RESUMEN
Host defense peptides (HDPs), also named antimicrobial peptides (AMPs), are increasingly being recognized for serving multiple functions in protecting the host from infection and disease. Previous studies have shown that various HDPs can also neutralize lipopolysaccharide (LPS, endotoxin), as well as lipoteichoic acid (LTA), inducing macrophage activation. However, antimicrobial activity is usually accompanied by systemic toxicity which makes it difficult to use HDPs as antiendotoxin agents. Here we report that key parameters can uncouple these two functions yielding nontoxic peptides with potent LPS and LTA neutralization activities in vitro and in animal models. The data reveal that peptide length, the number, and the placement of positive charges are important parameters involved in LPS neutralization. Crucially, the peptide exhibited a separation between its membrane-disrupting and antimicrobial properties, effectively decoupling them from its ability to neutralize LPS. This essential distinction prevented systemic toxicity and led to the peptide's complete rescue of mice suffering from severe septic shock in two distinct models. Strong binding to LPS, changes in structure, and oligomerization state upon LPS binding were important factors that determined the activity of the peptides. In the face of the increasing threat of septic shock worldwide, it is crucial to grasp how we can neutralize harmful substances like LPS. This knowledge is vital for creating nontoxic treatments for sepsis.
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Antibiotic-resistant bacterial infections have increased the prevalence of sepsis and septic shock mortality worldwide and have become a global concern. Antimicrobial peptides (AMPs) show remarkable properties for developing new antimicrobial agents and host response modulatory therapies. A new series of AMPs derived from pexiganan (MSI-78) were synthesized. The positively charged amino acids were segregated at their N- and C-termini, and the rest of the amino acids created a hydrophobic core surrounded by positive charges and were modified to simulate the lipopolysaccharide (LPS). The peptides were investigated for their antimicrobial activity and LPS-induced cytokine release inhibition profile. Various biochemical and biophysical methods were used, including attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, microscale thermophoresis (MST), and electron microscopy. Two new AMPs, MSI-Seg-F2F and MSI-N7K, preserved their neutralizing endotoxin activity while reducing toxicity and hemolytic activity. Combining all of these properties makes the designed peptides potential candidates to eradicate bacterial infection and detoxify LPS, which might be useful for sepsis treatment.
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The HIV envelope protein gp160 comprises two subunits, gp120 and gp41, responsible for receptor binding and membrane fusion during viral entry, respectively. In the course of the membrane fusion process, gp41 undergoes a conformational change, leading to the formation of a six-helix bundle (SHB), which ultimately drives membrane fusion. The gp41 C-terminal and N-terminal heptad repeats (CHR and NHR) interact with one another to form the SHB, and this step can be targeted by peptide inhibitors, which are used in the clinic to mitigate HIV infection. Here, we discover the calcium interaction motifs (CIMs) in the gp41 CHR and NHR regions via NMR spectroscopy. We find that the assembly of the CHR-NHR SHB is facilitated in Ca2+-containing media and impaired in CIM mutants. Of note, the clinically approved, gp41-derived fusion inhibitor T20, which does not contain the CIM motif, exhibits reduced inhibitory efficiency when challenged with calcium. This finding could have important implications for the development of better fusion inhibitors for HIV.
Asunto(s)
Infecciones por VIH , VIH-1 , Secuencia de Aminoácidos , Calcio/metabolismo , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Humanos , Fusión de MembranaRESUMEN
Lung infection is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients and is mainly dominated by Pseudomonas aeruginosa. Treatment of CF-associated lung infections is problematic because the drugs are vulnerable to multidrug-resistant pathogens, many of which are major biofilm producers like P. aeruginosa. Antimicrobial peptides (AMPs) are essential components in all life forms and exhibit antimicrobial activity. Here we investigated a series of AMPs (d,l-K6L9), each composed of six lysines and nine leucines but differing in their sequence composed of l- and d-amino acids. The d,l-K6L9 peptides showed antimicrobial and antibiofilm activities against P. aeruginosa from CF patients. Furthermore, the data revealed that the d,l-K6L9 peptides are stable and resistant to degradation by CF sputum proteases and maintain their activity in a CF sputum environment. Additionally, the d,l-K6L9 peptides do not induce bacterial resistance. Overall, these findings should assist in the future development of alternative treatments against resistant bacterial biofilms.
