RESUMO
SignificanceSimilar to mammalian TLR4/MD-2, the Toll9/MD-2-like protein complex in the silkworm, Bombyx mori, acts as an innate pattern-recognition receptor that recognizes lipopolysaccharide (LPS) and induces LPS-stimulated expression of antimicrobial peptides such as cecropins. Here, we report that papiliocin, a cecropin-like insect antimicrobial peptide from the swallowtail butterfly, competitively inhibits the LPS-TLR4/MD-2 interaction by directly binding to human TLR4/MD-2. Structural elements in papiliocin, which are important in inhibiting TLR4 signaling via direct binding, are highly conserved among insect cecropins, indicating that its TLR4-antagonistic activity may be related to insect Toll9-mediated immune response against microbial infection. This study highlights the potential of papiliocin as a potent TLR4 antagonist and safe peptide antibiotic for treating gram-negative sepsis.
Assuntos
Anti-Infecciosos Locais/farmacologia , Peptídeos Antimicrobianos/farmacologia , Borboletas/imunologia , Imunidade Inata/efeitos dos fármacos , Proteínas de Insetos/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , Animais , Anti-Infecciosos Locais/química , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Feminino , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Receptor 4 Toll-Like/metabolismoRESUMO
Carbapenem-resistant Acinetobacter baumannii (CRAB) is a well-known harmful bacterium that causes severe health disorders and dysregulates the host immune response associated with inflammation. Upon examining the suppressive activity of natural flavonoid rhamnetin on various pro-inflammatory cytokines in a CRAB-induced septic shock mouse model, we found that rhamnetin inhibited the production of IL-1ß and IL-18, two pro-inflammatory cytokines associated with pyroptotic cell death, a process dependent on caspase-1. In this study, we investigated the antioxidant and anti-apoptotic activities of rhamnetin and the underlying mechanism of action in a CRAB infection. In the CRAB-induced septic shock mouse model, rhamnetin reduced the level of lipopolysaccharide (LPS) in lung lysates, resulting in the inhibition of TLR4-mediated inflammatory signaling. Notably, rhamnetin reduced intracellular reactive oxygen species (ROS) generation in macrophages and inhibited apoptotic and pyroptotic cell injury induced by CRAB infection. Therefore, rhamnetin inhibited LPS-induced pro-inflammatory mediators, hindering apoptotic and pyroptotic processes and contributing to a recovery effect in CRAB-induced sepsis mice by suppressing oxidative stress. Taken together, our study presents the potential role of rhamnetin in protecting against oxidative damage induced by CRAB infection through a TLR4 and ROS-mediated pyroptotic pathway, showing an alternative mechanism for sepsis prevention. Therefore, rhamnetin is a promising therapeutic candidate for treating CRAB-induced sepsis.
Assuntos
Acinetobacter baumannii , Sepse , Choque Séptico , Camundongos , Animais , Espécies Reativas de Oxigênio/farmacologia , Lipopolissacarídeos/toxicidade , Receptor 4 Toll-Like , Sepse/induzido quimicamente , Sepse/tratamento farmacológico , Citocinas/farmacologia , Carbapenêmicos/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Testes de Sensibilidade MicrobianaRESUMO
Enterococcus faecalis has recently shown signs of high antibiotic resistance. These bacteria can endure extremes of temperature and this may be due to the high thermostability of its proteins. E. faecalis has two acyl carrier proteins (ACPs), AcpA (EfAcpA), which is essential for de novo fatty acid synthesis (FAS), and EfAcpB, which plays an auxiliary role in the incorporation of exogenous fatty acids. Structural studies on EfAcpA and its interaction with FAS enzymes have not yet been reported. Here, we investigated the structures of EfAcpA using NMR spectroscopy, showing that EfAcpA consists of three α-helices with a long α2α3 loop, while the other ACPs have four α-helices. CD experiments showed that the melting temperature of EfAcpA is 76.3 °C and the Ala mutation for Ile10 reduced it dramatically by 29.5 °C. Highly conserved Ile10 of EfAcpA mediates compact intramolecular packing and promotes high thermostability. A docking simulation of EfAcpA and ß-ketoacyl-ACP synthase III (EfKAS III) showed that the α2α3 loop of EfAcpA contributes to specific protein-protein interactions (PPI) with EfKAS III. Unconserved charged residues, Lys52 and Glu54, in the α2α3 loop of EfAcpA formed specific electrostatic interactions with Asp 226 and Arg217 of EfKAS III, respectively. Binding interactions between EfAcpA and EfKASIII may provide insights for designing PPI inhibitors targeting FAS in E. faecalis to overcome its antibacterial resistance.
