RESUMO
Antimicrobial peptides (AMPs) are short peptides that play crucial roles in diverse biological processes and have various functional activities against target organisms. Due to the abuse of chemical antibiotics and microbial pathogens' increasing resistance to antibiotics, AMPs have the potential to be alternatives to antibiotics. As such, the identification of AMPs has become a widely discussed topic. A variety of computational approaches have been developed to identify AMPs based on machine learning algorithms. However, most of them are not capable of predicting the functional activities of AMPs, and those predictors that can specify activities only focus on a few of them. In this study, we first surveyed 10 predictors that can identify AMPs and their functional activities in terms of the features they employed and the algorithms they utilized. Then, we constructed comprehensive AMP datasets and proposed a new deep learning-based framework, iAMPCN (identification of AMPs based on CNNs), to identify AMPs and their related 22 functional activities. Our experiments demonstrate that iAMPCN significantly improved the prediction performance of AMPs and their corresponding functional activities based on four types of sequence features. Benchmarking experiments on the independent test datasets showed that iAMPCN outperformed a number of state-of-the-art approaches for predicting AMPs and their functional activities. Furthermore, we analyzed the amino acid preferences of different AMP activities and evaluated the model on datasets of varying sequence redundancy thresholds. To facilitate the community-wide identification of AMPs and their corresponding functional types, we have made the source codes of iAMPCN publicly available at https://github.com/joy50706/iAMPCN/tree/master. We anticipate that iAMPCN can be explored as a valuable tool for identifying potential AMPs with specific functional activities for further experimental validation.
Assuntos
Peptídeos Catiônicos Antimicrobianos , Aprendizado Profundo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Peptídeos Antimicrobianos , Antibacterianos , AlgoritmosRESUMO
The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.
Assuntos
Nanopartículas , Polimixina B , Polimixina B/farmacologia , Lipossomos/farmacologia , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Klebsiella pneumoniae , Polissacarídeos Bacterianos/farmacologia , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana MúltiplaRESUMO
Antimicrobial peptides (AMPs) are a unique and diverse group of molecules that play a crucial role in a myriad of biological processes and cellular functions. AMP-related studies have become increasingly popular in recent years due to antimicrobial resistance, which is becoming an emerging global concern. Systematic experimental identification of AMPs faces many difficulties due to the limitations of current methods. Given its significance, more than 30 computational methods have been developed for accurate prediction of AMPs. These approaches show high diversity in their data set size, data quality, core algorithms, feature extraction, feature selection techniques and evaluation strategies. Here, we provide a comprehensive survey on a variety of current approaches for AMP identification and point at the differences between these methods. In addition, we evaluate the predictive performance of the surveyed tools based on an independent test data set containing 1536 AMPs and 1536 non-AMPs. Furthermore, we construct six validation data sets based on six different common AMP databases and compare different computational methods based on these data sets. The results indicate that amPEPpy achieves the best predictive performance and outperforms the other compared methods. As the predictive performances are affected by the different data sets used by different methods, we additionally perform the 5-fold cross-validation test to benchmark different traditional machine learning methods on the same data set. These cross-validation results indicate that random forest, support vector machine and eXtreme Gradient Boosting achieve comparatively better performances than other machine learning methods and are often the algorithms of choice of multiple AMP prediction tools.
