical applications.
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Berberine, the main bioactive component of many medicinal plants belonging to various genera such as Berberis, Coptis, and Hydrastis is a multifunctional compound. Among the numerous interesting biological properties of berberine is broad antimicrobial activity including a range of Gram-positive and Gram-negative bacteria. With the aim of identifying berberine analogues possibly endowed with higher lead-likeness and easier synthetic access, the molecular simplification approach was applied to the secondary metabolite and a series of analogues were prepared and screened for their antimicrobial activity against Gram-positive and Gram-negative bacterial test species. Rewardingly, the berberine simplified analogues displayed 2-20-fold higher potency with respect to berberine. Since our berberine simplified analogues may be easily synthesized and are characterized by lower molecular weight than the parent compound, they are further functionalizable and should be more suitable for oral administration. Molecular docking simulations suggested FtsZ, a well-known protein involved in bacterial cell division, as a possible target.
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The increasing onset of multidrug-resistant bacteria has propelled microbiology research towards antimicrobial peptides as new possible antibiotics from natural sources. Antimicrobial peptides are short peptides endowed with a broad range of activity against both Gram-positive and Gram-negative bacteria and are less prone to trigger resistance. Besides their activity against planktonic bacteria, many antimicrobial peptides also show antibiofilm activity. Biofilms are ubiquitous in nature, having the ability to adhere to virtually any surface, either biotic or abiotic, including medical devices, causing chronic infections that are difficult to eradicate. The biofilm matrix protects bacteria from hostile environments, thus contributing to the bacterial resistance to antimicrobial agents. Biofilms are very difficult to treat, with options restricted to the use of large doses of antibiotics or the removal of the infected device. Antimicrobial peptides could represent good candidates to develop new antibiofilm drugs as they can act at different stages of biofilm formation, on disparate molecular targets and with various mechanisms of action. These include inhibition of biofilm formation and adhesion, downregulation of quorum sensing factors, and disruption of the pre-formed biofilm. This review focuses on the proprieties of antimicrobial and antibiofilm peptides, with a particular emphasis on their mechanism of action, reporting several examples of peptides that over time have been shown to have activity against biofilm.
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Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Secuencia de Aminoácidos , Antibacterianos/química , Pruebas de Sensibilidad Microbiana , Péptidos/química , Péptidos/farmacologíaRESUMEN
BACKGROUND: The comprehension of the mechanism of action of antimicrobial peptides is fundamental for the design of new antibiotics. Studies performed looking at the interaction of peptides with bacterial cells offer a faithful picture of what really happens in nature. METHODS: In this work we focused on the interaction of the peptide Temporin L with E. coli cells, using a variety of biochemical and biophysical techniques that include: functional proteomics, docking, optical microscopy, TEM, DLS, SANS, fluorescence. RESULTS: We identified bacterial proteins specifically interacting with the peptides that belong to the divisome machinery; our data suggest that the GTPase FtsZ is the specific peptide target. Docking experiments supported the FtsZ-TL interaction; binding and enzymatic assays using recombinant FtsZ confirmed this hypothesis and revealed a competitive inhibition mechanism. Optical microscopy and TEM measurements demonstrated that, upon incubation with the peptide, bacterial cells are unable to divide forming long necklace-like cell filaments. Dynamic light scattering studies and Small Angle Neutron Scattering experiments performed on treated and untreated bacterial cells, indicated a change at the nanoscale level of the bacterial membrane. CONCLUSIONS: The peptide temporin L acts by a non-membrane-lytic mechanism of action, inhibiting the divisome machinery. GENERAL SIGNIFICANCE: Identification of targets of antimicrobial peptides is pivotal to the tailored design of new antimicrobials.
