RESUMO
Endotoxin is a kind of lipopolysaccharide that exits on the cell wall of Gram-negative bacteria. It can cause fever, shock or even death when is delivered into human body. So, it is necessary to control the endotoxin contamination for biopharmaceutical products that are mainly administered by intravenous route. Limulus Amebocyte Lysate (LAL)-based tests are usually used to detect endotoxin content in biologics formulations. However, an undesirable phenomenon called "Low Endotoxin Recovery (LER)" often occurs in formulation buffers that usually contain chelating component, such as sodium citrate, and amphiphilic surfactant, such as Tween-20. The occurrence of this LER phenomenon may interfere with endotoxin detection and cause false negative results. In this study, we compared the effect of different sample treatment methods on endotoxin detection and found that the LER phenomenon was better controlled under the conditions of low pH (pH = 5.0), low temperature (2-8 °C) and in the presence of divalent cations in the solution. In addition, although the endotoxin activity was found to have decreased due to LER phenomenon, the particle size distribution of endotoxin determined by dynamic light scattering (DLS) in LER solution did not change obviously, which is different from previous hypothesis about LER phenomenon in literature that the particle size of endotoxin aggregates would decrease under LER conditions. These findings provide some insights into different sample treatment methods for endotoxin detection and give a better understanding and solution on minimizing the LER phenomenon.
Assuntos
Métodos Analíticos de Preparação de Amostras/métodos , Endotoxinas/isolamento & purificação , Bactérias Gram-Negativas/química , Métodos Analíticos de Preparação de Amostras/instrumentação , Animais , Cátions Bivalentes/química , Endotoxinas/química , Endotoxinas/farmacologia , Caranguejos Ferradura , Concentração de Íons de Hidrogênio , Teste do Limulus , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Tamanho da Partícula , Tensoativos/químicaRESUMO
From arid, high desert soil samples collected near Bend, Oregon, 19 unique bacteria were isolated. Each strain was identified by 16S rRNA gene sequencing, and their organic extracts were tested for antibacterial and antiproliferative activities. Noteworthy, six extracts (30 %) exhibited strong inhibition resulting in less than 50 % cell proliferation in more than one cancer cell model, tested at 10â µg/mL. Principal component analysis (PCA) of LC/MS data revealed drastic differences in the metabolic profiles found in the organic extracts of these soil bacteria. In total, fourteen potent antibacterial and/or cytotoxic metabolites were isolated via bioactivity-guided fractionation, including two new natural products: a pyrazinone containing tetrapeptide and 7-methoxy-2,3-dimethyl-4H-chromen-4-one, as well as twelve known compounds: furanonaphthoquinone I, bafilomycin C1 and D, FD-594, oligomycin A, chloramphenicol, MY12-62A, rac-sclerone, isosclerone, tunicamycin VII, tunicamycin VIII, and (6S,16S)-anthrabenzoxocinone 1.264-C.
Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Produtos Biológicos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Solo/química , Antibacterianos/química , Antibacterianos/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Análise de Componente Principal , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Microbiologia do SoloRESUMO
TLR4 location, and bacterial species-derived lipopolysaccharides, play a significant role in the downstream activation of transcription factors, accessory molecules, and products. Here, this is demonstrated through the use of classically-activated and alternatively-activated macrophages. We show that, when polarized, human macrophages differentially express and localize TLR4, resulting in biased recognition and subsequent signalling of LPS derived from Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica. Analysis of activation demonstrated that in classically activated macrophages, P. aeruginosa signals from the plasma membrane via TLR4 to p65 dependent on TAK1 and TBK1 signalling. E. coli signals dependent or independent of the endosome, utilizing both TAK1- and TBK1-signalling to induce P65 and IRF3 inducible genes and cytokines. S. enterica however, only induces P65 and IRF3 phosphorylation through signalling via the endosome. This finding outlines clear signalling mechanisms by which innate immune cells, such as macrophages, can distinguish between bacterial species and initiate specialized responses through TLR4.
