RESUMO
AIM: Antimicrobial resistance (AMR) has become a global concern. Developing novel antimicrobials is one of the most effective approaches in tackling AMR. Considering its relatively low cost and risk, drug repurposing has been proposed as a valuable approach for novel antimicrobial discovery. The aim of this study was to screen for antimicrobial compounds against Streptococcus suis, an important zoonotic bacterial pathogen, from an Food and Drug Administration (FDA)-approved drug library. METHODS AND RESULTS: In this study, we tested the antimicrobial activity of 1815 FDA-approved drugs against S. suis. Sixty-seven hits were obtained that showed a growth inhibition of more than 98%. After excluding already known antibiotics and antiseptics, 12 compounds were subjected to minimal inhibition concentration (MIC) assessment against S. suis. This showed that pralatrexate, daunorubicin (hydrochloride), teniposide, aclacinomycin A hydrochloride and floxuridine gave a relatively low MIC, ranging from 0.85 to 5.25 µg/ml. Apart from pralatrexate, the remaining four drugs could also inhibit the growth of antimicrobial-resistant S. suis. It was also demonstrated that these four drugs had better efficacy against Gram-positive bacteria than Gram-negative bacteria. Cytotoxicity assays showed that floxuridine and teniposide had a relatively high 50% cytotoxic concentration (CC50 ). The in vivo efficacy of floxuridine was analysed using a Galleria mellonella larvae infection model, and the results showed that floxuridine was effective in treating S. suis infection in vivo. CONCLUSIONS: Five compounds from the FDA-approved drug library showed high antimicrobial activity against S. suis, among which floxuridine displayed potent in vivo efficacy that is worth further development. SIGNIFICANCE AND IMPACT OF STUDY: Our study identified several antimicrobial compounds that are effective against S. suis, which provides a valuable starting point for further antimicrobial development.
Assuntos
Anti-Infecciosos , Preparações Farmacêuticas , Infecções Estreptocócicas , Streptococcus suis , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Infecciosos/farmacologia , Anti-Infecciosos/uso terapêutico , Humanos , Testes de Sensibilidade Microbiana , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Estados Unidos , United States Food and Drug AdministrationRESUMO
In this work, we prepared silver nanowires (AgNWs) via the polyol method in the presence or absence of single wall carbon nanotubes (CNTs) and tested their physicochemical, antibacterial and cytotoxic properties. Results showed that the introduction of CNTs lead to the formation of AgNWs at lower temperature, but the final product characteristics of AgNWs and AgNWs-CNT were not significantly different. AgNWs exhibited antibacterial properties against all the studied bacterial species via the formation of oxygen reactive species (ROS) and membrane damage. Furthermore, AgNWs exhibited a dose-dependent and time-dependent toxicity at concentrations ≥ 10 µg/mL. Fibroblasts appeared to be more resistant than human colorectal adenocarcinoma (Caco-2) and osteoblasts to the toxicity of AgNWs. The cytotoxicity of AgNWs was found to be related to the formation of ROS, but not to membrane damage. Overall, these results suggest that AgNWs are potential antibacterial agents against E. coli, S. aureus, MRSA and S. saprophyticus, but their dosage needs to be adjusted according to the route of administration.