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Péptidos Antimicrobianos , Escherichia coli , Antibacterianos/metabolismo , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/metabolismo , Péptidos Catiónicos Antimicrobianos/farmacología , División Celular , Proteínas del Citoesqueleto/metabolismo , Escherichia coli/metabolismoRESUMEN
Antimicrobial peptides (AMPs) play a key role in the innate immunity, the first line of defense against bacteria, fungi, and viruses. AMPs are small molecules, ranging from 10 to 100 amino acid residues produced by all living organisms. Because of their wide biodiversity, insects are among the richest and most innovative sources for AMPs. In particular, the insect Hermetia illucens (Diptera: Stratiomyidae) shows an extraordinary ability to live in hostile environments, as it feeds on decaying substrates, which are rich in microbial colonies, and is one of the most promising sources for AMPs. The larvae and the combined adult male and female H. illucens transcriptomes were examined, and all the sequences, putatively encoding AMPs, were analysed with different machine learning-algorithms, such as the Support Vector Machine, the Discriminant Analysis, the Artificial Neural Network, and the Random Forest available on the CAMP database, in order to predict their antimicrobial activity. Moreover, the iACP tool, the AVPpred, and the Antifp servers were used to predict the anticancer, the antiviral, and the antifungal activities, respectively. The related physicochemical properties were evaluated with the Antimicrobial Peptide Database Calculator and Predictor. These analyses allowed to identify 57 putatively active peptides suitable for subsequent experimental validation studies.
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Dípteros/inmunología , Dípteros/metabolismo , Larva/inmunología , Larva/metabolismo , Proteínas Citotóxicas Formadoras de Poros/inmunología , Proteínas Citotóxicas Formadoras de Poros/farmacología , Algoritmos , Animales , Antifúngicos , Antineoplásicos , Antivirales , Fenómenos Químicos , Femenino , Inmunidad Innata , Aprendizaje Automático , Masculino , Proteínas Citotóxicas Formadoras de Poros/genética , Proteínas Citotóxicas Formadoras de Poros/metabolismo , TranscriptomaRESUMEN
Living organisms have developed specific defence mechanisms to counteract hostile environmental conditions. Alkylation stress response mechanisms also occur in Mycobacterium tuberculosis (MTB) the pathogen responsible for tuberculosis. The effect of alkylating agents on the cellular growth of MTB was investigated using methyl methanesulfonate (MMS) as methyl donor demonstrating that limited doses of alkylating agents might affect MTB cell viability. A global investigation of Mycobacterium smegmatis response to alkylating stress was then pursued by differential proteomics to identify the most affected cellular pathways. Quantitative analysis of proteomic profiles demonstrated that most of the proteins upregulated in the presence of alkylating agents are involved in biofilm formation and/or cell wall biosynthesis. Tailored experiments confirmed that under stress conditions M. smegmatis elicits physical defence mechanisms by increasing biofilm formation. Among the upregulated proteins, we identified the GlmU bifunctional enzyme as a possible factor involved in biofilm production. Experiments with both conditional deletion and overexpressing glmU mutants demonstrated that down regulation of GlmU decreased M. smegmatis capabilities to produce biofilm whereas overexpression of the enzyme increased biofilm formation. These results were supported by inhibition of GlmU acetyltransferase activity with two different inhibitors, suggesting the involvement of this enzyme in the M. smegmatis defence mechanisms.
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Acetiltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Metilmetanosulfonato/farmacología , Complejos Multienzimáticos/metabolismo , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium tuberculosis/crecimiento & desarrollo , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/genética , Alquilación , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Perfilación de la Expresión Génica , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/genética , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Ácido N-Acetilneuramínico/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismoRESUMEN
DNA methylation damage can be induced by endogenous and exogenous chemical agents, which has led every living organism to develop suitable response strategies. We investigated protein expression profiles of Escherichia coli upon exposure to the alkylating agent methyl-methane sulfonate (MMS) by differential proteomics. Quantitative proteomic data showed a massive downregulation of enzymes belonging to the glycolytic pathway and fatty acids degradation, strongly suggesting a decrease of energy production. A strong reduction in the expression of the N-acetylneuraminate lyases (NanA) involved in the sialic acid metabolism was also observed. Using a null NanA mutant and DANA, a substrate analog acting as competitive inhibitor, we demonstrated that down regulation of NanA affects biofilm formation and adhesion properties of E. coli MV1161. Exposure to alkylating agents also decreased biofilm formation and bacterial adhesion to Caco-2 eukaryotic cell line by the adherent invasive E. coli (AIEC) strain LF82. Our data showed that methylation stress impairs E. coli adhesion properties and suggest a possible role of NanA in biofilm formation and bacteria host interactions.