Asunto(s)
Antiinfecciosos , Fibrosis Quística , Infecciones por Pseudomonas , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Antiinfecciosos/uso terapéutico , Péptidos Antimicrobianos , Biopelículas , Fibrosis Quística/tratamiento farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosaRESUMEN
Antimicrobial peptides (AMPs) have the potential to treat multidrug-resistant bacterial infections. However, the clinical application of AMPs is prevented by their toxicity and poor proteolytic stability. Here, a site-specific approach is used to generate new AMPs to improve their efficacy against bacterial pathogens while reducing their toxicity. We modified and generated a new series of antimicrobial peptides from the leucine- and lysine-rich antimicrobial peptide Amp1L (LKLLKKLLKKLLKLL) by the site-specific incorporation of an isopeptide bond while retaining the peptide's size, sequence, charge, and molecular weight. This single bond switch provides the peptides with a weak helical conformation, strong antimicrobial activity, resistance to proteolytic degradation, low toxicity, and lower hemolytic activity. This new site-specific approach offers a powerful tool for developing potent and nontoxic antimicrobial drugs.
Asunto(s)
Antiinfecciosos , Péptidos Catiónicos Antimicrobianos , Antibacterianos/farmacología , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Antimicrobianos , Bacterias/metabolismo , Farmacorresistencia Bacteriana Múltiple , Pruebas de Sensibilidad MicrobianaRESUMEN
The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the two-component systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (Moskowitz, S. M.; Antimicrob. Agents Chemother. 2012, 56, 1019-1030; Cheng, H. Y.; J. Biomed. Sci. 2010, 17, 60). Our data suggested that the two systems have opposing effects on the properties of Salmonella enterica. The knockout of PhoPQ made the bacteria more susceptible to AMPs by making the surface less rigid, more polarized, and permeable with a slightly more negatively charged cell wall. In addition, the periplasmic space is thinner. In contrast, the knockout of PmrAB did not affect its susceptibility, while it made the bacterial outer layer very rigid, less polarized, and less permeable than the other two mutants, with a negatively charged cell wall similar to the WT. Overall, the data suggest that the coexistence of systems with opposing effects on the biophysical properties of the bacteria contribute to their membrane flexibility, which, on the one hand, is important to accommodate changing environments and, on the other hand, may inhibit the development of meaningful resistance to AMPs.
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Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Infecciones por Salmonella/tratamiento farmacológico , Salmonella enterica/efectos de los fármacos , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Periplasma/efectos de los fármacos , Infecciones por Salmonella/metabolismo , Infecciones por Salmonella/microbiología , Salmonella enterica/aislamiento & purificación , Salmonella enterica/metabolismo , SerogrupoRESUMEN
Antimicrobial peptides (AMPs), which can be modified to kill a broad spectrum of microoganisms or a specific microorganism, are considered as promising alternatives to combat the rapidly widespread, resistant bacterial infections. However, there are still several obstacles to overcome. These include toxicity, stability, and the ability to interfere with the immune response and bacterial resistance. To overcome these challenges, we herein replaced the regular peptide bonds with isopeptide bonds to produce new AMPs based on the well-known synthetic peptides Amp1L and MSI-78 (pexiganan). Two new peptides Amp1EP and MSIEP were generated while retaining properties such as size, sequence, charge, and molecular weight. These new peptides have reduced toxicity toward murine macrophage (RAW 264.7) cells, human monocytic (THP-1) cells, and human red blood cells (hRBCs) and enhanced the stability toward proteolytic degradation. Importantly, the new peptides do not repress the pro-inflammatory cytokine and hence should not modulate the immune response. Structurally, the new peptides, Amp1EP and MSIEP, have a structure of random coils in contrast to the helical structures of the parental peptides as revealed by circular dichroism (CD) analysis. Their mode of action, assessed by flow cytometry, includes permeabilization of the bacterial membrane. Overall, we present here a new approach to modulate AMPs to develop antimicrobial peptides for future therapeutic purposes.
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Bacterias , Animales , Dicroismo Circular , Humanos , Ratones , Proteínas Citotóxicas Formadoras de PorosRESUMEN
The outcome of an antibiotic treatment on the growth capacity of bacteria is largely dependent on the initial population size (Inoculum Effect). We characterized and built a model of this effect in E. coli cultures using a large variety of antimicrobials, including conventional antibiotics, and for the first time, cationic antimicrobial peptides (CAMPs). Our results show that all classes of antimicrobial drugs induce an inoculum effect, which, as we explain, implies that the dynamic is bistable: For a range of anti-microbial densities, a very small inoculum decays whereas a larger inoculum grows, and the threshold inoculum depends on the drug concentration. We characterized three distinct classes of drug-induced bistable growth dynamics and demonstrate that in rich medium, CAMPs correspond to the simplest class, bacteriostatic antibiotics to the second class, and all other traditional antibiotics to the third, more complex class. These findings provide a unifying universal framework for describing the dynamics of the inoculum effect induced by antimicrobials with inherently different killing mechanisms.