Assuntos
Antibacterianos/toxicidade , Nanocompostos/toxicidade , Nanotubos de Carbono/toxicidade , Nanofios/toxicidade , Antibacterianos/química , Células CACO-2 , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Nanocompostos/química , Nanotubos de Carbono/química , Nanofios/química , Espécies Reativas de Oxigênio/metabolismo , Salmonella/efeitos dos fármacos , Prata/químicaRESUMO
The Escherichia coli sensor kinase EnvZ modulates porin expression in response to various stimuli, including extracellular osmolarity, the presence of procaine and interaction with an accessory protein, MzrA. Two major outer membrane porins, OmpF and OmpC, act as passive diffusion-limited pores that allow compounds, including certain classes of antibiotics such as ß-lactams and fluoroquinolones, to enter the bacterial cell. Even though the mechanisms by which EnvZ detects and processes the presence of various stimuli are a fundamental component of microbial physiology, they are not yet fully understood. Here, we assess the role of TM1 during signal transduction in response to the presence of extracellular osmolarity. Various mechanisms of transmembrane communication have been proposed including rotation of individual helices within the transmembrane domain, dynamic movement of the membrane-distal portion of the cytoplasmic domain and regulated intra-protein unfolding. To assess these possibilities, we have created a library of single-Cys-containing EnvZ proteins in order to facilitate sulfhydryl-reactivity experimentation. Our results demonstrate that the major TM1-TM1' interface falls along a single surface consisting of residue positions 19, 23, 26, 30 and 34. In addition, we show that Cys substitutions within the N- and C-terminal regions of TM1 result in drastic changes to EnvZ signal output. Finally, we demonstrate that core residues within TM1 are responsible for both TM1 dimerisation and maintenance of steady-state signal output. Overall, our results suggest that no major rearrangement of the TM1-TM1' interface occurs during transmembrane communication in response to extracellular osmolarity. We conclude by discussing these results within the frameworks of several proposed models for transmembrane communication.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas de Escherichia coli/química , Complexos Multienzimáticos/química , Regulação Alostérica , Substituição de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Cisteína/química , Dimerização , Proteínas de Escherichia coli/genética , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Concentração Osmolar , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Transdução de Sinais/fisiologiaRESUMO
Aromatic tuning, i.e. repositioning aromatic residues found at the cytoplasmic end of transmembrane (TM) domains within bacterial receptors, has been previously shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor EnvZ of Escherichia coli. In the case of Tar, changes in signal output consistent with the vertical position of the native Trp-Tyr aromatic tandem within TM2 were observed. In contrast, within EnvZ, where a Trp-Leu-Phe aromatic triplet was repositioned, the surface that the triplet resided upon was the major determinant governing signal output. However, these studies failed to determine whether moving the aromatic residues was sufficient to physically reposition the TM helix within a membrane. Recent coarse-grained molecular dynamics (CG-MD) simulations predicted displacement of Tar TM2 upon moving the aromatic residues at the cytoplasmic end of the helix. Here, we demonstrate that repositioning the Trp-Tyr tandem within Tar TM2 displaces the C-terminal boundary of the helix relative to the membrane. In a similar analysis of EnvZ, an abrupt initial displacement of TM2 was observed but no subsequent movement was seen, suggesting that the vertical position of TM2 is not governed by the location of the Trp-Leu-Phe triplet. Our results also provide another set of experimental data, i.e. the resistance of EnvZ TM2 to being displaced upon aromatic tuning, which could be useful for subsequent refinement of the initial CG-MD simulations. Finally, we discuss the limitations of these methodologies, how moving flanking aromatic residues might impact steady-state signal output and the potential to employ aromatic tuning in other bacterial membrane-spanning receptors.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Membrana Celular/química , Citoplasma/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Complexos Multienzimáticos/química , Receptores de Superfície Celular/química , Motivos de Aminoácidos , Membrana Celular/metabolismo , Citoplasma/metabolismo , Escherichia coli/metabolismo , Leucina/química , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Fenilalanina/química , Estrutura Secundária de Proteína , Alinhamento de Sequência , Transdução de Sinais , Relação Estrutura-Atividade , Triptofano/químicaRESUMO
Colon cancer is among the most lethal and prevalent malignant tumors in the world, and the lack of effective therapies highlights the need for novel therapeutic approaches. Schisandrin B (Sch B), a lignan extracted from the fruit ofSchisandra chinensis, has been reported for its anticancer properties. However, to date, no studies have been done to characterize the exact molecular mechanisms underlying the antitumorigenic effects of Sch B in colon cancer. This study aimed to explore the antitumorigenic effects of Sch B in colon cancer and to understand the underlying therapeutic mechanism. A comprehensive analysis of the molecular mechanism underlying the antitumorigenic effects of Sch B on human colon cancer cells was performed using a combination of Raman spectroscopy, RNA-seq, computational docking, and molecular biological experiments. The in vivo efficacy was evaluated by a mouse xenograft model. Sch B reduced cell proliferation and triggered apoptosis in human colon cancer cell lines. Raman spectroscopy, computational, RNA-seq, and molecular and cellular studies revealed that Sch B activated unfolded protein responses by interacting with CHOP and upregulating CHOP, which thereby induced apoptosis. CHOP knockdown alleviated the Sch B-induced reduction in cell viability and apoptosis. Sch B reduced colon tumor growth in vivo. Our findings demonstrated that Sch B induced apoptosis and inhibited cell proliferation and tumor growth in vitro and in vivo. These results provided an essential background for clinical trials examining the effects of Sch B in patients with colon cancer.