Assuntos
Proteína de Transporte de Acila , Enterococcus faecalis , Ácidos Graxos , Proteína de Transporte de Acila/química , Ácidos Graxos/biossíntese , Proteínas de Bactérias/químicaRESUMO
In sepsis, the persistence of uncontrolled inflammatory response of infected host cells eventually leads to severe lung and organ failure and, ultimately, death. Carbapenem-resistant Acinetobacter baumannii (CRAB), causative bacteria of sepsis and lung failure in acute cases, belongs to a group of critical pathogens that cannot be eradicated using the currently available antibiotics. This underlines the necessity of developing new modes of therapeutics that can control sepsis at the initial stages. In this study, we investigated the anti-inflammatory activities in vitro and in vivo and the antiseptic effects of rhamnetin, a naturally occurring flavonoid. We found that among its isoforms, the potency of rhamnetin was less explored but rhamnetin possessed superior anti-inflammatory activity with least cytotoxicity. Rhamnetin showed significant anti-inflammatory effects in lipopolysaccharide-, CRAB-, and Escherichia coli (E. coli)-stimulated mouse macrophages by inhibiting the release of interleukin-6 and nitric oxide. In a mouse model of sepsis infected with clinically isolated CRAB or E. coli, rhamnetin significantly reduced the bacterial burden in the organs. In addition, normalized pro-inflammatory cytokine levels in lung lysates and histological analysis of lung tissue indicated alleviation of lung damage. This study implies that a potent natural product such as rhamnetin could be a future therapeutic for treating carbapenem-resistant gram-negative sepsis.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Sepse , Camundongos , Animais , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Flavonoides/farmacologia , Escherichia coli , Carbapenêmicos/farmacologia , Carbapenêmicos/uso terapêutico , Sepse/tratamento farmacológico , Sepse/microbiologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Modelos Animais de Doenças , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Testes de Sensibilidade MicrobianaRESUMO
Constant remodeling is necessary for bacterial cell growth and bacterial morphogenesis; peptidoglycan (PG) is a crucial component in this process. Murein DD-endopeptidase (MepS), initially annotated as Spr from E. coli K12, is a NlpC/P60 family endopeptidase, which cleaves the meso-diaminopimelate (DAP)-D-Ala peptide bond of PG. The Cys68, His119, His131 triad form the active site residues. MepS has autolytic activity, which is strictly regulated by a periplasmic degradation system comprising the NlpI/Prc protease complex. MepS is essential for maintaining the cell viability, and therefore, it is a potential target for developing antibiotics. This study aimed to understand the structural basis of substrate recognition and degradation. We determined the high-resolution structures of MepS, after mutating Cys68 to serine (MepS-C68S) to improve stability. We further found that citrate and L-malate molecules bind to the active site of MepS-C68S; this is in line with the recurrent observation of organic acids binding to PG endopeptidases. The presence of conserved residues on the surface revealed the potential peptide binding sites of MepS. We modelled a cross-linked peptide model of meso-DAP-D-Ala-meso-DAP, bound to the active site groove of MepS-C68S. Two conserved tyrosine residues, Tyr56 and Tyr147 appeared to be essential for the recognition of peptides. Our structural discoveries could provide insights for the design of novel antibiotics targeting MepS.