RESUMEN
Bacterial resistance to antibiotics is a major concern worldwide, leading to an extensive search for alternative drugs. Promising candidates are antimicrobial peptides (AMPs), innate immunity molecules, shown to be highly efficient against multidrug resistant bacteria. Therefore, it is essential to study bacterial resistance mechanisms against them. For that purpose, we used experimental evolution, and isolated a Salmonella enterica serovar typhimurium-resistant line to the AMP 4DK5L7. This AMP displayed promising features including widespread activity against Gram-negative bacteria and protection from proteolytic degradation. However, the resistance that evolved in the isolated strain was particularly high. Whole genome sequencing revealed that five spontaneous mutations had evolved. Of these, three are novel in the context of acquired AMP resistance. Two mutations are related to the AcrAB-TolC multidrug efflux pump. One occurred in AcrB, the substrate-binding domain of the system, and the second in RamR, a transcriptional regulator of the system. Together, the mutations increased the minimal inhibitory concentration (MIC) by twofold toward this AMP. Moreover, the mutation in AcrB induced hypersusceptibility toward ampicillin and colistin. The last mutation occurred in Skp, a periplasmic chaperone that participates in the biogenesis of outer membrane proteins (OMPs). This mutation increased the MIC by twofold to 4DK5L7 and by fourfold to another AMP, seg5D. Proteomic analysis revealed that the mutation abolished Skp expression, reduced OMP abundance, and increased DegP levels. DegP, a protease that was reported to have an additional chaperone activity, escorts OMPs through the periplasm along with Skp, but is also associated with AMP resistance. In conclusion, our data demonstrate that both loss of Skp and manipulation of the AcrAB-TolC system are alternative strategies of AMP acquired resistance in Salmonella typhimurium and might represent a common mechanism in other Gram-negative bacteria.
RESUMEN
Recruited blood monocytes contribute to the establishment, perpetuation, and resolution of tissue inflammation. Specifically, in the inflamed intestine, monocyte ablation was shown to ameliorate colitis scores in preclinical animal models. However, the majority of intestinal macrophages that seed the healthy gut are also monocyte derived. Monocyte ablation aimed to curb inflammation would therefore likely interfere with intestinal homeostasis. In this study, we used a TLR2 trans-membrane peptide that blocks TLR2 dimerization that is critical for TLR2/1 and TLR2/6 heterodimer signaling to blunt inflammation in a murine colitis model. We show that although the TLR2 peptide treatment ameliorated colitis, it allowed recruited monocytes to give rise to macrophages that lack the detrimental proinflammatory gene signature and reduced potentially damaging neutrophil infiltrates. Finally, we demonstrate TLR blocking activity of the peptide on in vitro cultured human monocyte-derived macrophages. Collectively, we provide a significantly improved anti-inflammatory TLR2 peptide and critical insights in its mechanism of action toward future potential use in the clinic.