RESUMO
Baseline signal output and communication between the periplasmic and cytoplasmic domains of the Escherichia coli aspartate chemoreceptor Tar(Ec) are both strongly influenced by residues at the C-terminus of transmembrane helix 2 (TM2). In particular, the cytoplasmic aromatic anchor, composed of residues Trp-209 and Tyr-210 in wild-type Tar(Ec), is important for determining the CheA kinase-stimulating activity of the receptor and its ability to respond to chemoeffector-induced stimuli. Here, we have studied the effect on Tar(Ec) function of the six-residue sequence at positions 207-212. Moving various combinations of aromatic residues among these positions generates substantial changes in receptor activity. Trp has the largest effect on function, both in maintaining normal activity and in altering activity when it is moved. Tyr has a weaker effect, and Phe has the weakest; however, all three aromatic residues can alter signal output when they are placed in novel positions. We also find that Gly-211 plays an important role in receptor function, perhaps because of the flexibility it introduces into the TM2-HAMP domain connector. The conservation of this Gly residue in the high-abundance chemoreceptors of E. coli and Salmonella enterica suggests that it may be important for the nuanced, bidirectional transmembrane signaling that occurs in these proteins.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Ácido Aspártico/metabolismo , Quimiotaxia/fisiologia , Citoplasma/metabolismo , Proteínas de Escherichia coli/genética , Glicina/metabolismo , Metilação , Mutação , Fenilalanina/metabolismo , Estrutura Terciária de Proteína , Receptores de Superfície Celular/genética , Transdução de Sinais , Tirosina/metabolismoRESUMO
Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.
Assuntos
Ácido Aspártico/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Maltose/metabolismo , Proteínas de Membrana/química , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Quimiotaxia , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Transdução de SinaisRESUMO
HAMP domains convert an extracellular sensory input into an intracellular signaling response in a wide variety of membrane-embedded bacterial proteins. These domains are almost invariably found adjacent to the inner leaflet of the cell membrane. We therefore examined the interaction of peptides corresponding to either AS1 or AS2 of four different, well-characterized HAMP domains with several membrane model systems. The proteins included an Archaeoglobus fulgidus protein (Af1503), the Escherichia coli osmosensor EnvZ(Ec), the E. coli nitrate/nitrite sensor NarX(Ec), and the aspartate chemoreceptor of E. coli (Tar(Ec)). Far-UV CD and NMR spectroscopy were used to monitor the induction of secondary structure upon association with neutral or acidic large unilamellar vesicles (LUVs) and bicelles. We observed significant increases in α-helicity within AS1 from NarX(Ec) and Tar(Ec) but not in AS1 from the other proteins. To characterize these interactions further, we determined the solution structure of AS1 from Tar(Ec) associated with acidic bicelles. The bulk of AS1 formed an amphipathic α-helix, whereas the N-terminal control cable, the region between TM2 and AS1, remained unstructured. We observed that the conserved prolyl residue found in AS1 of many membrane-adjacent HAMP domains defined the boundary between the unstructured and helical regions. In addition, two positively charged residues that flank the hydrophobic surface of AS1 are thought to facilitate electrostatic interactions with the membrane. We interpret these results within the context of the helix-interaction model for HAMP signaling and propose roles for AS1-membrane interactions during the membrane assembly and transmembrane communication of HAMP-containing receptors.