RESUMO
Carbapenem-resistant A. baumannii (CRAB) infection can cause acute host reactions that lead to high-fatality sepsis, making it important to develop new therapeutic options. Previously, we developed a short 9-meric peptide, Pro9-3D, with significant antibacterial and cytotoxic effects. In this study, we attempted to produce safer peptide antibiotics against CRAB by reversing the parent sequence to generate R-Pro9-3 and R-Pro9-3D. Among the tested peptides, R-Pro9-3D had the most rapid and effective antibacterial activity against Gram-negative bacteria, particularly clinical CRAB isolates. Analyses of antimicrobial mechanisms based on lipopolysaccharide (LPS)-neutralization, LPS binding, and membrane depolarization, as well as SEM ultrastructural investigations, revealed that R-Pro9-3D binds strongly to LPS and impairs the membrane integrity of CRAB by effectively permeabilizing its outer membrane. R-Pro9-3D was also less cytotoxic and had better proteolytic stability than Pro9-3D and killed biofilm forming CRAB. As an LPS-neutralizing peptide, R-Pro9-3D effectively reduced LPS-induced pro-inflammatory cytokine levels in RAW 264.7 cells. The antiseptic abilities of R-Pro9-3D were also investigated using a mouse model of CRAB-induced sepsis, which revealed that R-Pro9-3D reduced multiple organ damage and attenuated systemic infection by acting as an antibacterial and immunosuppressive agent. Thus, R-Pro9-3D displays potential as a novel antiseptic peptide for treating Gram-negative CRAB infections.
Assuntos
Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Farmacorresistência Bacteriana/genética , Peptídeos/farmacologia , Infecções por Acinetobacter/genética , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/patogenicidade , Anti-Infecciosos Locais/farmacologia , Biofilmes/efeitos dos fármacos , Carbapenêmicos/efeitos adversos , Carbapenêmicos/farmacologia , Humanos , Testes de Sensibilidade MicrobianaRESUMO
Fatty acid synthesis is essential for bacterial viability. Thus, fatty acid synthases (FASs) represent effective targets for antibiotics. Nevertheless, multidrug-resistant bacteria, including the human opportunistic bacteria, Acinetobacter baumannii, are emerging threats. Meanwhile, the FAS pathway of A. baumannii is relatively unexplored. Considering that acyl carrier protein (ACP) has an important role in the delivery of fatty acyl intermediates to other FAS enzymes, we elucidated the solution structure of A. baumannii ACP (AbACP) and, using NMR spectroscopy, investigated its interactions with ß-ketoacyl ACP synthase III (AbKAS III), which initiates fatty acid elongation. The results show that AbACP comprises four helices, while Ca2+ reduces the electrostatic repulsion between acid residues, and the unconserved F47 plays a key role in thermal stability. Moreover, AbACP exhibits flexibility near the hydrophobic cavity entrance from D59 to T65, as well as in the α1α2 loop region. Further, F29 and A69 participate in slow exchanges, which may be related to shuttling of the growing acyl chain. Additionally, electrostatic interactions occur between the α2 and α3-helix of ACP and AbKAS III, while the hydrophobic interactions through the ACP α2-helix are seemingly important. Our study provides insights for development of potent antibiotics capable of inhibiting A. baumannii FAS protein-protein interactions.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Acinetobacter baumannii/enzimologia , Acinetobacter baumannii/metabolismo , Proteína de Transporte de Acila/química , Antibacterianos/química , Sítios de Ligação , Cálcio/química , Dicroísmo Circular , Resistência Microbiana a Medicamentos , Ácidos Graxos/química , Interações Hidrofóbicas e Hidrofílicas , Espectroscopia de Ressonância Magnética , Metais/química , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas , Eletricidade EstáticaRESUMO