Assuntos
Algoritmos , Biologia Computacional/métodos , Aprendizado de Máquina , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Bactérias/classificação , Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Bases de Dados Factuais , Fungos/classificação , Fungos/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/classificação , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Máquina de Vetores de Suporte , Vírus/efeitos dos fármacosRESUMO
Inflammation is a pathological feature of kidney injury and its progression correlates with the development of kidney fibrosis which can lead to kidney function impairment. This project investigated the regulatory function of WNT1-inducible signaling pathway protein 1 (WISP1) in kidney inflammation. Administration of recombinant WISP1 protein to healthy mice induced kidney inflammation (macrophage accrual and production of tumor necrosis factor α (TNF-α), CCL2 and IL-6), which could be prevented by inhibition of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB). Furthermore, inhibition of WISP1, by gene knockdown or neutralising antibody, could inhibit cultured macrophages producing inflammatory cytokines following stimulation with lipopolysaccharides (LPSs) and kidney fibroblasts proliferating in response to TNFα, which both involved NF-κB signaling. Kidney expression of WISP1 was found to be increased in mouse models of progressive kidney inflammation-unilateral ureter obstruction (UUO) and streptozotocin (STZ)-induced diabetic nephropathy (DN). Treatment of UUO mice with WISP1 antibody reduced the kidney inflammation in these mice. Therefore, pharmacological blockade of WISP1 exhibits potential as a novel therapy for inhibiting inflammation in kidney disease.
Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Nefropatias Diabéticas/etiologia , Inflamação , NF-kappa B/metabolismo , Animais , Proteínas de Sinalização Intercelular CCN/genética , Proteínas de Sinalização Intercelular CCN/imunologia , Diabetes Mellitus Experimental/patologia , Fibrose , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos C57BL , Ratos , Transdução de Sinais , Obstrução UreteralRESUMO
Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.
Assuntos
Farmacorresistência Bacteriana/genética , Proteínas de Membrana/genética , Staphylococcus aureus Resistente à Meticilina/genética , Infecções Estafilocócicas/genética , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Antibacterianos/farmacologia , Daptomicina/farmacologia , Farmacorresistência Bacteriana/imunologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/imunologia , Staphylococcus aureus Resistente à Meticilina/metabolismo , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.
Assuntos
Terapia Genética , Receptores de Fatores de Crescimento do Endotélio Vascular/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Neovascularização Retiniana/genética , Neovascularização Retiniana/terapia , Animais , Hipóxia Celular , Dependovirus/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Células HEK293 , Humanos , Injeções Intravítreas , Domínios Proteicos , Ratos Sprague-Dawley , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/metabolismo , Transgenes , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Fibrosis is a pathological feature of chronic kidney disease and its progression correlates with declining renal function. Kidney fibrosis is driven by multiple profibrotic factors. This project examined the regulatory function of WNT1-inducible-signaling pathway protein 1 (WISP1) in the development of kidney fibrosis. Induction of WISP1 by transforming growth factor beta 1 (TGF-ß1), and the role of WISP1 in TGF-ß1/Smad signaling and fibrotic responses, was examined in multiple kidney cells. Kidney expression of WISP1 was examined in mouse models of unilateral ureter obstruction (UUO) and streptozotocin-induced diabetic nephropathy. WISP1 antibody was administered to UUO mice during the induction of kidney injury and the impact on kidney fibrosis was examined. WISP1 expression was upregulated in both mouse models. TGF-ß1-induced expression of WISP1 and profibrotic genes in cultured kidney cells via TGF-ßR1. Recombinant WISP1-induced expression of TGF-ßR1 in kidney cells. Suppression of WISP1 by shRNA or neutralizing antibody reduced TGF-ß1-mediated activation of Smad3, fibrotic gene expression, and fibroblast proliferation. Treatment with WISP1 antibody inhibited the development of kidney fibrosis in UUO mice. WISP1 mediates the profibrotic effects of TGF-ß1 in kidney cells and in kidney disease. Pharmacological blockade of WISP1 exhibits potential as a novel therapy for inhibiting kidney fibrosis.