Assuntos
Proteínas de Membrana/metabolismo , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/química , Fosfolipídeos/metabolismo , Ligação Proteica , Espectrofotometria UltravioletaRESUMO
Tetraspanins are found in multicellular eukaryotes and are generally thought to act as scaffolding proteins, localizing multiple proteins to a specific region of the cell membrane. Activities for tetraspanins have been identified in several fundamental processes such as motility, cell adhesion, proliferation and viral entry. Tetraspanins are also key players in cancer development and progression. However, structural and biochemical information on tetraspanins is decidely limited, due in no small part to the difficulties associated with expressing eukaryotic membrane proteins. In this study, we have used GFP fusions of a library of human tetraspanin proteins to identify growth conditions for expression in Escherichia coli. Three tetraspanin-GFP proteins could be produced at high enough levels to allow subsequent purification, paving the way for future structural and biochemical studies.
Assuntos
Antígenos de Superfície/biossíntese , Escherichia coli , Proteínas de Neoplasias/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tetraspanina 24/biossíntese , Uroplaquina Ib/biossíntese , Antígenos de Superfície/isolamento & purificação , Soluções Tampão , Cromatografia de Afinidade , Cromatografia em Gel , Detergentes/química , Humanos , Proteínas de Neoplasias/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Solubilidade , Tetraspanina 24/isolamento & purificação , Uroplaquina Ib/isolamento & purificaçãoRESUMO
Dental caries are a worldwide endemic chronic disease affecting people of all ages. Due to the limitations of daily used oral hygiene products, there is an unmet need for new, effective, safe, and economic oral products. We have recently demonstrated that N-(2(2,6-diaminohexanamide)-chitosan (CS3H Lys) has enhanced antibacterial properties against Streptococcus mutans, the main cariogenic bacterium, and here we investigated the effect of fluoridation of this polymer (CS3H Lys F) on its antibacterial properties and the ability to protect teeth from acid demineralization. We further formulated this polymer into mouthwash preparations and studied their cytocompatibility and physicochemical stability over 6 months. CS3H Lys F was 1.6-fold more effective than the highest tested oral NaF dose in preventing acid demineralization. CS3H Lys F has a 3- to 5-fold lower minimum inhibitory concentration value against S. mutants than the values reported for chitosan polymers and showed negligible cell toxicity. The mouthwashes were stable at both 25 and 40 °C. Further work is under way towards other CS3H Lys F oral hygiene products such as a toothpaste.
RESUMO
The development of antibacterial resistance imposes the development of novel materials to relieve the burden of infection. Chitosan, a material of natural and sustainable origin, possesses ideal characteristics to translate into a novel biomaterial with antibacterial properties, as it already has these properties and it allows easy and scalable chemical modification to enhance its activity. The aim of the present work was that of producing low molecular weight chitosans that have higher solubility and can remain protonated at physiological pH, thus enhancing the antimicrobial action. This was achieved by reacting acid hydrolysed low molecular weight chitosan with 2-bromoethyleneamine hydrobromide or Fmoc-Lys(Fmoc)-OH to elicit N-(2-ethylamino)-chitosan and N-2(2,6-diaminohexanamide)-chitosan polymers. The latter derivative, CS3H Lys, that was synthesised for the first time, showed superior efficacy against Staphylococcus aureus, supporting further studies for its inclusion in implant coating materials to tackle the burden of orthopaedic implant-associated infections.