Assuntos
Bactérias Gram-Negativas/química , Lipopolissacarídeos , Macrófagos/imunologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/isolamento & purificação , Lipopolissacarídeos/farmacologia , Transdução de Sinais/imunologia , Especificidade da Espécie , Células THP-1RESUMO
ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Bacillus subtilis/química , Biologia Computacional/métodos , Cristalografia por Raios X , Firmicutes/química , Firmicutes/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Domínios ProteicosRESUMO
Infectious diseases are a major cause of morbidity and mortality worldwide, exacerbated by increasing antibiotic resistance in many bacterial species. The development of drugs with new modes of action is essential. A leading strategy is antivirulence, with the aim to target bacterial proteins that are important in disease causation and progression but do not affect growth, resulting in reduced selective pressure for resistance. Immunophilins, a superfamily of peptidyl-prolyl cis-trans isomerase (PPIase) enzymes have been shown to be important for virulence in a broad-spectrum of pathogenic bacteria. This Perspective will provide an overview of the recent advances made in understanding the role of each immunophilin family, cyclophilins, FK506 binding proteins (FKBPs), and parvulins in bacteria. Inhibitor design and medicinal chemistry strategies for development of novel drugs against bacterial FKBPs will be discussed. Furthermore, drugs against human cyclophilins and parvulins will be reviewed in their current indication as antiviral and anticancer therapies.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Dobramento de Proteína/efeitos dos fármacos , Animais , Antibacterianos/administração & dosagem , Infecções Bacterianas/tratamento farmacológico , Infecções Bacterianas/metabolismo , Proteínas de Bactérias/química , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The synthesis of a library of bacterial phosphoglycolipid, PGL-1, is described. Key features of the synthesis include regioselective esterification of the primary alcohol of the diacylglycerol moiety and an H-phosphonate method to install the phosphate in PGL-1 in comparison with earlier reported procedures. A representative set of PGL-1 analogues was prepared and evaluated for their biological activities. Results showed that the immunological activity of PGL-1 is dependent on the chain lengths of the fatty acids.
Assuntos
Glicolipídeos/farmacologia , Bactérias Gram-Negativas/química , Fatores Imunológicos/farmacologia , Organofosfatos/farmacologia , Animais , Relação Dose-Resposta a Droga , Glicolipídeos/síntese química , Glicolipídeos/química , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Interleucina-6/antagonistas & inibidores , Interleucina-6/biossíntese , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Estrutura Molecular , Organofosfatos/síntese química , Organofosfatos/química , Estereoisomerismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Lanthipeptides are intriguing peptides known since 1928, the year of penicillin's discovery. At that time, they were known as lantibiotics due to their (methyl)lanthionine amino acids and antibacterial activity. Their body of knowledge expanded tremendously over the last few years. Our analysis reveals that Bacteroidetes has a high state of clusters encoding the biosynthesis of class I lanthipeptides. We show that some strains of Pedobacter have a number of LanBs/genome comparable to that of some Actinobacteria. The case study selected was Pedobacter lusitanus NL19. Its clusters identified encode LanBs associated with LanCs as well as orphan LanBs. The first are concomitant with LanT transporters typical of class II lanthipeptides (and not class I), making their clusters into a hybrid class I and class II type. So far, this kind of operon was described only once and is involved in the production of pinensins, the first lanthipeptide with antifungal activity. A particular feature of pinensins is their splitted LanBs and we found that these enzymes are also widely encoded in Bacteroides. The function of a high percentage of proteins predicted to play a role in the production of Pedobacter lanthipeptides is unknown. Other major fraction of these proteins is expected to be enrolled in signal-transduction pathways. We demonstrate that the occurrence of lanthipeptides clusters in the genomes of Gram-negative bacteria is higher than previously reported. More importantly, we show that their genetic background is highly diverse, which is an undeniable foreshadowing of novel peptide structures, biochemistry and biological function.
Assuntos
Bacteriocinas/química , Bacteroidetes/genética , Família Multigênica , Pedobacter/química , Pedobacter/genética , Peptídeos/química , Antibacterianos/química , Bacteriocinas/biossíntese , Bacteroidetes/química , Genoma Bacteriano , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/genética , Biossíntese Peptídica , Peptídeos/isolamento & purificaçãoRESUMO
An FcεRI-IgE-based genetically encoded microfluidic cell sensor was constructed for fast Gram-negative bacterial screening in food samples. CD14-Fcε IgE, produced by the gene engineered antibodies (GEAs) technology, was used for the recognition of the target bacteria or lipopolysaccharide (LPS). Stable cell lines expressing GCaMP6s, a genetically encoded indicator of calcium flux, were first established for monitoring mast cell activation and improving detection sensitivity. The microfluidic system was designed to improve automation and control the reaction time. Once Gram-negative bacteria bound to the CD14-Fcε IgE on the RBL-2H3 cell surface, RBL-2H3 cell receptor (FcεRI)-induced Ca2+ signaling pathway was immediately activated to release Ca2+. The elevated intracellular Ca2+ triggers GCaMP6s for reporting the presence of Gram-negative bacteria. The developed biosensor was able to detect 80 CFU mL-1 Gram-negative bacteria within 2.5 min in pure culture samples. The biosensor was used to detect Gram-negative bacteria in pork samples. With its short screening time and easy operation, the proposed biosensor shows promise in future applications of foodborne pathogen testing in 1 h to 1 day.