Assuntos
Proteínas de Sinalização Intercelular CCN/metabolismo , Nefropatias Diabéticas/metabolismo , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Sinalização Intercelular CCN/genética , Linhagem Celular , Proliferação de Células , Células Cultivadas , Nefropatias Diabéticas/patologia , Fibroblastos/fisiologia , Fibrose , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas/genética , Ratos , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Multidrug-resistant Pseudomonas aeruginosa presents a global medical challenge, and polymyxins are a key last-resort therapeutic option. Unfortunately, polymyxin resistance in P. aeruginosa has been increasingly reported. The present study was designed to define metabolic differences between paired polymyxin-susceptible and -resistant P. aeruginosa strains using untargeted metabolomics and lipidomics analyses. The metabolomes of wild-type P. aeruginosa strain K ([PAK] polymyxin B MIC, 1 mg/liter) and its paired pmrB mutant strains, PAKpmrB6 and PAKpmrB12 (polymyxin B MICs of 16 mg/liter and 64 mg/liter, respectively) were characterized using liquid chromatography-mass spectrometry, and metabolic differences were identified through multivariate and univariate statistics. PAKpmrB6 and PAKpmrB12, which displayed lipid A modifications with 4-amino-4-deoxy-l-arabinose, showed significant perturbations in amino acid and carbohydrate metabolism, particularly the intermediate metabolites from 4-amino-4-deoxy-l-arabinose synthesis and the methionine salvage cycle pathways. The genomics result showed a premature termination (Y275stop) in speE (encoding spermidine synthase) in PAKpmrB6, and metabolomics data revealed a decreased intracellular level of spermidine in PAKpmrB6 compared to that in PAKpmrB12 Our results indicate that spermidine may play an important role in high-level polymyxin resistance in P. aeruginosa Interestingly, both pmrB mutants had decreased levels of phospholipids, fatty acids, and acyl-coenzyme A compared to those in the wild-type PAK. Moreover, the more resistant PAKpmrB12 mutant exhibited much lower levels of phospholipids than the PAKpmrB6 mutant, suggesting that the decreased phospholipid level was associated with polymyxin resistance. In summary, this study provides novel mechanistic information on polymyxin resistance in P. aeruginosa and highlights its impacts on bacterial metabolism.
Assuntos
Antibacterianos/farmacologia , Polimixinas/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana , Lipídeo A/metabolismo , Metabolômica , Testes de Sensibilidade Microbiana , Fosfolipídeos/metabolismo , Polimixina B/farmacologia , Infecções por PseudomonasRESUMO
The regulatory role of a novel miRNA, miR-378, was determined in the development of fibrosis through repression of the MAPK1 pathway, miR-378 and fibrotic gene expression was examined in streptozotocin (STZ)-induced diabetic mice at 18 weeks or in unilateral ureteral obstruction (UUO) mice at 7 days. miR-378 transfection of proximal tubular epithelial cells, NRK52E and mesangial cells was assessed with/without endogenous miR-378 knockdown using the locked nucleic acid (LNA) inhibitor. NRK52E cells were co-transfected with the mothers against decapentaplegic homolog 3 (SMAD3) CAGA reporter and miR-378 in the presence of transforming growth factor-ß (TGF-ß1) was assessed. Quantitative polymerase chain reaction (qPCR) showed a significant reduction in miR-378 (P<0.05) corresponding with up-regulated type I collagen, type IV collagen and α-smooth muscle actin (SMA) in kidneys of STZ or UUO mice, compared with controls. TGF-ß1 significantly increased mRNA expression of type I collagen (P<0.05), type IV collagen (P<0.05) and α-SMA (P<0.05) in NRK52E cells, which was significantly reduced (P<0.05) following miR-378 transfection and reversed following addition of the LNA inhibitor of endogenous miR-378 Overexpression of miR-378 inhibited mesangial cell expansion and proliferation in response to TGF-ß1, with LNA-miR-378 transfection reversing this protective effect, associated with cell morphological alterations. The protective function of MAPK1 on miR-378 was shown in kidney cells treated with the MAPK1 inhibitor, selumetinib, which inhibited mesangial cell hypertrophy in response to TGF-ß1. Taken together, these results suggest that miR-378 acts via regulation of the MAPK1 pathway. These studies demonstrate the protective function of MAPK1, regulated by miR-378, in the induction of kidney cell fibrosis and mesangial hypertrophy.