Owing to the challenges faced by conventional therapeutics, novel peptide antibiotics against multidrug-resistant (MDR) gram-negative bacteria need to be urgently developed. We had previously designed Pro9-3 and Pro9-3D from the defensin of beetle Protaetia brevitarsis; they showed high antimicrobial activity with cytotoxicity. Here, we aimed to develop peptide antibiotics with bacterial cell selectivity and potent antibacterial activity against gram-negative bacteria. We designed 10-meric peptides with increased cationicity by adding Arg to the N-terminus of Pro9-3 (Pro10-1) and its D-enantiomeric alteration (Pro10-1D). Among all tested peptides, the newly designed Pro10-1D showed the strongest antibacterial activity against Escherichia coli, Acinetobacter baumannii, and MDR strains with resistance against protease digestion. Pro10-1D can act as a novel potent peptide antibiotic owing to its outstanding inhibitory activities against bacterial film formation with high bacterial cell selectivity. Dye leakage and scanning electron microscopy revealed that Pro10-1D targets the bacterial membrane. Pro10-1D inhibited inflammation via Toll Like Receptor 4 (TLR4)/Nuclear factor-κB (NF-κB) signaling pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Furthermore, Pro10-1D ameliorated multiple-organ damage and attenuated systemic infection-associated inflammation in an E. coli K1-induced sepsis mouse model. Overall, our results suggest that Pro10-1D can potentially serve as a novel peptide antibiotic for the treatment of gram-negative sepsis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Besouros/metabolismo , Defensinas/química , Infecções por Escherichia coli/tratamento farmacológico , Lipopolissacarídeos/efeitos adversos , Choque/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/crescimento & desenvolvimento , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Membrana Celular/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Estabilidade de Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/metabolismo , Feminino , Proteínas de Insetos/química , Camundongos , Testes de Sensibilidade Microbiana , NF-kappa B/metabolismo , Células RAW 264.7 , Choque/tratamento farmacológico , Choque/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
Thermotoga maritima, a deep-branching hyperthermophilic bacterium, expresses an extraordinarily stable Thermotoga maritima acyl carrier protein (Tm-ACP) that functions as a carrier in the fatty acid synthesis system at near-boiling aqueous environments. Here, to understand the hyperthermal adaptation of Tm-ACP, we investigated the structure and dynamics of Tm-ACP by nuclear magnetic resonance (NMR) spectroscopy. The melting temperature of Tm-ACP (101.4 °C) far exceeds that of other ACPs, owing to extensive ionic interactions and tight hydrophobic packing. The D59 residue, which replaces Pro/Ser of other ACPs, mediates ionic clustering between helices III and IV. This creates a wide pocket entrance to facilitate the accommodation of long acyl chains required for hyperthermal adaptation of the T. maritima cell membrane. Tm-ACP is revealed to be the first ACP that harbor an amide proton hyperprotected against hydrogen/deuterium exchange for I15. The hydrophobic interactions mediated by I15 appear to be the key driving forces of the global folding process of Tm-ACP. Our findings provide insights into the structural basis of the hyperthermal adaptation of ACP, which might have allowed T. maritima to survive in hot ancient oceans.
Assuntos
Proteína de Transporte de Acila/química , Adaptação Biológica , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Modelos Moleculares , Temperatura , Thermotoga maritima/fisiologia , Proteína de Transporte de Acila/genética , Proteína de Transporte de Acila/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Conformação Proteica , Estabilidade Proteica , Desdobramento de Proteína , Relação Estrutura-Atividade , Temperatura de TransiçãoRESUMO
Originally annotated as the initiator of fatty acid synthesis (FAS), ß-ketoacyl-acyl carrier protein synthase III (KAS III) is a unique component of the bacterial FAS system. Novel variants of KAS III have been identified that promote the de novo use of additional extracellular fatty acids by FAS. These KAS III variants prefer longer acyl-groups, notably octanoyl-CoA. Acinetobacter baumannii, a clinically important nosocomial pathogen, contains such a multifunctional KAS III (AbKAS III). To characterize the structural basis of its substrate specificity, we determined the crystal structures of AbKAS III in the presence of different substrates. The acyl-group binding cavity of AbKAS III and co-crystal structure of AbKAS III and octanoyl-CoA confirmed that the cavity can accommodate acyl groups with longer alkyl chains. Interestingly, Cys264 formed a disulfide bond with residual CoA used in the crystallization, which distorted helices at the putative interface with acyl-carrier proteins. The crystal structure of KAS III in the alternate conformation can also be utilized for designing novel antibiotics.