Assuntos
Quitosana , Ortopedia , Infecções Estafilocócicas , Antibacterianos/química , Antibacterianos/farmacologia , Quitosana/química , Humanos , Complicações Pós-Operatórias , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureusRESUMO
Transmembrane helix 2 (TM2) of the Tar chemoreceptor undergoes an inward piston-like displacement of 1 to 3 Å upon binding aspartate. This signal is transmitted to the kinase-control module via the HAMP domain. Within Tar, the HAMP domain forms a parallel four-helix bundle consisting of a dimer of two amphipathic helices connected by a flexible linker. In the nuclear magnetic resonance structure of an archaeal HAMP domain, residues corresponding to the MLLT sequence between Arg-214 at the end of TM2 and Pro-219 of Tar are an N-terminal helical extension of AS1. We modified this region to test whether it behaves as a continuous helical connection between TM2 and HAMP. First, one to four Gly residues were inserted between Thr-218 and Pro-219. Second, the MLLT sequence was replaced with one to nine Gly residues. Third, the sequence was shortened or extended with residues compatible with helix formation. Cells expressing receptors in which the MLLT sequence was shortened to MLL or in which the MLLT sequence was replaced by four Gly residues performed good aspartate chemotaxis. Other mutant receptors supported diminished aspartate taxis. Most mutant receptors had biased signal outputs and/or abnormal patterns of adaptive methylation. We interpret these results to indicate that a strong, permanent helical connection between TM2 and the HAMP domain is not necessary for normal transmembrane signaling.
Assuntos
Células Quimiorreceptoras/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Modelos Moleculares , Mutação , Fragmentos de Peptídeos , Conformação Proteica , Estrutura Terciária de Proteína/fisiologia , Receptores de Superfície CelularRESUMO
A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Cinética , Óperon Lac/genética , Proteínas de Fusão de Membrana/biossíntese , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/metabolismo , Regiões Promotoras Genéticas/genética , Proteoma/metabolismo , Proteínas Virais/metabolismoRESUMO
Eukaryote-like serine/threonine kinases (STKs) and cognate phosphatases (STPs) comprise an important regulatory system in many bacterial pathogens. The complexity of this regulatory system has not been fully understood due to the presence of multiple STKs/STPs in many bacteria and their multiple substrates involved in many different physiological and pathogenetic processes. Streptococci are the best materials for the study due to a single copy of the gene encoding STK and its cognate STP. Although several studies have been done to investigate the roles of STK and STP in zoonotic Streptococcus suis, respectively, few studies were performed on the coordinated regulatory roles of this system. In this study, we carried out a systemic study on STK/STP in S. suis by using a comparative phenotypic, proteomic, and phosphoproteomic analysis. Mouse infection assays revealed that STK played a much more important role in S. suis pathogenesis than STP. The ∆stk and ∆stp∆stk strains, but not ∆stp, showed severe growth retardation. Moreover, both ∆stp and ∆stk strains displayed defects in cell division, but they were abnormal in different ways. The comparative proteomics and phosphoproteomics revealed that deletion of stk or stp had a significant influence on protein expression. Interestingly, more virulence factors were found to be downregulated in ∆stk than ∆stp. In ∆stk strain, a substantial number of the proteins with a reduced phosphorylation level were involved in cell division, energy metabolism, and protein translation. However, only a few proteins showed increased phosphorylation in ∆stp, which also included some proteins related to cell division. Collectively, our results show that both STP and STK are critical regulatory proteins for S. suis and that STK seems to play more important roles in growth, cell division, and pathogenesis.