Assuntos
Nefropatias Diabéticas/metabolismo , Rim/patologia , Sistema de Sinalização das MAP Quinases , Células Mesangiais/patologia , MicroRNAs/metabolismo , Animais , Benzimidazóis , Células Cultivadas , Nefropatias Diabéticas/patologia , Fibrose , Humanos , Hipertrofia , Masculino , Camundongos Endogâmicos C57BL , Ratos , Fator de Crescimento Transformador beta1RESUMO
The advancement of microRNA (miRNA) therapies has been hampered by difficulties in delivering miRNA to the injured kidney in a robust and sustainable manner. Using bioluminescence imaging in mice with unilateral ureteral obstruction (UUO), we report that mesenchymal stem cells (MSCs), engineered to overexpress miRNA-let7c (miR-let7c-MSCs), selectively homed to damaged kidneys and upregulated miR-let7c gene expression, compared with nontargeting control (NTC)-MSCs. miR-let7c-MSC therapy attenuated kidney injury and significantly downregulated collagen IVα1, metalloproteinase-9, transforming growth factor (TGF)-ß1, and TGF-ß type 1 receptor (TGF-ßR1) in UUO kidneys, compared with controls. In vitro analysis confirmed that the transfer of miR-let7c from miR-let7c-MSCs occurred via secreted exosomal uptake, visualized in NRK52E cells using cyc3-labeled pre-miRNA-transfected MSCs with/without the exosomal inhibitor, GW4869. The upregulated expression of fibrotic genes in NRK52E cells induced by TGF-ß1 was repressed following the addition of isolated exosomes or indirect coculture of miR-let7c-MSCs, compared with NTC-MSCs. Furthermore, the cotransfection of NRK52E cells using the 3'UTR of TGF-ßR1 confirmed that miR-let7c attenuates TGF-ß1-driven TGF-ßR1 gene expression. Taken together, the effective antifibrotic function of engineered MSCs is able to selectively transfer miR-let7c to damaged kidney cells and will pave the way for the use of MSCs for therapeutic delivery of miRNA targeted at kidney disease.
Assuntos
Exossomos/metabolismo , Nefropatias/genética , Nefropatias/patologia , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Actinas/metabolismo , Animais , Transporte Biológico , Engenharia Celular , Colágeno/metabolismo , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Vesículas Extracelulares/metabolismo , Fibrose , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Nefropatias/metabolismo , Nefropatias/terapia , Masculino , Camundongos , Ratos , Transdução GenéticaRESUMO
A fundamental understanding of the effect of amphiphilic protein encapsulation on the nanostructure of the bicontinuous cubic phase is crucial to progressing biomedical and biological applications of these hybrid protein-lipid materials, including as drug delivery vehicles, as biosensors, biofuel cells and for in meso crystallization. The relationship between the lipid nanomaterial and the encapsulated protein, however, remains poorly understood. In this study, we investigated the effect of incorporating the five transmembrane and lipo-proteins which make up the ß-barrel assembly machinery from Gram-negative bacteria within a series of bicontinuous cubic phases. The transmembrane ß-barrel BamA caused an increase in lattice parameter of the cubic phase upon encapsulation. By contrast, the mainly hydrophilic lipo-proteins BamB-E caused the cubic phase lattice parameters to decrease, despite their large size relative to the diameter of the cubic phase water channels. Analysis of the primary amino acid sequence was used to rationalize this effect, based on specific interactions between aromatic amino acids within the proteins and the polar-apolar interface. Other factors that were found to have an effect were lateral bilayer pressure and rigidity within the lipid bilayer, water channel diameter, and size and structure of the lipo-proteins. The data presented suggest that hydrophilic bioactive molecules can be selectively encapsulated within the cubic phase by using a lipid anchor or aromatic amino acids, for drug delivery or biosensing applications.