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Acinetobacter baumannii/enzimologia , Sequência de Aminoácidos , Ácidos Graxos/biossíntese , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/patogenicidade , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cisteína/química , Cisteína/metabolismo , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Difração de Raios XRESUMO
Propionibacterium acnes is an anaerobic gram-positive bacterium found in the niche of the sebaceous glands in the human skin, and is a causal pathogen of inflammatory skin diseases as well as periprosthetic joint infection. To gain effective control of P. acnes, a deeper understanding of the cellular metabolism mechanism involved in its ability to reside in this unique environment is needed. P. acnes exhibits typical cell membrane features of gram-positive bacteria, such as control of membrane fluidity by branched-chain fatty acids (BCFAs). Branching at the iso- or anteiso-position is achieved by incorporation of isobutyryl- or 2-methyl-butyryl-CoA via ß-ketoacyl acyl carrier protein synthase (KAS III) from fatty acid synthesis. Here, we determined the crystal structure of P. acnes KAS III (PaKAS III) at the resolution of 1.9â¯Å for the first time. Conformation-sensitive urea polyacrylamide gel electrophoresis and tryptophan fluorescence quenching experiments confirmed that PaKAS III prefers isobutyryl-CoA as the acetyl-CoA, and the unique shape of the active site cavity complies with incorporation of branched-short chain CoAs. The determined structure clearly illustrates how BCFA synthesis is achieved in P. acnes. Moreover, the unique shape of the cavity required for the branched-chain primer can be invaluable in designing novel inhibitors of PaKAS III and developing new specifically targeted antibiotics.
Assuntos
3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Proteínas de Bactérias/metabolismo , Ácidos Graxos/metabolismo , Propionibacterium acnes/metabolismo , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , Acil Coenzima A/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Vias Biossintéticas , Cristalografia por Raios X , Ácidos Graxos/química , Modelos Moleculares , Propionibacterium acnes/química , Propionibacterium acnes/enzimologia , Conformação Proteica , Alinhamento de SequênciaRESUMO
Acyl carrier protein (ACP) is highly conserved across taxa and plays key roles in the fatty acid synthesis system by mediating acyl group delivery and shuttling. Here, we compared the structural and dynamic features of human type Ι ACP (hACP) and Escherichia coli type II ACP (EcACP). Analysis of chemical shift perturbations upon octanoyl group attachment showed perturbations in hACP only near acyl-group attachment sites, whereas EcACP showed the perturbation at residues in the hydrophobic cavity. This difference confirmed that hACP does not sequester the acyl chain in the hydrophobic cavity, which is blocked by hydrophobic triad residues (L34, L39, and V64). Moreover, hACP showed more flexible backbone dynamics than EcACP, especially in the front of α1α2 loop. We further investigated the interactions of hACP with Streptomyces coelicolor ACP synthase (ScAcpS), which is used to convert apo mammalian ACP to the holo form. Similar to protein-protein interface (PPI) found in hACP-hAcpS crystal structure, docking simulation and binding affinity measurements showed that the hydrophobic residues in universal recognition helix II of hACP contribute mainly to ScAcpS binding with binding affinity of 9.2⯱â¯9.1â¯×â¯104â¯M. In contrast, interaction found in EcACP-EcAcpS crystal structure is dominated by electrostatic interactions. These results suggest that ScAcpS has relatively relaxed substrate specificity and a similar charge distribution to hAcpS. These fundamental differences of the charge distribution in hAcpS, ScAcpS and EcAcpS largely affect the interaction with hACP. These findings can provide a useful resource for development of novel antibiotics inhibiting PPI in bacterial FAS proteins with specificity.
Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Streptomyces coelicolor/metabolismo , Proteína de Transporte de Acila/química , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/metabolismo , Humanos , Simulação de Acoplamento Molecular , Conformação Proteica , Mapas de Interação de Proteínas , Alinhamento de Sequência , Streptomyces coelicolor/químicaRESUMO
Recently, bioactive peptides have attracted attention for their therapeutic applications in the pharmaceutical industry. Among them, antimicrobial peptides are candidates for new antibiotic drugs. Since pseudin-2 (Ps), isolated from the skin of the paradoxical frog Pseudis paradoxa, shows broad-spectrum antibacterial activity with high cytotoxicity, we previously designed Ps-K18 with a Lys substitution for Leu18 in Ps, which showed high antibacterial activity and low toxicity. Here, we examined the potency of Ps-K18, aiming to develop antibiotics derived from bioactive peptides for the treatment of Gram-negative sepsis. We first investigated the antibacterial mechanism of Ps-K18 based on confocal micrographs and field emission scanning electron microscopy, confirming that Ps-K18 targets the bacterial membrane. Anti-inflammatory mechanism of Ps-K18 was investigated by secreted alkaline phosphatase reporter gene assays and RT-PCR, which revealed that Ps-K18 activates innate defense via Toll-like receptor 4-mediated nuclear factor-kappa B signaling pathways. Moreover, we investigated the antiseptic effect of Ps-K18 using a lipopolysaccharide or Escherichia coli K1-induced septic shock mouse model. Ps-K18 significantly reduced bacterial growth and inflammatory responses in the septic shock model. Ps-K18 showed low renal and liver toxicity and attenuated lung damage effectively. This study suggests that Ps-K18 is a potent peptide antibiotic that could be applied therapeutically to Gram-negative sepsis.
Assuntos
Proteínas de Anfíbios/química , Anti-Infecciosos Locais/farmacologia , Anti-Inflamatórios/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Endotoxemia/tratamento farmacológico , Endotoxemia/microbiologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/ultraestrutura , Humanos , Macrófagos , Camundongos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Phloretin is a natural chalcone with antibacterial and anti-inflammatory effects. This study investigated the anti-acne activity of phloretin against Propionibacterium acnes-induced skin infection and the potential target proteins of its anti-inflammatory and antibacterial effects. Phloretin potently inhibited the growth of P. acnes and P. acnes-induced Toll-like receptor (TLR) 2-mediated inflammatory signaling in human keratinocytes. Secreted embryonic alkaline phosphatase assay confirmed that the anti-inflammatory activity of phloretin is associated with the P. acnes-stimulated TLR2-mediated NF-κB signaling pathway. Phloretin significantly decreased the level of phosphorylated c-Jun N-terminal kinase (JNK), showing a binding affinity of 1.184 × 10-5 M-1. We also found that phloretin binds with micromolar affinity to P. acnes ß-ketoacyl acyl carrier protein (ACP) synthase III (KAS III), an enzyme involved in fatty acid synthesis. Conformation-sensitive native polyacrylamide gel electrophoresis showed that phloretin reduced KAS III-mediated 3-ketoacyl ACP production by over 66%. A docking study revealed that phloretin interacts with the active sites of JNK1 and KAS III, suggesting their involvement in P. acnes-induced inflammation and their potential as targets for the antibacterial activity of phloretin. These results demonstrate that phloretin may be useful in the prevention or treatment of P. acnes infection.
Assuntos
Antibacterianos/farmacologia , Infecções por Bactérias Gram-Positivas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Floretina/farmacologia , Propionibacterium acnes/efeitos dos fármacos , Dermatopatias Bacterianas/metabolismo , Dermatopatias Bacterianas/microbiologia , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/antagonistas & inibidores , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Antibacterianos/química , Sítios de Ligação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Relação Dose-Resposta a Droga , Infecções por Bactérias Gram-Positivas/tratamento farmacológico , Humanos , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Floretina/química , Propionibacterium acnes/enzimologia , Propionibacterium acnes/imunologia , Ligação Proteica , Dermatopatias Bacterianas/tratamento farmacológico , Relação Estrutura-Atividade , Receptor 2 Toll-Like/metabolismoRESUMO
Isorhamnetin is a flavonoid that is abundant in the fruit of Hippophae rhamnoides L. It is widely studied for its ability to modulate inflammatory responses. In this study, we evaluated the potential of isorhamnetin to prevent gram-negative sepsis. We investigated its efficacy using an Escherichia coli-induced sepsis model. Our study reveals that isorhamnetin treatment significantly enhances survival and reduces proinflammatory cytokine levels in the serum and lung tissue of E. coli-infected mice. Further, isorhamnetin treatment also significantly reduces the levels of aspartate aminotransferase, alanine amino transferase and blood urea nitrogen, suggesting that it can improve liver and kidney function in infected mice. Docking studies reveal that isorhamnetin binds deep in the hydrophobic binding pocket of MD-2 via extensive hydrophobic interactions and hydrogen bonding with Tyr102, preventing TLR4/MD-2 dimerization. Notably, binding and secreted alkaline phosphatase reporter gene assays show that isorhamnetin can interact directly with the TLR4/MD-2 complex, thus inhibiting the TLR4 cascade, which eventually causes systemic inflammation, resulting in death due to cytokine storms. We therefore presume that isorhamnetin could be a suitable therapeutic candidate to treat bacterial sepsis.