Assuntos
Técnicas Biossensoriais/métodos , Contaminação de Alimentos/análise , Bactérias Gram-Negativas/isolamento & purificação , Imunoglobulina E/imunologia , Técnicas Analíticas Microfluídicas/métodos , Receptores de IgE/metabolismo , Animais , Basófilos/metabolismo , Técnicas Biossensoriais/instrumentação , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Linhagem Celular Tumoral , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/imunologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoglobulina E/genética , Imunoglobulina E/metabolismo , Dispositivos Lab-On-A-Chip , Receptores de Lipopolissacarídeos/genética , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Técnicas Analíticas Microfluídicas/instrumentação , Carne de Porco/microbiologia , Ratos , Receptores de IgE/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , SuínosRESUMO
Lack of new antibiotics and increasing antimicrobial resistance are among the main concerns of healthcare communities nowadays, and these concerns necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids-a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe 1)â a concise total synthesis of cystobactamid 507, 2)â the identification of the bioactive conformation using noncovalently bonded rigid analogues, and 3)â the first structure-activity relationship (SAR) study for cystobactamid 507 leading to new analogues with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor-groove binding as part of the drug-target interaction without showing significant intercalation. By designing a new analogue of cystobactamid 919-2, we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria.
Assuntos
Antibacterianos/síntese química , Asparagina/análogos & derivados , Bactérias Gram-Negativas/efeitos dos fármacos , Nitrocompostos/química , Antibacterianos/química , Antibacterianos/farmacologia , Asparagina/química , Asparagina/farmacologia , Bactérias Gram-Negativas/química , Testes de Sensibilidade Microbiana , Conformação Molecular , Nitrocompostos/farmacologia , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Hybridization is a useful strategy to bond the advantages of different peptides into novel constructions. We designed a series of AMPs based on the structures of a synthetic AMP KFA3 and a naturally-occurred host defense peptide substance P (SP) to obtain peptides retaining the high antibacterial activity of KFA3 and the immunomodulatory activity and low cytotoxicity of SP. METHODS: Two repeats of KFA and different C terminal fragments of SP were hybridized, generating a series of novel AMPs (KFSP1-8). The antibacterial activities, host cell toxicity and immunomodulation were measured. The antibacterial mechanisms were investigated. RESULTS: Hybrid peptides KFSP1-4 exerted substantial antibacterial activities against Gram-negative bacteria of standard strains and clinical drug-resistant isolates including E.coli, A.baumannii and P.aeruginosa, while showing little toxicity towards host cells. Compared with KFA3, moderate reduction in α-helix content and the interruption in α-helix continuality were indicated in CD spectra analysis and secondary-structure simulation in these peptides. Membrane permeabilization combined with time-kill studies and FITC-labeled imaging, indicated a selective membrane interaction of KFSP1 with bacteria cell membranes. By specially activating NK1 receptor, the hybrid peptides kept the ability of SP to induce intracellular calcium release and ERK1/2 phosphorylation, but unable to stimulate NF-κB phosphorylation. KFSP1 facilitated the survival of mouse macrophage RAW264.7, directly interacting with LPS and inhibiting the LPS-induced NF-κB phosphorylation and TNF-α expression. CONCLUSION: Hybridization is a useful strategy to bond the advantages of different peptides. KFSP1 and its analogs are worth of advanced efforts to explore their potential applications as novel antimicrobial agents.
Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Oligopeptídeos/farmacologia , Substância P/farmacologia , Animais , Antibacterianos/síntese química , Antibacterianos/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Bactérias Gram-Negativas/química , Células Hep G2 , Humanos , Fatores Imunológicos/síntese química , Fatores Imunológicos/química , Lipopolissacarídeos/antagonistas & inibidores , Camundongos , Oligopeptídeos/síntese química , Oligopeptídeos/química , Células RAW 264.7 , Relação Estrutura-Atividade , Substância P/síntese química , Substância P/químicaRESUMO
Model membranes are a valuable tool to investigate the mechanism of interaction between antibiotic compounds and bacterial membranes. However, the development of supported lipid bilayer (SLB) models for Gram-negative and Gram-positive bacteria has been challenging because of the high charge and spontaneous curvature of the lipids that make up these membranes. Here we describe a method for preparing mimetic Gram-negative inner membrane and Gram-positive membrane SLBs, including asymmetric SLBs (asy-SLBs) that contain a fluorescent tracer only in the upper leaflet of the membrane. We quantified the dynamics of the lipids in these membranes with fluorescence correlation spectroscopy (FCS) and found that lipid diffusion is slower in Gram-negative SLBs/asySLBs than in Gram-positive SLBs/asySLBs. Peptide binding to these membranes was also characterized using colistin, a Gram-negative specific antibiotic. Interactions between colistin and membrane lipids phosphatidylethanolamine (PE) or cardiolipin (TOCL) were probed with pulsed-interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS). Overall, our data provide unique insight into the diffusion dynamics of lipids in Gram-negative and Gram-positive membranes as well as a novel platform for investigating the mechanism of interaction between antibiotic peptides and bacterial membrane lipids.
Assuntos
Cardiolipinas/análise , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química , Bicamadas Lipídicas/química , Peptídeos/química , Fosfatidiletanolaminas/análise , Sítios de Ligação , Espectrometria de FluorescênciaRESUMO
The structures of bacterial cell surface glycans are remarkably diverse. In spite of this diversity, the general strategies used for their assembly are limited. In one of the major processes, found in both Gram-positive and Gram-negative bacteria, the glycan is polymerized in the cytoplasm on a polyprenol lipid carrier and exported from the cytoplasm by an ATP-binding cassette (ABC) transporter. The ABC transporter actively participates in determining the chain length of the glycan substrate, which impacts functional properties of the glycoconjugate products. A subset of these systems employs an additional elaborate glycan capping strategy that dictates the size distribution of the products. The hallmarks of prototypical capped glycan systems are a chain-terminating enzyme possessing a coiled-coil molecular ruler and an ABC transporter possessing a carbohydrate-binding module, which recognizes the glycan cap. To date, detailed investigations are limited to a small number of prototypes, and here, we used our current understanding of these processes for a bioinformatics census of other examples in available genome sequences. This study not only revealed additional instances of existing terminators but also predicted new chemistries as well as systems that diverge from the established prototypes. These analyses enable some new functional hypotheses and offer a roadmap for future research.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Biologia Computacional , Bactérias Gram-Negativas/química , Bactérias Gram-Positivas/química , Polissacarídeos Bacterianos/química , Polissacarídeos Bacterianos/metabolismo , Sítios de Ligação , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Modelos MolecularesRESUMO
Mass spectrometry-based quantitative proteomic analysis has proven valuable for clinical and biotechnology-related research and development. Improvements in sensitivity, resolution, and robustness of mass analyzers have also added value. However, manual sample preparation protocols are often a bottleneck for sample throughput and can lead to poor reproducibility, especially for applications where thousands of samples per month must be analyzed. To alleviate these issues, we developed a "cells-to-peptides" automated workflow for Gram-negative bacteria and fungi that includes cell lysis, protein precipitation, resuspension, quantification, normalization, and tryptic digestion. The workflow takes 2 h to process 96 samples from cell pellets to the initiation of the tryptic digestion step and can process 384 samples in parallel. We measured the efficiency of protein extraction from various amounts of cell biomass and optimized the process for standard liquid chromatography-mass spectrometry systems. The automated workflow was tested by preparing 96 Escherichia coli samples and quantifying over 600 peptides that resulted in a median coefficient of variation of 15.8%. Similar technical variance was observed for three other organisms as measured by highly multiplexed LC-MRM-MS acquisition methods. These results show that this automated sample preparation workflow provides robust, reproducible proteomic samples for high-throughput applications.