Assuntos
Bicamadas Lipídicas/química , Nanoestruturas/química , Proteínas/química , Proteínas da Membrana Bacteriana Externa/química , Materiais Biomiméticos/química , Colesterol/química , Proteínas de Escherichia coli/química , Lipoproteínas/química , Modelos Moleculares , Nanocápsulas/química , Nanotecnologia , Conformação Proteica em Folha beta , Proteínas Recombinantes/químicaRESUMO
The lipopeptide surfactin produced by certain strains of Bacillus subtillis is a potent biosurfactant with high amphiphilicity and a strong tendency for self-aggregation. Surfactin possesses a number of valuable biological properties such as antiviral, antibacterial, antifungal, and hemolytic activities. Owing to these properties, in addition to the general advantages of biosurfactants over synthetic surfactants, surfactin has potential biotechnological and biomedical applications. Here, the aggregation properties of surfactin in solution together with its behavior at the water/air interface were studied using classical molecular dynamics simulations (MD) at three different pH values. Validation of the MD structural data was performed by comparing neutron reflectivity and volume fraction profiles computed from the simulations with their experimental counterparts. Analysis of the MD trajectories supported conclusions about the distribution, conformations, and interactions of surfactin in solution and at the water-air interface. Considering altogether, the work presented provides atomistic models for the rationalization of some of the structural and dynamic characteristics as well as the modes of action of surfactin at different pH values.
Assuntos
Lipopeptídeos/química , Peptídeos Cíclicos/química , Água/química , SoluçõesRESUMO
Lipidic bicontinuous cubic mesophases with encapsulated amphiphilic proteins are widely used in a range of biological and biomedical applications, including in meso crystallization, as drug delivery vehicles for therapeutic proteins, and as biosensors and biofuel cells. However, the effect of amphiphilic protein encapsulation on the cubic phase nanostructure is not well-understood. In this study, we illustrate the effect of incorporating the bacterial amphiphilic membrane protein Ag43, and its individual hydrophobic ß(43) and hydrophilic α(43) domains, in bicontinuous cubic mesophases. For the monoolein, monoalmitolein, and phytantriol cubic phases with and without 8% w/w cholesterol, the effect of the full length amphiphilic protein Ag43 on the cubic phase nanostructure was more significant than the sum of the individual hydrophobic ß(43) and hydrophilic α(43) domains. Several factors were found to potentially influence the impact of the hydrophobic ß(43) domain on the cubic phase internal nanostructure. These include the size of the hydrophobic ß(43) domain relative to the thickness of the lipid bilayer, as well as its charge and diameter. The size of the hydrophilic α(43) domain relative to the water channel radius of the cubic mesophase was also found to be important. The secondary structure of the Ag43 proteins was affected by the hydrophobic thickness and physicochemical properties of the lipid bilayer and the water channel diameter of the cubic phase. Such structural changes may be small but could potentially affect membrane protein function.
Assuntos
Lipídeos/química , Proteínas de Membrana/química , Dicroísmo Circular , Interações Hidrofóbicas e HidrofílicasRESUMO
Annexin V is of crucial importance for detection of the phosphatidylserine of apoptotic cell membranes. However, the manner in which different amounts of phosphatidylserine at the membrane surface at different stages of apoptosis contribute to binding of annexin V is unclear. We have used a quartz crystal microbalance combined with dissipative monitoring (QCM-D) and neutron reflectivity to characterize binding of human annexin V to supported bilayers of different phospholipid composition. We created model apoptotic bilayers of 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine and 1-palmitoyl-2-oleoyl-sn-glycerophosphoserine (POPS) in the ratios 19:1, 9:1, 6.7:1, 4:1, 3:1, and 2:1 (w/w) in the presence of 2.5 mM CaCl2. QCM-D data revealed that annexin V bound less to supported fluid lipid bilayers with higher POPS content (>25 % POPS). Neutron reflectivity was used to further characterize the detailed composition of lipid bilayers with membrane-bound annexin V. Analysis confirmed less annexin V binding with higher POPS content, that bound annexin V formed a discrete layer above the lipid bilayer with little effect on the overall structure of the membrane, and that the thickness and volume fraction of the annexin V layer varied with POPS content. From these results we show that the POPS content of the outer surface of lipid bilayers affects the structure of membrane-bound annexin V.