Assuntos
Escherichia coli/patogenicidade , Quercetina/análogos & derivados , Sepse/tratamento farmacológico , Sepse/etiologia , Animais , Feminino , Inflamação/tratamento farmacológico , Inflamação/etiologia , Inflamação/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Quercetina/uso terapêutico , Sepse/microbiologia , Ressonância de Plasmônio de Superfície , Receptor 4 Toll-Like/metabolismoRESUMO
Cold-shock proteins (Csps) are expressed at lower-than-optimum temperatures, and they function as RNA chaperones; however, no structural studies on psychrophilic Csps have been reported. Here, we aimed to investigate the structure and dynamics of the Csp of psychrophile Colwellia psychrerythraea 34H, ( Cp-Csp). Although Cp-Csp shares sequence homology, common folding patterns, and motifs, including a five ß-stranded barrel, with its thermophilic counterparts, its thermostability (37 °C) was markedly lower than those of other Csps. Cp-Csp binds heptathymidine with an affinity of 10-7 M, thereby increasing its thermostability to 50 °C. Nuclear magnetic resonance spectroscopic analysis of the Cp-Csp structure and backbone dynamics revealed a flexible structure with only one salt bridge and 10 residues in the hydrophobic cavity. Notably, Cp-Csp contains Tyr51 instead of the conserved Phe in the hydrophobic core, and its phenolic hydroxyl group projects toward the surface. The Y51F mutation increased the stability of hydrophobic packing and may have allowed for the formation of a K3-E21 salt bridge, thereby increasing its thermostability to 43 °C. Cp-Csp exhibited conformational exchanges in its ribonucleoprotein motifs 1 and 2 (754 and 642 s-1), and heptathymidine binding markedly decreased these motions. Cp-Csp lacks salt bridges and has longer flexible loops and a less compact hydrophobic cavity resulting from Tyr51 compared to mesophilic and thermophilic Csps. These might explain the low thermostability of Cp-Csp. The conformational flexibility of Cp-Csp facilitates its accommodation of nucleic acids at low temperatures in polar oceans and its function as an RNA chaperone for cold adaptation.
Assuntos
Alteromonadaceae/química , Proteínas de Bactérias/química , Proteínas e Peptídeos de Choque Frio/química , Alteromonadaceae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas e Peptídeos de Choque Frio/metabolismo , Temperatura Alta , Interações Hidrofóbicas e Hidrofílicas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Alinhamento de Sequência , Timidina/análogos & derivados , Timidina/metabolismo , Tirosina/química , Tirosina/metabolismoRESUMO
Bacterial fatty acid synthesis (FAS) has been extensively studied as a potential target of antimicrobials. In FAS, FabD mediates transacylation of the malonyl group from malonyl-CoA to acyl-carrier protein (ACP). The mounting threat of nosocomial infection by multidrug-resistant Acinetobacter baumannii warrants a deeper understanding of its essential cellular mechanisms, which could lead to effective control of this highly competent pathogen. The molecular mechanisms involved in A. baumannii FAS are poorly understood, and recent research has suggested that Pseudomonas aeruginosa, a closely related nosocomial pathogen of A. baumannii, utilizes FAS to produce virulence factors. In this study, we solved the crystal structure of A. baumannii FabD (AbFabD) to provide a platform for the development of new antibacterial agents. Analysis of the structure of AbFabD confirmed the presence of highly conserved active site residues among bacterial homologs. Binding constants between AbFabD variants and A. baumannii ACP (AbACP) revealed critical conserved residues Lys195 and Lys200 involved in AbACP binding. Computational docking of a potential inhibitor, trifluoperazine, revealed a unique inhibitor-binding pocket near the substrate-binding site. The structural study presented herein will be useful for the structure-based design of potent AbFabD inhibitors.