RESUMO
One-dimensional nanostructures, such as silver nanowires (AgNWs), have attracted considerable attention owing to their outstanding electrical, thermal and antimicrobial properties. However, their application in the prevention of infections linked to bone tissue regeneration intervention has not yet been explored. Here we report on the development of an innovative scaffold prepared from chitosan, composite hydroxyapatite and AgNWs (CS-HACS-AgNWs) having both bioactive and antibacterial properties. In vitro results highlighted the antibacterial potential of AgNWs against both gram-positive and gram-negative bacteria. The CS-HACS-AgNWs composite scaffold demonstrated suitable Ca/P deposition, improved gel strength, reduced gelation time, and sustained Ag⺠release within therapeutic concentrations. Antibacterial studies showed that the composite formulation was capable of inhibiting bacterial growth in suspension, and able to completely prevent biofilm formation on the scaffold in the presence of resistant strains. The hydrogels were also shown to be biocompatible, allowing cell proliferation. In summary, the developed CS-HACS-AgNWs composite hydrogels demonstrated significant potential as a scaffold material to be employed in bone regenerative medicine, as they present enhanced mechanical strength combined with the ability to allow calcium salts deposition, while efficiently decreasing the risk of infections. The results presented justify further investigations into the potential clinical applications of these materials.
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PMMA-based cements are the most used bone cements in vertebroplasty and total hip arthroplasty. However, they present several drawbacks, including susceptibility to bacterial infection, monomer leakage toxicity, and high polymerization temperature, which can all lead to damage to the surrounding tissues and their failure. In the present study, silver nanowires (AgNWs) have been introduced to bestow antibacterial properties; chitosan (CS) to promote porosity and to reduce the polymerization temperature, without negatively affecting the mechanical performance; and methacryloyl chitosan (CSMCC) to promote cross-linking with methyl methacrylate (MMA) and reduce the quantity of monomer required for polymerization. Novel PMMA cements were formulated containing AgNWs (0 and 1% w/w) and CS or CSMCC at various concentrations (0, 10, 20, and 30% w/w), testing two different ratios of powder and MMA (P/L). Mechanical, thermal, antibacterial, and cytotoxic properties of the resulting composite cements were tested. Cements with concentrations of CS > 10% presented a significantly reduced polymerization temperature. The mechanical performances were affected for concentrations > 20% with a P/L concentration equal to 2:1. Concentrations of AgNWs as low as 1% w/w conferred antimicrobial activity against S. aureus, whereas biofilm formation on the surface of the cements was increased when CS was included in the preparation. The combination of CS and AgNWs allowed a higher concentration of Ag+ to be released over time with enhanced antimicrobial activity. Inclusion of AgNWs did not affect cell viability on the scaffolds. In conclusion, a combination of CS and AgNWs may be beneficial for reducing both polymerization temperature and biofilm formation, without significantly affecting mesenchymal stem cell proliferation on the scaffolds. No advantages have been noticed as a result of the reducing P/L ratio or using CSMCC instead of CS.
RESUMO
The Tar chemoreceptor-CheA-CheW ternary complex of Escherichia coli is a transmembrane allosteric enzyme in which binding of ligands to the periplasmic domain modulates the activity of CheA kinase. Kinase activity is also affected by reversible methylation of four glutamyl residues in the cytoplasmic domain of the receptor. E. coli Tar contains 553 residues. Residues 549-553 comprise the NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. The crystal structure of the similar Tsr chemoreceptor predicts that residues 263-289 and 490-515 of Tar form the most membrane-proximal portion of the extended CD1-CD2 four-helix bundle of the cytoplasmic domain. The last methylation site, Glu-491, is in the C19 heptad, and the N22-19 and C22-19 heptads are present in all classes of bacterial transmembrane chemoreceptors. Residues 516-548 probably serve as a flexible tether for the NWETF pentapeptide. Here, we present a mutational analysis of residues 505-548. The more of this region that is deleted, the less sensitive Tar is to inhibition by aspartate. Tar deleted from residue 505 through the NWETF sequence stimulates CheA in vitro but is not inhibited by aspartate. Thus, interaction of the last two heptads (C21 and C22) of CD2 with the first two heptads (N22 and N21) of CD1 must be important for transmitting an inhibitory signal from the HAMP domain to the four-helix bundle. The R514A, K523A, R529A, R540A, and R542A substitutions, singly or together, increase the level of activation of CheA in vitro, whereas the R505A substitution decreases the level of CheA stimulation by 40% and lowers the aspartate K(i) 7-fold. The R505E substitution completely abolishes stimulation of CheA in vitro. Glu-505 may interact electrostatically with Asp-273 to destabilize the "on" signaling state by loosening the four-helix bundle.