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Colitis/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Macrófagos/inmunología , Receptor Toll-Like 2/metabolismo , Animales , Células Cultivadas , Dimerización , Modelos Animales de Enfermedad , Homeostasis , Humanos , Inflamación , Ratones , Ratones Endogámicos C57BL , Transducción de SeñalRESUMEN
The human immunodeficiency virus enters its host cells by membrane fusion, initiated by the gp41 subunit of its envelope protein. gp41 has also been shown to bind T-cell receptor (TCR) complex components, interfering with TCR signaling leading to reduced T-cell activation. This immunoinhibitory activity is suggested to occur during the membrane fusion process and is attributed to various membranotropic regions of the gp41 ectodomain and to the transmembrane domain. Although extensively studied, the cytosolic region of gp41, termed the cytoplasmic tail (CT), has not been examined in the context of immune suppression. Here we investigated whether the CT inhibits T-cell activation in different T-cell models by utilizing gp41-derived peptides and expressed full gp41 proteins. We found that a conserved region of the CT, termed lentiviral lytic peptide 2 (LLP2), specifically inhibits the activation of mouse, Jurkat, and human primary T-cells. This inhibition resulted in reduced T-cell proliferation, gene expression, cytokine secretion, and cell surface expression of CD69. Differential activation of the TCR signaling cascade revealed that CT-based immune suppression occurs downstream of the TCR complex. Moreover, LLP2 peptide treatment of Jurkat and primary human T-cells impaired Akt but not NFκB and ERK1/2 activation, suggesting that immune suppression occurs through the Akt pathway. These findings identify a novel gp41 T-cell suppressive element with a unique inhibitory mechanism that can take place post-membrane fusion.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Secuencias de Aminoácidos , Animales , Proliferación Celular , Citocinas/genética , Citocinas/metabolismo , Expresión Génica , Proteína gp41 de Envoltorio del VIH/química , Humanos , Células Jurkat , Ratones , Ratones Endogámicos C57BL , Fosforilación , Dominios Proteicos , Proteínas Proto-Oncogénicas c-akt/química , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células RAW 264.7 , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/virología , Serina-Treonina Quinasas TOR/química , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.
Asunto(s)
Glicoproteínas/metabolismo , Infecciones por Hantavirus/metabolismo , Fiebre Hemorrágica con Síndrome Renal/metabolismo , Multimerización de Proteína , Virus Puumala/fisiología , Fracciones Subcelulares/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Membrana Celular/virología , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Glicoproteínas/química , Aparato de Golgi/metabolismo , Aparato de Golgi/virología , Células HEK293 , Infecciones por Hantavirus/virología , Fiebre Hemorrágica con Síndrome Renal/virología , Humanos , Proteínas Luminiscentes/metabolismo , Fracciones Subcelulares/virología , Células Vero , Proteínas del Envoltorio Viral/químicaRESUMEN
The ability of the Lentivirus HIV-1 to inhibit T-cell activation by its gp41 fusion protein is well documented, yet limited data exists regarding other viral fusion proteins. HIV-1 utilizes membrane binding region of gp41 to inhibit T-cell receptor (TCR) complex activation. Here we examined whether this T-cell suppression strategy is unique to the HIV-1 gp41. We focused on T-cell modulation by the gp21 fusion peptide (FP) of the Human T-lymphotropic Virus 1 (HTLV-1), a Deltaretrovirus that like HIV infects CD4+ T-cells. Using mouse and human in-vitro T-cell models together with in-vivo T-cell hyper activation mouse model, we reveal that HTLV-1's FP inhibits T-cell activation and unlike the HIV FP, bypasses the TCR complex. HTLV FP inhibition induces a decrease in Th1 and an elevation in Th2 responses observed in mRNA, cytokine and transcription factor profiles. Administration of the HTLV FP in a T-cell hyper activation mouse model of multiple sclerosis alleviated symptoms and delayed disease onset. We further pinpointed the modulatory region within HTLV-1's FP to the same region previously identified as the HIV-1 FP active region, suggesting that through convergent evolution both viruses have obtained the ability to modulate T-cells using the same region of their fusion protein. Overall, our findings suggest that fusion protein based T-cell modulation may be a common viral trait.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/inmunología , Virus Linfotrópico T Tipo 1 Humano/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Proteínas Virales de Fusión/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Secuencia de Aminoácidos , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Activación de Linfocitos , Fusión de Membrana , Ratones , Ratones Endogámicos C57BL , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
Multidrug resistant bacteria possess various mechanisms that can sense environmental stresses such as antibiotics and antimicrobial peptides and rapidly respond to defend themselves. Two known defense strategies are biofilm formation and lipopolysaccharide (LPS) modification. Though LPS modifications are observed in biofilm-embedded bacteria, their effect on biofilm formation is unknown. Using biochemical and biophysical methods coupled with confocal microscopy, atomic force microscopy, and transmission electron microscopy, we show that biofilm formation is promoted in a Pseudomonas aeruginosa PAO1 strain with a loss of function mutation in the arnB gene. This loss of function prevents the addition of the positively charged sugar 4-amino-4-deoxy-l-arabinose to lipid A of LPS under restrictive magnesium conditions. The data reveal that the arnB mutant, which is susceptible to antimicrobial peptides, forms a biofilm that is more robust than that of the wild type. This is in line with the observations that the arnB mutant exhibits outer surface properties such as hydrophobicity and net negative charge that promote the formation of biofilms. Moreover, when grown under Mg2+ limitation, both the wild type and the arnB mutant exhibited a reduction in the level of membrane-bound polysaccharides. The data suggest that the loss of polysaccharides exposes the membrane and alters its biophysical properties, which in turn leads to more biofilm formation. In summary, we show for the first time that blocking a specific lipid A modification promotes biofilm formation, suggesting a trade-off between LPS remodeling and resistance mechanisms of biofilm formation.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas , Biopelículas/efectos de los fármacos , Lípido A , Polisacáridos Bacterianos , Pseudomonas aeruginosa/fisiología , Péptidos Catiónicos Antimicrobianos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/crecimiento & desarrollo , Lípido A/genética , Lípido A/metabolismo , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismoRESUMEN
Cell surfaces are densely populated with various proteins. Aggregation of these proteins to nanoscale clusters can be critical for various cellular functions such as signaling, motility and division. Quantitative characterization of corresponding structures and their changes might be useful to understand these basic cell processes and serve as an early marker of cellular stress or diseases. Atomic force microscopy (AFM) allows high-resolution imaging of cell surface structures, resolving fine details of these structures. Moreover, AFM enables simultaneous imaging of cell surface morphology and mapping of its' mechanical characteristics. This review focuses on applying the fractal dimension measure as a sensitive method to quantify single cell surface structures and their changes from AFM images.
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Membrana Celular/química , Membrana Celular/ultraestructura , Fractales , Microscopía de Fuerza Atómica , Propiedades de SuperficieRESUMEN
Recently, a single study revealed a new complex composed of Toll-like receptor 4 (TLR4), TLR6, and CD36 induced by fibrillary Aß peptides, the hallmark of Alzheimer's disease. Unlike TLRs located on the plasma membrane that dimerize on the membrane after ligand binding to their extracellular domain, the TLR4-TLR6-CD36 complex assembly has been suggested to be induced by intracellular signals from CD36, similar to integrin inside-out signaling. However, the assembly site of TLR4-TLR6-CD36 and the domains participating in Aß-induced signaling is still unknown. By interfering with TLR4-TLR6 dimerization using a TLR4-derived peptide, we show that receptor assembly is abrogated within the plasma membrane. Furthermore, we reveal that the transmembrane domains of TLR4 and TLR6 have an essential role in receptor dimerization and activation. Inhibition of TLR4-TLR6 assembly was associated with reduced secretion of proinflammatory mediators from microglia cells, ultimately rescuing neurons from death. Our findings support TLR4-TLR6 dimerization induced by Aß. Moreover, we shed new light on TLR4-TLR6 assembly and localization and show the potential of inhibiting TLR4-TLR6 dimerization as a treatment of Alzheimer's disease.
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Péptidos beta-Amiloides/toxicidad , Antígenos CD36/metabolismo , Microglía/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 6/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/antagonistas & inhibidores , Animales , Antígenos CD36/química , Antígenos CD36/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Transferencia Resonante de Energía de Fluorescencia , Inmunoprecipitación , Ligandos , Ratones , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/patología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/efectos de los fármacos , Neuronas/inmunología , Neuronas/patología , Nootrópicos/química , Nootrópicos/metabolismo , Nootrópicos/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Multimerización de Proteína/efectos de los fármacos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/química , Receptor Toll-Like 4/genética , Receptor Toll-Like 6/antagonistas & inhibidores , Receptor Toll-Like 6/química , Receptor Toll-Like 6/genéticaRESUMEN
The HIV gp160 envelope fusion protein is situated in the viral membrane and mediates virus entry into its host cell. Increasing evidence suggests that virtually all parts of the HIV envelope are structurally and functionally dependent on membranes. Protein-lipid interactions and membrane properties influence the dynamics of a manifold of gp160 biological activities such as membrane fusion, immune suppression and gp160 incorporation into virions during HIV budding and assembly. In the following we will summarize our current understanding of this interdependence between membrane interaction, structural conformation and functionality of the different gp160 domains. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/química , Proteínas gp160 de Envoltorio del VIH/química , Proteína gp41 de Envoltorio del VIH/química , VIH-1/química , Microdominios de Membrana/química , Esfingomielinas/química , Secuencia de Aminoácidos , Expresión Génica , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/genética , Proteínas gp160 de Envoltorio del VIH/inmunología , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/inmunología , VIH-1/inmunología , Interacciones Huésped-Patógeno , Humanos , Fusión de Membrana , Microdominios de Membrana/inmunología , Microdominios de Membrana/virología , Conformación Proteica , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Esfingomielinas/inmunología , Linfocitos T/inmunología , Linfocitos T/virología , Ensamble de Virus/inmunología , Liberación del Virus/inmunologíaRESUMEN
Multidrug-resistant bacteria are a growing problem worldwide. One extensively studied resistance mechanism is biofilm colonization-microbial colonies formed by many Gram-positive and Gram-negative bacteria species. Cationic antimicrobial peptides (AMPs) are innate immune system molecules serving as a first line of defense in fighting invading pathogens. The AMPs' underlying mechanism and biophysical properties required for anti-biofilm activity are not fully known. Here we present protocols for investigating AMPs' biological activity against major stages of biofilm life cycle, namely, planktonic stage (MIC assay), initial adhesion to surfaces (bacterial attachment assay), and formation or degradation of sessile microcolonies (biofilm formation and degradation assays). Furthermore, we demonstrate experiments that allow determination and comparison between peptide biophysical properties (secondary structure, hydrophobicity, and oligomerization) and how they affect their mechanism (peptide-binding assays) of anti-biofilm activity.