Assuntos
Células/química , Técnicas Microbiológicas/métodos , Peptídeos/isolamento & purificação , Proteômica/métodos , Manejo de Espécimes/métodos , Fluxo de Trabalho , Automação , Proteínas de Bactérias/análise , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/química , Proteínas Fúngicas/análise , Proteínas Fúngicas/isolamento & purificação , Fungos/química , Bactérias Gram-Negativas/química , Humanos , Peptídeos/análise , Manejo de Espécimes/normasRESUMO
OBJECTIVE: Major depression (MDD) and a lifetime history of MDD are characterized by increased nitrosylation, while bipolar disorder type 1 (BP1), but not BP2, is accompanied by highly increased levels of oxidative stress and nitric oxide (NO) production. Nevertheless, it is unknown whether nitrosylation is involved in BP and whether there are differences in nitrosylation between BP1 and BP2. METHODS: Serum IgM antibodies directed against nitroso (NO)-adducts were examined in MDD, BP1, BP2 and healthy controls, namely IgM responses to NO-cysteine, NO-tryptophan (NOW), NO-arginine and NO-albumin (SBA) in association with IgA/IgM responses to LPS of Gram-negative bacteria, IgG responses to oxidized low-density lipoprotein (ox-LDL) and serum peroxides. RESULTS: Serum IgM levels against NO adducts were significantly higher in BP1 and MDD as compared with healthy controls, whereas BP2 patients occupied an intermediate position. IgM responses to NO-albumin were significantly higher in BP1 and MDD than in BP2 patients. There were highly significant associations between the IgM responses to NO-adducts and IgG responses to ox-LDL and IgA/IgM responses to Gram-negative bacteria. CONCLUSIONS: BP1 and MDD are characterized by an upregulation of the nitrosylome (the proteome of nitrosylated proteins) and increased IgM responses to nitrosylated conjugates. Increased nitrosylation may be driven by increased bacterial translocation and is associated with lipid peroxidation processes. Innate-like (B1 and marginal zone) B cells and increased nitrosylation may play a key role in the major affective disorders through activation of immune-inflammatory and oxidative pathways, cardiovascular comorbidity and impairments in antioxidant defenses, neuro-glial interactions, synaptic plasticity, neuroprotection, neurogenesis.
Assuntos
Transtorno Bipolar/metabolismo , Transtorno Depressivo Maior/metabolismo , Imunoglobulina M/imunologia , Processamento de Proteína Pós-Traducional , Proteoma/metabolismo , Regulação para Cima , Adolescente , Adulto , Idoso , Translocação Bacteriana/fisiologia , Biomarcadores/química , Biomarcadores/metabolismo , Transtorno Bipolar/classificação , Feminino , Bactérias Gram-Negativas/química , Humanos , Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Nitrosação , Proteoma/química , Proteoma/imunologia , Adulto JovemRESUMO
Outer membrane vesicles (OMVs) are vesicular nano-particles produced by Gram-negative bacteria that are recently being explored as vaccine vector. The fact that OMVs can be efficiently produced by a hypervesiculating Salmonella typhimurium strain, are packed with naturally-occurring adjuvants like lipopolysaccharides (LPS), and can be engineered to express any antigen of choice, makes them ideal candidates for vaccinology. However, it is unclear whether OMVs induce dendritic cell (DC)-mediated antigen-specific T cell responses and how immune activation is coordinated. Here, we show that OMVs induce maturation of human monocyte-derived DCs, murine bone marrow-derived DCs and CD11c+ splenic DCs. OMV-induced DC maturation was dependent on the presence of LPS and the myeloid differentiation primary response 88 (MyD88) adapter protein downstream of toll-like receptor signaling. Importantly, OMVs did not induce pyroptosis/cell death, but instead provided a significant survival benefit in DCs over non-stimulated DCs. OMVs displaying a sizeable ovalbumin fragment at the vesicle surface induce potent cross-presentation in BMDCs and splenic CD11c+ DCs to OTI CD8+ T cells, dependent on MyD88. Interestingly, the OMV-induced preference to cross-presentation was only partly dependent on the BATF3-dependent CD8a+ professional cross-presenting DC subset. Hence, an OMV-specific programming of DCs that induces maturation and provides a survival benefit for antigen presentation to T cells is identified. Additionally, for the first time, antigen-specific and potent cross-presentation of antigen-loaded OMVs to CD8+ T cells is demonstrated. These data provide mechanistical insight into the processes needed for the DC-mediated cross-presentation of OMV-derived antigens to CD8+ T cells with implications for therapeutic strategies. STATEMENT OF SIGNIFICANCE: Bacteria are primarily known to cause disease. However, recent research has focused on using engineered bacteria and its byproducts as vaccine agents. In particular, outer membrane vesicles (OMVs) have shown promise in eliciting potent immunity against a variety of pathogens. While most vaccines rely on the generation of antibodies, the control of viral replication and tumor growth is driven by cytotoxic CD8+ T cells induced by dendritic cells (DCs). As such, there is a dire need for vaccines that use DCs to elicit CD8+ T cell responses. Studying OMVs as engineered biomaterial and its interaction with DCs allows tailored induction of immunity. This study includes important findings on OMV-dendritic cell interactions and for the first time supports OMVs as vehicles for the induction of antigen-specific CD8+ T cell responses. Additionally, important mechanistical insight into the molecular pathways needed for the cross-presentation of OMV-derived antigens to CD8+ T cells is provided.