Assuntos
Ácido Aspártico/química , Ácido Aspártico/fisiologia , Proteínas de Escherichia coli/antagonistas & inibidores , Oligopeptídeos/fisiologia , Receptores de Aminoácido/fisiologia , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Arginina/genética , Ácido Aspártico/genética , Células Quimiorreceptoras/metabolismo , Metilação de DNA/genética , Análise Mutacional de DNA , Escherichia coli K12/química , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Estrutura Terciária de Proteína/genética , Receptores de Aminoácido/química , Receptores de Aminoácido/genética , Receptores de Superfície Celular , Deleção de SequênciaRESUMO
Aromatic tuning facilitates stimulus-independent modulation of receptor output. The methodology is based upon the affinity of amphipathic aromatic residues, namely Trp and Tyr, for the polar-hydrophobic interfaces found within biological membranes. Here, we describe the application of aromatic tuning within the aspartate chemoreceptor of Escherichia coli (Tar). We have also employed the method within other related proteins, such as sensor histidine kinases (SHKs), and therefore hope that other research groups find it useful to modulate signal output from their receptor of interest.
Assuntos
Aminoácidos Aromáticos/genética , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Quimiotaxia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Histidina Quinase/genética , Histidina Quinase/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Mutagênese Sítio-Dirigida , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Triptofano/genética , Tirosina/genéticaRESUMO
Multidrug resistance (MDR) is a globally relevant problem that requires novel approaches. Two-component systems are a promising, yet untapped target for novel antibacterials. They are prevalent in bacteria and absent in mammals, and their activity can be modulated upon perception of various stimuli. Screening pre-existing compound libraries could reveal small molecules that inhibit stimulus-perception by virulence-modulating receptors, reduce signal output from essential receptors or identify artificial stimulatory ligands for novel SHKs that are involved in virulence. Those small molecules could possess desirable therapeutic properties to combat MDR. We propose that a modular screening platform in which the periplasmic domain of the targeted receptors are fused to the cytoplasmic domain of a well-characterized receptor that governs fluorescence reporter genes could be employed to rapidly screen currently existing small molecule libraries. Here, we have examined two previously created Tar-EnvZ chimeras and a novel NarX-EnvZ chimera. We demonstrate that it is possible to couple periplasmic stimulus-perceiving domains to an invariable cytoplasmic domain that governs transcription of a dynamic fluorescent reporter system. Furthermore, we show that aromatic tuning, or repositioning the aromatic residues at the end of the second transmembrane helix (TM2), modulates baseline signal output from the tested chimeras and even restores output from a nonfunctional NarX-EnvZ chimera. Finally, we observe an inverse correlation between baseline signal output and the degree of response to cognate stimuli. In summary, we propose that the platform described here, a fluorescent Escherichia coli reporter strain with plasmid-based expression of the aromatically tuned chimeric receptors, represents a synthetic biology approach to rapidly screen pre-existing compound libraries for receptor-modulating activities.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Two-component signaling circuits (TCSs) govern the majority of environmental, pathogenic and industrial processes undertaken by bacteria. Therefore, controlling signal output from these circuits in a stimulus-independent manner is of central importance to synthetic microbiologists. Aromatic tuning, or repositioning the aromatic residues commonly found at the cytoplasmic end of the final TM helix has been shown to modulate signal output from the aspartate chemoreceptor (Tar) and the major osmosensor (EnvZ) of Escherichia coli. Aromatic residues are found in a similar location within other bacterial membrane-spanning receptors, suggesting that aromatic tuning could be harnessed for a wide-range of applications. Here, a brief synopsis of the data underpinning aromatic tuning, the initial successes with the method and the inherent advantages over those previously employed for modulating TCS signal output are presented.