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Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Biopelículas/efectos de los fármacos , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Adhesión Bacteriana/efectos de los fármacos , Interacciones Hidrofóbicas e Hidrofílicas , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía Confocal , Multimerización de Proteína , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The persistence of HIV in resting memory CD4+ T cells at a latent state is considered as the major barrier on the path to achieve a cure for HIV. Proteasome inhibitors (PIs) were previously reported as latency reversing agents (LRAs) but the mechanism underlying this function is yet unclear. Here we demonstrate that PIs reactivate latent HIV ex vivo without global T cell activation, and may facilitate host innate immune responses. Mechanistically, latent HIV reactivation induced by PIs is mediated by heat shock factor 1 (HSF1) via the recruitment of the heat shock protein (HSP) 90-positive transcriptional elongation factor b (p-TEFb) complex. Specifically, HSP90 downstream HSF1 gives positive feedback to the reactivation process through binding to cyclin-dependent kinase 9 (CDK9) and preventing it from undergoing degradation by the proteasome. Overall, these findings suggest proteasome inhibitors as potential latency reversing agents. In addition, HSF1/HSP90 involved in HIV transcription elongation, may serve as therapeutic targets in HIV eradication.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Quinasa 9 Dependiente de la Ciclina/metabolismo , VIH-1/fisiología , Proteínas HSP90 de Choque Térmico/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Quinasa 9 Dependiente de la Ciclina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Proteínas HSP90 de Choque Térmico/genética , Factores de Transcripción del Choque Térmico , Humanos , Masculino , Complejo de la Endopetidasa Proteasomal/genética , Elongación de la Transcripción Genética/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiologíaRESUMEN
Pseudomonas aeruginosa is the major microorganism colonizing the respiratory epithelium in cystic fibrosis (CF) sufferers. The widespread use of available antibiotics has drastically reduced their efficacy, and antimicrobial peptides (AMPs) are a promising alternative. Among them, the frog skin-derived AMPs, i.e., Esc(1-21) and its diastereomer, Esc(1-21)-1c, have recently shown potent activity against free-living and sessile forms of P. aeruginosa Importantly, this pathogen also escapes antibiotics treatment by invading airway epithelial cells. Here, we demonstrate that both AMPs kill Pseudomonas once internalized into bronchial cells which express either the functional or the ΔF508 mutant of the CF transmembrane conductance regulator. A higher efficacy is displayed by Esc(1-21)-1c (90% killing at 15 µM in 1 h). We also show the peptides' ability to stimulate migration of these cells and restore the induction of cell migration that is inhibited by Pseudomonas lipopolysaccharide when used at concentrations mimicking lung infection. This property of AMPs was not investigated before. Our findings suggest new therapeutics that not only eliminate bacteria but also can promote reepithelialization of the injured infected tissue. Confocal microscopy indicated that both peptides are intracellularly localized with a different distribution. Biochemical analyses highlighted that Esc(1-21)-1c is significantly more resistant than the all-l peptide to bacterial and human elastase, which is abundant in CF lungs. Besides proposing a plausible mechanism underlying the properties of the two AMPs, we discuss the data with regard to differences between them and suggest Esc(1-21)-1c as a candidate for the development of a new multifunctional drug against Pseudomonas respiratory infections.