Assuntos
Apresentação de Antígeno , Antígenos de Bactérias , Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Vesículas Extracelulares/imunologia , Bactérias Gram-Negativas , Lipopolissacarídeos , Nanopartículas/química , Animais , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Vesículas Extracelulares/química , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/imunologia , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/imunologia , Camundongos , Camundongos Knockout , Monócitos/imunologiaRESUMO
Polychlorinated pesticides (PPs) were classified as persistent organic pollutants because of their toxicity, limited degradability in the environment, bioaugmentation, and accumulation in animal tissues. PPs accumulate in the environment mainly in the soils and water sediments where they are toxic to the decomposer organisms including soil bacteria and fungi. Therefore, there is an urgent need to search for the microorganisms capable of PP biodegradation which could be applied for soil bioremediation. The exact mechanism of PP microbial toxicity is unknown; however, there is evidence that it can be membrane related. To shed light on the interactions of PPs with microbial membranes, we applied Langmuir monolayers formed by phospholipids as model biomembranes. The model membranes were formed by phospholipids typical to microbial membranes: cardiolipins and phosphatidylglycerols the main components of Gram positive bacteria membranes, phosphatidylcholine typical to fungal membranes, as well as phosphatidylethanolamine found in the inner membranes of Gram negative bacteria. For the studies, the most ecotoxic PPs and their water-soluble metabolites were chosen. The monolayers were studied with the application of mutually complementary techniques: Langmuir technique, grazing incidence X-ray diffraction, and PM-IRRAS spectroscopy. It turned out that the cyclodiene PPs are more membrane active than monocyclic PPs and that the possibility of their incorporation is strictly related to the phospholipid structure. The membranes prepared with cardiolipin turned out to be especially resistant to PP incorporation. Regarding the metabolites, pentachlorophenol turned out to be especially structure breaking, affecting the molecular organization of all of the investigated phospholipids.
Assuntos
Bactérias Gram-Negativas/química , Hidrocarbonetos Clorados/química , Membranas Intracelulares/química , Modelos Biológicos , Praguicidas/química , Fosfolipídeos/química , Animais , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/metabolismo , Hidrocarbonetos Clorados/metabolismo , Membranas Intracelulares/metabolismo , Estrutura Molecular , Tamanho da Partícula , Praguicidas/metabolismo , Fosfolipídeos/metabolismo , Propriedades de SuperfícieRESUMO
Lipopolysaccharide (LPS) is a major microbial mediator for tissue injury and sepsis resulting from Gram-negative bacterial infection. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute-phase proteins, but the relationship between SAA expression and LPS-induced tissue injury remains unclear. Here, we report that mice with inducible transgenic expression of human SAA1 are partially protected against inflammatory response and lung injury caused by LPS and cecal ligation and puncture (CLP). In comparison, transgenic SAA1 does not attenuate TNFα-induced lung inflammation and injury. The SAA1 expression level correlates inversely with the endotoxin concentrations in serum and lung tissues since SAA1 binds directly to LPS to form a complex that promotes LPS uptake by macrophages. Disruption of the SAA1-LPS interaction with a SAA1-derived peptide partially reduces the protective effect and exacerbates inflammation. These findings demonstrate that acute-phase SAA provides innate feedback protection against LPS-induced inflammation and tissue injury.