Assuntos
Acinetobacter baumannii/genética , Proteína de Transporte de Acila S-Maloniltransferase/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Ácido Graxo Sintase Tipo II/genética , Acinetobacter baumannii/enzimologia , Proteína de Transporte de Acila S-Maloniltransferase/química , Proteína de Transporte de Acila S-Maloniltransferase/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Cristalografia por Raios X , Ácido Graxo Sintase Tipo II/química , Ácido Graxo Sintase Tipo II/metabolismo , Modelos Moleculares , Mutação , Domínios Proteicos , Homologia de Sequência de AminoácidosRESUMO
Sepsis is a systemic inflammatory response to pathogenic infection that currently has no specific pharmaceutical interventions. Instead, antibiotics administration is considered the best available option, despite increasing drug resistance. Alternative strategies are therefore urgently required to prevent sepsis and strengthen the host immune system. One such option is tamarixetin (4'- O-methylquercetin), a naturally occurring flavonoid derivative of quercetin that protects against inflammation. The purpose of this study was to determine whether the anti-inflammatory effects of tamarixetin protect against the specific inflammatory conditions induced in lipopolysaccharide (LPS) or Escherichia coli K1 models of sepsis. Our study showed that tamarixetin reduced the secretion of various inflammatory cytokines by dendritic cells after activation with LPS. It also promoted the secretion of the anti-inflammatory cytokine interleukin (IL)-10 and specifically increased the population of IL-10-secreting immune cells in LPS-activated splenocytes. Tamarixetin showed general anti-inflammatory effects in mouse models of bacterial sepsis and decreased bacteria abundance and endotoxin levels. We therefore conclude that tamarixetin has superior anti-inflammatory properties than quercetin during bacterial sepsis. This effect is associated with an increased population of IL-10-secreting immune cells and suggests that tamarixetin could serve as a specific pharmaceutical option to prevent bacterial sepsis.
Assuntos
Anti-Inflamatórios/farmacologia , Dissacarídeos/farmacologia , Interleucina-10/metabolismo , Quercetina/análogos & derivados , Sepse/tratamento farmacológico , Animais , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Escherichia coli/patogenicidade , Feminino , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Quercetina/farmacologia , Sepse/metabolismoRESUMO
Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8 °C, which is much higher than that of Escherichia coli ACP (67.2 °C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four α-helices (helix I (residues 3-17), helix II (residues 39-53), helix III (residues 60-64), and helix IV (residues 68-78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe(45) in helix II and Phe(18) in the α1α2 loop and a hydrogen bonding between Ser(15) in helix I and Ile(20) in the α1α2 loop, resulting in its high thermal stability. Phe(45)-mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser(58) in the α2α3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.
Assuntos
Proteína de Transporte de Acila/química , Proteína de Transporte de Acila/metabolismo , Enterococcus faecalis/metabolismo , Proteína de Transporte de Acila/genética , Cristalografia por Raios X , Enterococcus faecalis/química , Enterococcus faecalis/genética , Temperatura Alta , Ligação de Hidrogênio , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Estabilidade Proteica , Estrutura Secundária de ProteínaRESUMO
The structure and stability of membrane proteins can vary widely in different detergents and this variability has great practical consequences for working with membrane proteins. Nevertheless, the mechanisms that operate to alter the behavior of proteins in micelles are poorly understood and not predictable. Atomic simulations could provide considerable insight into these mechanisms. Building protein-micelle complexes for simulation is fraught with uncertainty, however, in part because it is often unknown how many detergent molecules are present in the complex. Here, we describe several convenient ways to employ Micelle Builder in CHARMM-GUI to rapidly construct protein-micelle complexes and performed simulations of the isolated voltage-sensor domain of voltage-dependent potassium-selective channel and an antimicrobial peptide papiliocin with varying numbers of detergents. We found that once the detergent number exceeds a threshold, protein-detergent interactions change very little and remain very consistent with experimental observations. Our results provide a platform for future studies of the interplays between protein structure and detergent properties at the atomic level. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.