Assuntos
Anexina A5/química , Apoptose , Bicamadas Lipídicas/química , Sequência de Aminoácidos , Anexina A5/metabolismo , Sítios de Ligação , Humanos , Bicamadas Lipídicas/metabolismo , Dados de Sequência Molecular , Ligação ProteicaRESUMO
A novel surface-enhanced Raman scattering (SERS) sensing system which operates by the self-assembly of Ag nanoparticles (AgNPs) onto the nanocomposite of AgNPs and graphene oxide (AgNP-GO) in the presence of two complementary DNAs has been developed. In this system, AgNP-GO serves as a SERS-active substrate. The AgNPs with the modification of non-fluorescent 4-mercaptobenzoic acid (4-MBA) act as highly efficient Raman probes for DNA hybridization. When probe DNAs on AgNP-GO are complementary to target DNAs on AgNPs functionalized with 4-MBA, the DNA hybridization occurring directs the self-assembly of AgNPs onto AgNP-GO, leading to the creation of SERS hot spots. Due to the fact that partial 4-MBA molecules are located in the region of the hot spots, their SERS signals are greatly enhanced, indicating successful DNA hybridization. It is noteworthy that the size of AgNPs contributes significantly to the enhancement of SERS activity. The detection limit of the target DNAs at the pM level can be achieved through the self-assembly of large sized AgNPs onto AgNP-GO. More importantly, the AgNP-AgNP-GO system shows reproducible SERS signals in proportion to the logarithm of the target DNA concentrations spanning from 10(-6) to 10(-12) M and the excellent capability for multiplex DNA detection.
RESUMO
The ß-barrel assembly machinery (BAM) complex in Gram-negative bacteria facilitates the assembly of ß-barrel proteins into the outer membrane. Understanding the protein-protein interactions within this complex is essential for unravelling its functional mechanisms. Here, we present the use of neutron reflectometry for investigating the organization of ß-barrel membrane protein complexes in the membrane environment. The spatial organization, protein positioning, protein-lipid interactions, and conformational changes within the complex can be elucidated by this method.
RESUMO
The waning pipeline of the useful antibacterial arsenal has necessitated the urgent development of more effective antibacterial strategies with distinct mechanisms to rival the continuing emergence of resistant pathogens, particularly Gram-negative bacteria, due to their explicit drug-impermeable, two-membrane-sandwiched cell wall envelope. Herein, we have developed multicomponent coassembled nanoparticles with strong bactericidal activity and simultaneous bacterial cell envelope targeting using a peptide coassembly strategy. Compared to the single-component self-assembled nanoparticle counterparts or cocktail mixtures of these at a similar concentration, coassembled multicomponent nanoparticles showed higher bacterial killing efficiency against Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli by several orders of magnitude (about 100-1,000,000-fold increase). Comprehensive confocal and electron microscopy suggest that the superior antibacterial activity of the coassembled nanoparticles proceeds via multiple complementary mechanisms of action, including membrane destabilization, disruption, and cell wall hydrolysis, actions that were not observed with the single nanoparticle counterparts. To understand the fundamental working mechanisms behind the improved performance of coassembled nanoparticles, we utilized a "dilution effect" system where the antibacterial components are intermolecularly mixed and coassembled with a non-antibacterial protein in the nanoparticles. We suggest that coassembled nanoparticles mediate enhanced bacterial killing activity by attributes such as optimized local concentration, high avidity, cooperativity, and synergy. The nanoparticles showed no cytotoxic or hemolytic activity against tested eukaryotic cells and erythrocytes. Collectively, these findings reveal potential strategies for disrupting the impermeable barrier that Gram-negative pathogens leverage to restrict antibacterial access and may serve as a platform technology for potential nano-antibacterial design to strengthen the declining antibiotic arsenal.