Assuntos
Infecções por Bactérias Gram-Negativas/genética , Inflamação/genética , Lesão Pulmonar/genética , Sepse/genética , Proteína Amiloide A Sérica/genética , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica/imunologia , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/microbiologia , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Inflamação/imunologia , Inflamação/microbiologia , Lipopolissacarídeos/química , Lipopolissacarídeos/farmacologia , Lesão Pulmonar/microbiologia , Lesão Pulmonar/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Camundongos Transgênicos , Sepse/imunologia , Sepse/microbiologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Gram-negative bacilli such as Pseudomonas spp., Pseudoalteromonas sp., Angiococcus sp., Archangium sp., Burkholderia spp., Chromobacterium sp., Chondromyces sp., Cystobacter sp., Jahnella sp., Janthinobacterium sp., Lysobacter spp., Paraliomyxa sp., Photobacterium spp., Photorhabdus sp., Pontibacter sp., Ruegeria sp., Serratia sp., Sorangium sp., Sphingomonas sp., and Xenorhabdus spp. produce an enormous array of short peptides of 30 residues or fewer that are potential pharmaceutical drugs and/or biocontrol agents. The need for novel lead antibiotic compounds is urgent due to increasing drug resistance, and this review summarises 150 Gram-negative bacilli-derived compounds reported since 2000, including 40 cyclic lipopeptides from Pseudomonas spp.; nine aromatic peptides; eight glycopeptides; 45 different cyclic lipopeptides; 24 linear lipopeptides; eight thiopeptides; one lasso peptide; ten typical cyclic peptides; and five standard linear peptides. The current and potential therapeutic applications of these peptides, including structures and antituberculotic, anti-cyanobacterial, antifungal, antibacterial, antiviral, insecticidal, and antiprotozoal activities are discussed.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/química , Bactérias Gram-Negativas/química , Peptídeos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Bactérias Gram-Negativas/metabolismo , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologiaRESUMO
Lipopolysaccharides (LPS, endotoxin) are complex and indispensable components of the outer membrane of most Gram-negative bacteria. They represent stimuli for many biological effects with pathophysiological character. Recombinant therapeutic proteins that are manufactured using biotechnological processes are prone to LPS contaminations due to their ubiquitous occurrence. The maximum endotoxin load of recombinant therapeutic proteins must be below the pyrogenic threshold. Certain matrices that are commonly used for recombinant therapeutic proteins show a phenomenon called "Low Endotoxin Recovery (LER)". LER is defined as the loss of detectable endotoxin activity over time using compendial Limulus amebocyte lysate (LAL) assays when undiluted products are spiked with known amount of endotoxin standards. Because LER poses potential risks that endotoxin contaminations in products may be underestimated or undetected by the LAL assay, the United States (U.S.) Food and Drug Administration's (FDA's) Center for Drug Evaluation and Research (CDER) has recently started requesting that companies conduct endotoxin spike/hold recovery studies to determine whether a given biological product causes LER. Here, we have performed an analysis of different LPS preparations with relevant detergents studying their acyl chain phase transition, their aggregate structures, their size distributions, and binding affinity with a particular anti-endotoxin peptide, and correlating it with the respective data in the macrophage activation test. In this way, we have worked out biophysical parameters that are important for an understanding of LER.
Assuntos
Bioensaio/métodos , Lipopolissacarídeos/química , Animais , Endotoxinas/química , Bactérias Gram-Negativas/química , Caranguejos Ferradura/química , Proteínas de Membrana/químicaRESUMO
Betulin derivatives containing a 1,2,3-triazole ring possess a wide spectrum of biological activities, including antiviral, anticancer, and antibacterial activity. A series of novel triazoles were prepared by the 1,3-dipolar cycloaddition reaction between the alkyne derivatives of betulin and organic azides. The chemical structures of the obtained compounds were defined by ¹H and 13C NMR, IR, and high-resolution mass spectrometry (HR-MS) analysis. The target triazoles were screened for their antiviral activity against DNA and RNA viruses. The cytotoxic activity of the obtained compounds 5a-k and 6a-h was determined using five human cancer cell lines (T47D, MCF-7, SNB-19, Colo-829, and C-32) by a WST-1 assay. The bistriazole 6b displayed a promising IC50 value (0.05 µM) against the human ductal carcinoma T47D (500-fold higher potency than cisplatin). The microdilution method was applied for an evaluation of the antimicrobial activity of all of the compounds. The triazole 5e containing a 3'-deoxythymidine-5'-yl moiety exhibited antibacterial activity against two gram-negative bacteria vz. Klebsiellapneumoniae and Escherichia coli (minimal inhibitory concentration (MIC) range of 0.95-1.95 µM).