Assuntos
Antibacterianos , Nanopartículas , Antibacterianos/farmacologia , Antibacterianos/química , Nanopartículas/química , Bactérias , Bactérias Gram-Negativas , Membrana Celular , Escherichia coli , Testes de Sensibilidade MicrobianaRESUMO
Over the past few decades, organic-inorganic halide perovskites (OIHPs) as novel photocatalyst materials have attracted intensive attention for an impressive variety of photocatalytic applications due to their excellent photophysical (chemical) properties. Regarding practical application and future commercialization, the air-water stability and photocatalytic performance of OIHPs need to be further improved. Accordingly, studying modification strategies and interfacial interaction mechanisms is crucial. In this review, the current progress in the development and photocatalytic fundamentals of OIHPs is summarized. Furthermore, the structural modification strategies of OIHPs, including dimensionality control, heterojunction design, encapsulation techniques, and so on for the enhancement of charge-carrier transfer and the enlargement of long-term stability, are elucidated. Subsequently, the interfacial mechanisms and charge-carrier dynamics of OIHPs during the photocatalytic process are systematically specified and classified via diverse photophysical and electrochemical characterization methods, such as time-resolved photoluminescence measurements, ultrafast transient absorption spectroscopy, electrochemical impedance spectroscopy measurements, transient photocurrent densities, and so forth. Eventually, various photocatalytic applications of OIHPs, including hydrogen evolution, CO2 reduction, pollutant degradation, and photocatalytic conversion of organic matter.
RESUMO
The multidrug-resistant Gram-negative bacteria has evolved into a worldwide threat to human health; over recent decades, polymyxins have re-emerged in clinical practice due to their high activity against multidrug-resistant bacteria. Nevertheless, the nephrotoxicity and neurotoxicity of polymyxins seriously hinder their practical use in the clinic. Based on the quantitative structure-activity relationship (QSAR), analogue design is an efficient strategy for discovering biologically active compounds with fewer adverse effects. To accelerate the polymyxin analogues discovery process and find the polymyxin analogues with high antimicrobial activity against Gram-negative bacteria, here we developed PmxPred, a GCN and catBoost-based machine learning framework. The RDKit descriptors were used for the molecule and residues representation, and the ensemble learning model was utilized for the antimicrobial activity prediction. This framework was trained and evaluated on multiple Gram-negative bacteria datasets, including Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and a general Gram-negative bacteria dataset achieving an AUROC of 0.857, 0.880, 0.756, 0.895 and 0.865 on the independent test, respectively. PmxPred outperformed the transfer learning method that trained on 10 million molecules. We interpreted our model well-trained model by analysing the importance of global and residue features. Overall, PmxPred provides a powerful additional tool for predicting active polymyxin analogues, and holds the potential elucidate the mechanisms underlying the antimicrobial activity of polymyxins. The source code is publicly available on GitHub (https://github.com/yanwu20/PmxPred).
Assuntos
Infecções por Bactérias Gram-Negativas , Polimixinas , Humanos , Polimixinas/farmacologia , Polimixinas/química , Antibacterianos/química , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Bactérias Gram-Negativas , Farmacorresistência Bacteriana Múltipla , Escherichia coli , Testes de Sensibilidade MicrobianaRESUMO
Polymyxins are often the only effective antibiotics against the "Critical" pathogen Acinetobacter baumannii. Worryingly, highly polymyxin-resistant A. baumannii displaying dependence on polymyxins has emerged in the clinic, leading to diagnosis and treatment failures. Here, we report that arginine metabolism is essential for polymyxin-dependent A. baumannii. Specifically, the arginine degradation pathway was significantly altered in polymyxin-dependent strains compared to wild-type strains, with critical metabolites (e.g., L-arginine and L-glutamate) severely depleted and expression of the astABCDE operon significantly increased. Supplementation of arginine increased bacterial metabolic activity and suppressed polymyxin dependence. Deletion of astA, the first gene in the arginine degradation pathway, decreased phosphatidylglycerol and increased phosphatidylethanolamine levels in the outer membrane, thereby reducing the interaction with polymyxins. This study elucidates the molecular mechanism by which arginine metabolism impacts polymyxin dependence in A. baumannii, underscoring its critical role in improving diagnosis and treatment of life-threatening infections caused by "undetectable" polymyxin-dependent A. baumannii.